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[ { "from": "human", "value": "Full Text: Mycoplasma pneumoniae is a common cause of upper and lower respiratory tract infections. It remains one of the most frequent causes of atypical pneumonia particu-larly among young adults. [1, 2, 3, 4, 5] Although it is highly transmissible, most infections caused by this organism are relatively minor and include pharyngitis, tracheobronchitis, bronchiolitis, and croup with one fifth of in-fections being asymptomatic. [6, 7] Only 3 -10% of infected subjects develop symptoms consistent with bronchopneumonia and mortality from infection is rare. [6, 7] The organism is fastidious and difficult to grow on cultures. Therefore, diagnosis of infections caused by this organism is usually confirmed with serological tests or polymerase chain reaction-gene amplification techniques. At King Abdulaziz University Hospital (KAUH), Jeddah, Saudi Arabia, the facility to perform Mycoplasma culture has been available since January 1997. As published information concerning M. pneumoniae infections in Saudi Arabia is scarce, [8, 9, 10] we wished to study the epidemiology and clinical features of cultureproven infections caused by this organism at this hospital.\nKAUH is a tertiary care teaching hospital with a bed capacity of 265 beds and annual admissions of 18000 to 19000 patients. Patients with M. pneumoniae positive cultures from respiratory specimens were identified over a 24-months\" period from January, 1997 through December, 1998 for this review.\nDuring the study period, respiratory specimens (sputum, nasopharyngeal aspiration, endotracheal secretion, and bronchoalveolar lavage) for M. pneumoniae culture were obtained from patients with upper or lower respiratory tract infections seen as inpatients or in the outpatient or emergency departments. Respiratory specimens were aslo Gram-stained and cultured for bacteria and viruses. M. pneumoniae serological tests for IgG or IgM were not available at KAUH during the study period. All positive culture results were obtained from the Microbiology laboratory records. Charts of patients were reviewed with standardized data collection. Information collected included patients' demographics, comorbidities, clinical manifestations, complications, and outcome.\nM. pneumoniae was cultured using the classic M. pneumoniae agar medium (M.P. agar) and the Pneumofast tray (Pneumofast ® , International Microbio, Signes, France). Specimens were processed according to the instructions of the manufacturer. The M.P. agars and Pneumofast trays were incubated anaerobically at 37°C and inspected daily for 4 weeks. The organism was identified based on typical colonial morphology (granular colonies, rarely fried-egg-like, 10-150 ∝ in diameter) on the M.P. agar medium and the change in the Pneumofast broth color from red to orange then to yellow (glucose fermentation) in the absence of turbidity of the broth. Antibiotic sensitivity profile on the Pneumofast tray was also used for identification according to the instructions of the manufacturer. Bacterial and viral cultures were performed using standard methods.\nM. pneumoniae isolates were considered community-acquired if they were recovered from unhospitalized patients or within 72 hours of admission to the hospital, and nosocomial if they were recovered beyond that period.\nPneumonia was diagnosed based on clinical symptoms and signs, along with radiographic evidence of pneumonia when possible. Severe pneumonia was defined as pneumonia associated with tachycardia (>140 /minute), tachypnoea (>30/minute), hypotension (Systolic blood pressure <90 mmHg), hypoxemia (arterial oxygen partial pressure <8 kPa or oxygen saturation <90%), and/or more than 2 areas of consolidation.\nOutcome of patients with M. pneumoniae infection was classified into 4 categories; uneventful recovery, recovery following complications, death due to M. pneumoniae infection, or death unrelated to M. pneumoniae infection.\nThe Statistical Package for Social Sciences (SPSS) program was used for data analysis. Comparison of categorical data was by Chi-square statistic or Fisher's exact test for small expected values.\nA total of 40 respiratory specimens from 40 patients were positive for M. pneumoniae over the 24-months study period. The demographic and epidemiological characteristics of the patients are summarized in Table 1 . Of all isolates, 37 (92.5%) were community-acquired and 3 (7.5%) were nosocomial. Thirty-three (82.5%) patients required admission to the hospital and the remaining 7 (17.5%) were treated as outpatients. Twenty-four isolates (60%) were associated with pneumonia, 14 (35%) with upper respiratory tract infections, and 2 (5%) with bronchiolitis. Of the 24 cases of pneumonia, 21 were confirmed radiologically and the remaining 3 were diagnosed clinically. The two cases of bronchiolitis occurred in 2 children, one and three years old. Thirty-one patients (77.5%) had comorbidities. Eleven patients (27.5%) had cardiopulmonary comorbidities (asthma, 8, lung fibrosis, 1, congestive heart failure, 1, congenial heart disease, 1), 9 patients (22.5%) were immunocompromised (malignancy, 7, steroid therapy, 3, Human immunodeficiency virus infection, 1), and 11 patients (27.5%) had other comorbidities (premature newborns, 2, and one each of myelodysplastic syndrome, myelopro-liferative disorder, sickle cell anemia, Evan's syndrome, Down syndrome, sarcoidosis, demyelinating disease, cerebral palsy, and spinal muscle atrophy). Organisms concomitantly isolated with M. pneumoniae from the respiratory tract included herpes simplex virus type 1 (2 occasions), adenovirus (2 occasions), cytomegalo virus (1 occasion), respiratory syncytial virus (1 occasion), and bacterial isolates (2 occasions: Acinetobacter species, 1, and Enter obacter cloacae, 1).\nClinical manifestations associated with M. pneumoniae infections are summarized in Table 2 . Pneumonia was more common than upper respiratory tract infections (57.5 % versus 27.5%, respectively). Immunocompromised patients were more likely to present with pneumonia as opposed to upper respiratory tract infection or bronchiolitis than non-immunocompromised patients (8/9 versus 16/31, P = 0.05). Similarly, there was a tendency for patients 60 years of age or older to present with pneumonia more frequently than those below 60 (4/4 versus 20/36, P = 0.1). Of the 24 patients with clinically or radiologically confirmed pneumonia, 19 (79.2%) had crepitations and only 6 (25%) had bronchial breath sounds on physical examination. Of the 16 patients in whom wheezes were detected, 9 (56.3%) were not known to have asthma or other obstructive airway disease. Table 3 . Of the 24 patients with pneumonia, 21 (87.5%) were admitted to the hospital, and 20 (83.3%) had comorbidities. All patients with upper respiratory tract infections (11 patients) or bronchiolitis (2 patients) had uneventful recovery. Of the 24 patients with pneumonia, 14 (58.3%) had uneventful recovery, 4 (16.7%) recovered following some complications (acute respiratory distress syndrome, 2, respiratory failure, 1, septic shock, 1), 3 (12.5%) died because of M pneumoniae infection, and 3 (12.5%) died due to underlying comorbidities. The 3 patients who died of M pneumoniae pneumonia had other comorbidities; one had congestive heart failure, the second had congenital heart disease, and the third was a 3months old infant born prematurely at 32 weeks of gestation who previously had 3 episodes of pneumonia due to other pathogens.\nMycoplasma pneumoniae is one of the most common causes of atypical pneumonia accounting for 5-23% of community-acquired pneumonia, [1, 2, 3, 4, 5] In a study of 511 children with acute respiratory tract infection in Riyadh, Saudi Arabia, Mycoplasma pneumoniae was found to be the second most common causative agent after Respiratory syncytial virus (RSV) accounting for 9% of all cases, [8] In a study of 112 adult patients with community acquired pneumonia admitted to a military hospital in Riyadh, Saudi Arabia, this organism accounted for 6% of all cases, [9] In another retrospective study of 567 pneumonic episodes in adult patients from Al-Qassim area, the organism accounted for 23% of all episodes, [10] The organism also causes other relatively minor infections such as pharyngitis, tracheobronchitis, bronchiolitis, and croup. It is transmitted from person-to-person by infected respiratory droplets during close contact. It is most common in school-aged children, military recruits, and college students. [11] Most cases occur singly or as family outbreaks. Larger outbreaks can also occur in closed populations such as military recruit camps or boarding schools, [12] Infection occurs most frequently during the fall and winter in temperate climates but may develop year-round, [13] The average incubation period is 3 weeks following exposure, [6] Although rare, complications are protean and may involve virtually any organ system such as the respiratory system (e.g.: pleurisy, pneumothorax, acute respiratory distress syndrome, lung abscess), the hematologic system (e.g.: hemolytic anemia, intravascular coagulation, thrombocytopenia), the dermatologic system (e.g.: maculopapular or urticarial rashes, erythema multiforme, erythema nodosum), the musculoskeletal system (e.g.: myalgias, arthralgias, arthritis), the cardiovascular system (e.g.: pericarditis, myocarditis), the nervous system (e.g.: meningoencephalitis, Guillain-Barre syndrome, neuropathies, acute psychosis), or the eye (optic disc edema, optic nerve atrophy, retinal exudation and hemorrhages). [6, 7, 14, 15, 16, 17, 18] Immunity following infection is not long lasting. [11] In our study, the infection affected all age groups but was most common in infants (32.5%) and preschool children (22.5%), and least common in adults aged 15 to 30 years (2.5%) and elderly above 70 years of age (5%). This contrasts with data from temperate countries where the infection is most common in school-aged children, and young adults. [11] One possible explanation for this difference is that infants and preschool children perhaps had more severe infections than did school-aged children, and young adults which prompted presentation of the former group to the hospital. The infection occurred year-round but was most common in the fall (35%), and spring (30%), and least common in the summer (10%). Most infections were community-acquired (92.5%).\nMore than one half of patients (57.5%) presented with pneumonia, and about a third (27.5%) presented with upper respiratory tract infection, Immunocompromised patients and patients 60 years of age or older were more likely to present with pneumonia as opposed to upper respiratory tract infection than non-immunocompromised patients or those below 60 years of age. Cough (82.5%), fever (75%), and malaise (58.8%) were the most common presenting symptoms. Cough was usually dry or slightly productive of white sputum and mild to moderate in severity. Most febrile patients had mild to mod- erate fever of 39°C or less; high-grade fever of more than 39°C was rare. Crepitations (60%), and wheezes (40%) were the most common signs. Wheezes were as common in patients with no history of obstructive airway disease (9 patients) as it was in those with such a history (7 patients). Bronchial breathing as a sign of consolidation was detected in only one fourth of patients with pneumonia, which is consistent with the known disparity between clinical and radiological signs of M pneumoniae pneumonia. Crepitations, however, were detected in the majority (79.2%) of patients. Pleuritic chest pain and pleural effusion were rare.\nMore than half (56.5%) of the patients with pneumonia had uneventful recovery. Mortality from M. pneumoniae pneumonia was high (12.5%) and occurred only in patients with underlying comorbidities. None of the 9 patients with no underlying comorbidities died of M pneumoniae pneumonia. The relatively high complications rate (16.7%) and mortality (12.5%) related to M. pneumoniae pneumonia are likely due to selection bias as most patients with pneumonia were sick enough to require admission to the hospital (21/24 or 87.5%) and most of them had comorbidities (20/24 or 83.3%).\nIn conclusion, our data shed some light on the epidemiology and clinical features of M pneumoniae infections in one of the Saudi tertiary care centers. The data are comparable to those of other countries except for the finding that infections were more common in infants and preschool children than in school children and young adults. Additionally, mortality attributable to M. pneumoniae pneumonia was relatively high in patients with comorbidities. It is hoped this information will assist clinicians in their approach and management of respiratory tract infections." }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: Since its discovery as a biological messenger molecule more than 10 years ago, the gaseous molecule nitric oxide (NO • ) is now well recognized for its involvement in diverse biological processes, including vasodilation, bronchodilation, neurotransmission, tumor surveillance, antimicrobial defense and regulation of inflammatory-immune processes [1] [2] [3] . In the respiratory tract, NO • is generated enzymically by three distinct isoforms of NO • synthase (NOS-1, NOS-2 and NOS-3) that are present to different extents in numerous cell types, including airway and alveolar epithelial cells, neuronal cells, macrophages, neutrophils, mast cells, and endothelial and smoothmuscle cells. In contrast with the other two NOS isoforms (NOS-1 and NOS-3), which are expressed constitutively and activated by mediator-induced or stress-induced cell activation, NOS-2 activity is primarily regulated transcriptionally and is commonly induced by bacterial products and pro-inflammatory cytokines. As such, inflammatory diseases of the respiratory tract, such as asthma, acute respiratory distress syndrome (ARDS) and bronchiectasis, are commonly characterized by an increased expression of NOS-2 within respiratory epithelial and inflammatory-immune cells, and a markedly elevated local production of NO • , presumably as an additional host defense mechanism against bacterial or viral infections. The drawback of such excessive NO • production is its accelerated metabolism to a family of potentially harmful reactive nitrogen species (RNS), including peroxynitrite (ONOO -) and nitrogen dioxide (NO 2 • ), especially in the presence of phagocyte-generated oxidants. The formation of such RNS is thought to be the prime reason why NO • can in many cases contribute to the etiology of inflammatory lung disease [4] [5] [6] . Despite extensive research into both pro-inflammatory and anti-inflammatory actions of NO • , the overall contribution of NO • to inflammatory conditions of the lung is not easily predicted and seems to depend on many factors, such as the site, time and degree of NO • production in relation to the local redox status, and the acute or chronic nature of the immune response. In addition, our current understanding of the pro-inflammatory or pro-injurious mechanisms of NO • or related RNS is incomplete; this commentary will focus primarily on these latter aspects.\nTo explore a role for NO • (or NOS) in infectious or inflammatory diseases, two general research approaches have been taken: the use of pharmacological inhibitors of NOS isoenzymes, and the targeted deletion of individual NOS enzymes in mice. Both approaches suffer from the shortcoming that animal models of respiratory tract diseases generally do not faithfully reflect human disease. The use of NOS inhibitors to determine the contribution of individual NOS isoenzymes is also hindered by problems related to specificity and pharmacokinetic concerns. However, the unconditional gene disruption of one or more NOS isoforms, leading to lifelong deficiency, can have a markedly different outcome from pharmacological inhibition at a certain stage of disease, as the involvement of individual NOS isoenzymes can be different depending on disease stage and severity. Despite these inherent limitations, studies with the targeted deletion of NOS isoforms have led to some insights, indicating a role for NO • and NOS-2 in the etiology of some inflammatory lung diseases. For instance, mice deficient in NOS-2 are less susceptible to lethality after intranasal inoculation with influenza A virus, suffer less lung injury after administration of endotoxin, and display reduced allergic eosinophilia in airways and lung injury in a model of asthma, than their wild-type counterparts [7] [8] [9] . However, although the contribution of NOS-2 is expected in inflammatory conditions, recent studies have determined that NOS-1, rather than NOS-2, seems to be primarily involved in the development of airway hyper-reactivity in a similar asthma model [10] . The linkage of NOS-1 to the etiology of asthma was more recently supported in asthmatic humans by an association of a NOS-1 gene polymorphism with this disease, although the physiological basis for this association remains unclear [11] .\nDespite the potential contribution of NOS-2-derived NO • to lung injury after endotoxemia, the sequestration of neutrophils in the lung and their adhesion to postcapillary and postsinusoidal venules after administration of endotoxin were found to be markedly increased in NOS-2-deficient mice, and NOS-2 deficiency did not alleviate endotoxininduced mortality. It therefore seems that the 'harmful' and 'protective' effects of NOS-2 might contend with each other within the same model, which makes the assessment of the potential role of NOS in human disease even more difficult. In this context, it is interesting to note that humans or animals with cystic fibrosis have subnormal levels of NOS-2 in their respiratory epithelium, related to a gene mutation in the cystic fibrosis transmembrane conductance regulator [12] . This relative absence of epithelial NOS-2 might be one of the contributing factors behind the excessively exuberant respiratory tract inflammatory response in patients with cystic fibrosis, even in the absence of detectable respiratory infections. Overall, the apparently contrasting findings associated with NOS deficiency, together with concerns about animal disease models used, make interpretations and conclusions with regard to human lung disease all the more difficult.\nPharmacological inhibitors of NOS have also been found to reduce oxidative injury in several animal models of lung injury, such as ischemia/reperfusion, radiation, paraquat toxicity, and endotoxemia (see, for example, [13] [14] [15] ). However, results are again not always consistent, and in some cases NOS inhibition has been found to worsen lung injury, indicating anti-inflammatory or protective roles for NO • . All in all, despite these inconsistencies, there is ample evidence from such studies to suggest a contributing role of NO • in various respiratory disease conditions, which continues to stimulate research into mechanistic aspects underlying such pro-inflammatory roles and modulation of NO • generation as a potential therapeutic target.\nAlthough the pro-inflammatory and injurious effects of NO • might be mediated by a number of diverse mechanisms, it is commonly assumed that such actions are largely due to the generation of reactive by-products generated during the oxidative metabolism of NO • ; these are collectively termed RNS. One of the prime suspects commonly implicated in the adverse or injurious properties of NO • is ONOO -, a potent oxidative species formed by its almost diffusion-limited reaction with superoxide (O 2\n•-), which is a product of activated phagocytes and of endothelial or epithelial cells [4, 5, 13] . The formation of ONOOseems highly feasible under conditions of elevated production of both NO • and O 2\n•in vivo, and its oxidative and cytotoxic potential is well documented [5, 6] . However, because the direct detection of ONOOunder inflammatory conditions is virtually impossible because of its instability and high reactivity, the formation of ONOOin vivo can be demonstrated only by indirect methods. Thus, many investigators have relied on the analysis of characteristic oxidation products in biological molecules, such as proteins and DNA, most notably free or protein-associated 3-nitrotyrosine, a product of tyrosine oxidation that can be formed by ONOO -(and several other RNS) but not by NO • itself (see, for commentary review reports primary research http://respiratory-research.com/content/1/2/067 example, [5] ). Indeed, elevated levels of 3-nitrotyrosine have been observed in many different inflammatory conditions of the respiratory tract [16] , which illustrates the endogenous formation of ONOOor related RNS in these cases. However, without known evidence for functional consequences of (protein) tyrosine nitration, the detection of 3-nitrotyrosine should not be regarded as direct proof of a pro-inflammatory role of NO • . Moreover, although the detection of 3-nitrotyrosine has in most cases been interpreted as conclusive evidence for the formation of ONOOin vivo (see, for example, [17] ), it should be realized that other RNS formed by alternative mechanisms might also contribute to endogenous tyrosine nitration. Indeed, it has recently become clear that the presence of inflammatory-immune cells, and specifically their heme peroxidases myeloperoxidase (MPO) and eosinophil peroxidase (EPO), can catalyze the oxidization of NO • and/or its metabolite NO 2 to more reactive RNS and thereby contribute to protein nitration [16, 18, 19] . This notion is further supported by the fact that 3-nitrotyrosine is commonly detected in tissues affected by active inflammation, mostly in and around these phagocytic cells and macrophages, which can also contain active peroxidases originating from apoptotic neutrophils or eosinophils. Hence, the detection of 3-nitrotyrosine in vivo cannot be used as direct proof of the formation of ONOO -, but merely indicates the formation of RNS by multiple oxidative pathways, possibly including ONOObut more probably involving the activity of phagocyte peroxidases [16, 20] . In this regard, a preliminary study with EPO-deficient mice has recently demonstrated the critical importance of EPO in the formation of 3-nitrotyrosine in a mouse model of asthma [21] . Future studies with animals deficient in MPO and/or EPO will undoubtedly help to clarify this issue.\nGiven the considerable interest in 3-nitrotyrosine as a collective marker of the endogenous formation of NO •derived RNS, the crucial question remains of whether the detection of 3-nitrotyrosine adequately reflects the toxic or injurious properties of NO • . The formation of ONOO -(or of other RNS that can induce tyrosine nitration) might in fact represent a mechanism of decreasing excessive levels of NO • that might exert pro-inflammatory actions by other mechanisms. For instance, NO • can promote the expression of pro-inflammatory cytokines or cyclo-oxygenase (responsible for the formation of inflammatory prostanoids) by mechanisms independent of ONOO - [22, 23] , and the removal of NO • would minimize these responses. Furthermore, although ONOOor related NO •derived oxidants can be cytotoxic or induce apoptosis, these effects might not necessarily relate to their ability to cause protein nitration (see, for example, [16]). For instance, the bactericidal and cytotoxic properties of ONOOare minimized by the presence of CO 2 , even though aromatic nitration and other radical-induced modifications are enhanced [5] . Similarly, the presence of NO 2 in the incubation medium decreases the cytotoxicity of MPO-derived hypochlorous acid (HOCl) toward epithelial cells or bacteria, despite increased tyrosine nitration of cellular proteins (A van der Vliet and M Syvanen, unpublished data). Thus, it would seem that the cytotoxic properties of NO • and/or its metabolites might instead be mediated through preferred reactions with other biological targets, and these might not necessarily be correlated with the degree of tyrosine nitration. The extent of nitrotyrosine immunoreactivity in bronchial biopsies of asthmatic patients was correlated directly with measured levels of exhaled NO • and inversely with the provocation concentration for methacholine (PC 20 ) and forced expiratory volume in 1 s [24] . However, an immunohistochemical analysis of nitrotyrosine and apoptosis in pulmonary tissue samples from lung transplant recipients did not identify patients with an imminent risk of developing obliterative bronchiolitis [25] . It is therefore still unclear to what degree tyrosine nitration relates to disease progression.\nSeveral studies with purified enzymes have suggested that nitration of critical tyrosine residues adversely affects enzyme activity, but there is as yet no conclusive evidence in vivo for biological or cellular changes as a direct result of tyrosine nitration [16, 20] . For instance, tyrosine nitration was suggested to have an effect on cellular pathways by affecting cytoskeletal proteins or tyrosine phosphorylation, thereby affecting processes involved in, for example, cell proliferation or differentiation [16, 26] . Recent studies have provided support for selective tyrosine nitration within certain proteins [27, 28] and of selective cellular targets for nitration by RNS (see, for example, [29, 30] ), and such specificity might indicate a potential physiological role for this protein modification. However, in none of these cases could tyrosine nitration be linked directly to changes in enzyme function. Chemical studies have indicated that tyrosine nitration by RNS accounts for only a minor fraction of oxidant involved, and reactions with other biological targets (thiols, selenoproteins, or transition metal ions) are much more prominent [5, 6] . Indeed, the extent of tyrosine nitration in vivo is very low (1-1000 per 10 6 tyrosine residues according to best estimates [16]), although different analytical methods used to detect 3-nitrotyrosine in biological systems have often given inconsistent results. It is important to note that recent rigorous studies have unveiled substantial sources of artifact during sample preparation, which might frequently have led to an overestimation of tyrosine nitration in vivo in previous studies [31] .\nOn the basis of current knowledge, the formation of 3-nitrotyrosine seems to be merely a marker of NO •derived oxidants, with as yet questionable pathophysiological significance. In view of the low efficiency of tyrosine nitration by biological RNS, and the endogenous presence of variable factors that influence protein nitration (antioxidants or other RNS scavengers), it seems unlikely that tyrosine nitration is a reliable mechanism of, for example, enzyme regulation. Nevertheless, the recent discovery of enzymic 'denitration' mechanisms that can reverse tyrosine nitration [32] merits further investigation of the possibility that tyrosine nitration might reflect a signaling pathway, for example analogous to tyrosine phosphorylation or sulfation.\nThe biological effects of NO • are mediated by various actions, either by NO • itself or by secondary RNS, and the overall biochemistry of NO • is deceptively complex. Moreover, the metabolism and chemistry of NO • depend importantly on local concentrations and pH; the recently described acidification of the airway surface in asthmatics [33] might significantly affect NO • metabolism in these patients. It is well known that interactions with the ion centers of iron or other transition metals are responsible for many of the signaling properties of NO • ; the activation of the heme enzyme guanylyl cyclase and the consequent formation of cGMP is involved not only in smooth-muscle relaxation but also in the activation of certain transcription factors, the expression of several pro-inflammatory and anti-inflammatory genes (including cytokines and cyclo-oxygenase), and the production of respiratory mucus [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] . In addition to such direct signaling properties, many actions of NO • might be due largely to secondary RNS that can react with multiple additional targets, in some cases forming nitroso or nitro adducts as potentially unique NO • -mediated signaling mechanisms. As discussed, the formation of protein nitrotyrosine has been postulated as a potential RNSspecific signaling pathway. Even more interest has been given to the reversible S-nitros(yl)ation of protein cysteine residues, which has been proposed to affect a number of redox-sensitive signaling pathways, for example by the activation of p21 ras or the inhibition of protein tyrosine phosphatases [35, 36] . Similar modifications of reactive cysteine residues in transcription factors such as nuclear factor-κB or of caspases contribute to the regulation of gene expression and apoptosis [37] [38] [39] . The precise mechanisms leading to protein S-nitrosylation in vivo are still not clarified, but might involve dinitrogen trioxide (formed during the autoxidation of NO • ), iron-nitrosyl complexes, and perhaps ONOO - [16] ; changes in NO • metabolism during inflammatory lung diseases undoubtedly affect such NO • -dependent signaling pathways. In addition, S-nitrosylation can be reversed by either enzymic (thioredoxin or glutaredoxin) or chemical (metals or oxidants) mechanisms, and evidence is increasing that this reversible modification is complementary to more widely accepted oxidant-dependent redox signaling pathways [40] . The reported alterations in S-nitrosothiol levels in tracheal secretions of patients with asthma or cystic fibrosis further point to altered NO • metabolism in these cases, and might provide new clues to the role of S-nitrosylation in controlling such disease processes [41, 42] . Unfortunately, technical limitations to detect S-nitrosylation in specific protein targets in vivo have limited a full understanding of this potential signaling pathway; further research in these areas can be expected to establish more clearly its significance in the pathophysiological properties of NO • .\nDespite the by now overwhelming evidence for the increased formation of NO • and NO • -derived oxidants in many different lung diseases, the exact contribution of NO • or its metabolites to inflammatory lung disease is still unclear. Indeed, NO • might have distinctly different roles in different stages of respiratory tract inflammatory diseases, being pro-inflammatory or pro-injurious in acute and severe stages but perhaps being protective and antiinflammatory in more stable conditions; it is uncertain whether NOS is a suitable therapeutic target in the management of inflammatory lung disease. Caution is clearly needed when interpreting observations of tyrosine nitration in animal models of disease or in human tissues, which does not automatically implicate ONOO -(as often thought), but rather indicates the formation of RNS by various mechanisms. Furthermore, animal models of chronic lung disease that usually reflect short-term or acute inflammation might not always be applicable to chronic airway diseases in humans. For instance, phagocyte degranulation, a common feature observed in association with human airway inflammatory diseases such as asthma, does not seem to occur in mouse models of asthma [43] . Therefore the importance of granule proteins, such as heme peroxidases, in the pathology of human airway diseases might not be adequately reflected in such animal models. More work with animal models more characteristic of human diseases or with biopsy materials from human subjects will be required to unravel the precise role of NO • in inflammatory lung disease, and might establish more clearly whether the pharmacological inhibition of NOS isoenzymes can be beneficial. This brings up the interesting paradox that, despite presumed adverse roles of NO • in such inflammatory lung diseases as septic shock and ARDS, NO • inhalation has been suggested as a potential therapeutic strategy to improve overall gas exchange [44] . Intriguingly, in a rat model of endotoxemia, inhalation of NO • was found to reduce neutrophilic inflammation and protein nitration [45] , again supporting the crucial involvement of inflammatory-immune cells in this protein modification.\nFor a better assessment of the role of NO • in respiratory tract diseases in humans, the production of RNS and/or characteristic markers would need to be more carefully monitored during various disease stages. Care should be given to analytical techniques, their quantitative capacity and the possibility of artifacts. The monitoring of exhaled NO • , although convenient and non-invasive, does not reflect the actual production or fate of NO • in the respiratory tract and is not well correlated with NOS activity in the lung [46] . We therefore need to continue research into the local biochemistry of NO • in the lung, taking into account the presence of secreted or phagocyte peroxidases and possible changes in local pH, as in asthmatic airways [33] , that might modulate NO • activity and metabolism. This might result in a better understanding of relationships between the various metabolic endproducts of NO • (NO 2 -, NO 3 -, or nitroso and nitro adducts) and its pro-inflammatory or injurious properties." }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: Surfactant protein-D (SP-D) is a member of the collagenous subfamily of calcium-dependent lectins (collectins) that includes pulmonary surfactant protein A (SP-A) and the serum mannose-binding lectin [1] [2] [3] . Collectins inter-act with a wide variety of microorganisms, lipids, and organic particulate antigens, and can modulate the function of immune effector cells and their responses to these ligands. This article reviews what is currently known about the sites of production, structure, function, and regulated expression of SP-D. Emphasis will be placed on functional attributes, known ligand interactions, and structure-function relationships believed to be important for host defense. For additional information on SP-A and other members of the collectin family, the reader is referred to other recent reviews [4] [5] [6] .\nSP-D is synthesized and secreted into the airspaces of the lung by the respiratory epithelium [1] . At the alveolar level, SP-D is constitutively synthesized and secreted by alveolar type II cells. More proximally in the lung, SP-D is secreted by a subset of bronchiolar epithelial cells, the non-ciliated Clara cells. Because SP-D is stored within the secretory granules of Clara cells [7, 8] , it seems likely that SP-D is subject to regulated secretion via granule exocy-tosis at this level of the respiratory tract. In some species, SP-D is also synthesized by epithelial cells and/or submucosal glands associated with the bronchi and trachea [9] . Although many alveolar macrophages show strong cytoplasmic and/or membrane labeling with antibody against SP-D, they do not contain detectable SP-D message.\nThe lung seems to be the major site of SP-D production. However, there is increasing evidence for extrapulmonary sites of expression as assessed with monoclonal or affinity-purified antibodies, reverse-transcriptase-mediated PCR (RT-PCR), and/or hybridization assays of tissues from humans and other large mammals [10 • ,11-14] (summarized in Table 1 ). It is difficult to entirely exclude crossreactions or amplification of related sequences; however, localization to many of these sites in human tissues was confirmed by using monoclonal antibodies in combination with RT-PCR with sequencing of the amplified products [10 • ]. Non-pulmonary expression seems to be largely restricted to cells lining epithelial surfaces or ducts and certain glandular epithelial cells that are in direct or indirect continuity with the environment. Notable exceptions to this generalization might include heart, brain, pancreatic islets, and testicular Leydig cells. SP-D has also been identified in amnionic epithelial cells by immunohistochemistry [15] ; however, it is unclear whether this is synthesized locally or derived from the lung by way of the amniotic fluid. Interestingly, in many of these sites SP-D microscopically co-localizes with gp-340, an SP-D binding protein and putative SP-D receptor [10 • ].\nSites of extrapulmonary expression have also been described in small mammals. In the rat, SP-D message was identified in RNA extracted from skin and blood vessel [16] , and both protein and message were identified in gastric mucosa [17] and mesentery [13] . Using RT-PCR, SP-D message has also been identified in mouse stomach, heart, and kidney [14] .\nSP-D (43 kDa, reduced) consists of at least four discrete structural domains: a short, N-terminal domain; a relatively long collagenous domain, a short amphipathic connecting peptide or coiled-coil neck domain, and a C-terminal, Ctype lectin carbohydrate recognition domain (CRD). Each molecule consists of trimeric subunits (3 × 43 kDa), which associate at their N-termini (Fig. 1) . Although most preparations of SP-D contain a predominance of dodecamers (that is, four trimeric subunits), the proportions of various oligomers vary between species. For example, rat lavage and recombinant rat SP-D are almost exclusively assembled as dodecamers (four trimers), whereas recombinant human SP-D is secreted as trimers, dodecamers and higher-order multimers [18] . SP-D isolated from the lavage of some patients with alveolar proteinosis consists predominantly of higher-order multimers, which can contain up to 32 (or more) trimeric subunits (Fig. 1 ).\nRecent crystallographic and mutagenesis studies suggest that the structural determinants of saccharide binding are similar to those originally described for mannose-binding lectin [19,20,21 • ,22 • ]. At least two bound calcium ions and two intrachain disulfide crosslinks stabilize the required tertiary structure, and Glu321 and Asn323 within the CRD participate in glucose/mannose type recognition. Interactions with at least one glycolipid ligand, phosphatidylinositol (PI), require the participation of the C-terminal end of the protein [23, 24] .\nA trimeric cluster of CRDs is necessary for high-affinity binding to carbohydrate ligands [21 • ,25]. The crystal structure of human SP-D suggests that the spatial distribution of CRDs within a trimeric subunit permits simultaneous and cooperative interactions with two or three glycoconjugates displayed on the surface of a particulate ligand [21 • ]. Furthermore, solid-phase binding studies have shown that monomeric CRDs have an approximately 10-fold lower binding affinity for multivalent ligands than trimeric CRDs.\nCrystallographic studies of human SP-D further suggest that the spatial organization of CRDs within a trimer is stabilized by interactions of the C-terminal sequence with the trimeric neck domain [21 • ,26]. Interestingly, the three CRDs show a deviation from threefold asymmetry, suggesting some flexibility of the CRDs in relation to the neck. Thus, the dependence of the binding of PI on the C-terminal sequence could reflect conformational effects, rather than the direct participation of this sequence in ligand interactions.\nThe collagen domain length of SP-D is highly conserved and lacks interruptions in the repeating Gly-X-Y sequence (in which X and Y are different amino acids). As for other collagenous proteins, this domain is enriched in imino acids and contains hydroxyproline. Unlike SP-A, SP-D also contains hydroxylysine. Although the collagen domain of rat, human, bovine, and mouse SP-D lacks cysteine residues, cDNA sequencing has identified a codon for cysteine within the collagen domain of pig SP-D [27 • ]; this suggests the possibility of alternative patterns of chain association and oligomeric assembly for pig SP-D.\nThe first translated exon of SP-D contains a highly conserved and unusually hydrophilic Gly-X-Y sequence that shows little homology with the remainder of the collagen sequence. The functional significance of this region is unknown. However, it has been suggested that this region contributes to oligomer assembly or mediates interactions with cellular receptors.\nThe collagen domain determines the maximal spatial separation of trimeric, C-terminal lectin domains within SP-D molecules, but might also contribute to normal oligomeric assembly and secretion. For example, deletion of the entire collagen domain of rat SP-D results in the secretion of trimers rather than dodecamers [28] . In addition, 2,2-dipyridyl, an inhibitor of prolyl hydroxylation that interferes with the formation of a stable collagen helix, causes the intracellular accumulation of 43 kDa monomers and dimers [29] . In any case, the complete conservation of the number of Gly-X-Y triplets suggests that the spatial separation of trimeric CRDs is critical for normal SP-D function.\nThe N-terminal peptide of the mature protein contains two conserved cysteine residues at positions 15 and 20. These residues participate in interchain disulfide crosslinks that stabilize the trimer, as well as the N-terminal association of four or more trimeric subunits. Stable oligomerization of trimeric subunits permits cooperative or bridging interactions between spatially separated binding sites on the same surface or on different particles.\nThe process of forming interchain disulfide bonds is complex, and appropriate crosslinking of the N-terminal domains might be rate limiting for secretion [30] . Subcellular fractionation studies suggest that interchain bonds form initially between the three chains of a trimeric subunit. Subsequent rearrangements within the rough endoplasmic reticulum might allow the covalent crosslinking of a single chain from one subunit and two crosslinked chains of another, with the associated elimination of free thiol groups. Mutant proteins that contain unpaired N-terminal cysteine residues are not secreted. However, it is unclear whether this results from abnormalities in disulfide bonding itself, or the failure to stabilize the required N-terminal conformation.\nThe collagen domain contains hydroxylysyl-derived glycosides and a single N-linked oligosaccharide. In most species (human, rat, mouse, and cow) the site of N-linked glycosylation is located near the N-terminal end of the collagenous domain. Recently, it was shown that pig SP-D has an additional potential site of N-linked glycosylation within the CRD [27 • ]. Although rat and human lung lavage SP-D seem to be sialylated, as suggested by charge heterogeneity and cleavage with highly purified neuraminidase, preparations of human amniotic fluid and bovine lavage SP-D recovered from amniotic fluid showed predominantly complex type biantennary structures and no sialic acid [31] .\nA variant form of SP-D (50 kDa) has been identified in lavage from a subset of human lavage samples; this protein shows O-linked glycosylation of threonyl residues within the N-terminal peptide domain [32 • ]. At present, the functional significance of these sugars is not known. The presence of O-linked glycosylation within the N-terminal domain might be predicted to interfere with normal dodecamer assembly. In this regard, the O-glycosylated 50 kDa form of human SP-D is recovered as trimeric subunits or smaller species.\nAs for many glycoproteins, the functional role of the attached carbohydrate is unknown. Mutational analysis has shown that the N-linked sugar on rat SP-D is not required for secretion, for dodecamer formation, or for interactions with a variety of microorganisms [29,33].\nConsistent with its designation as a 'mannose-type' C-type lectin, SP-D preferentially binds to simple and complex saccharides containing mannose, glucose, or inositol [34, 35] . SP-D also interacts with specific constituents of pulmonary surfactant including PI [36-38] and glucosylceramide [39] . Binding to glucosylceramide involves interactions of the carbohydrate-binding sequences of the CRD with the glucosyl moiety. However, the interaction of SP-D with PI involves interactions with the lipid, as well as CRD-dependent interactions with the inositol moiety [24, 40] .\nMicroorganisms are surfaced with a diverse and complex array of polysaccharides and glycoconjugates, and most classes of microorganism contain one or more sugars recognized by SP-D. However, the outcome of this interaction depends on the specific organism and can be modified by the conditions of microbial growth. The potential consequences of this interaction include the following: varying degrees of lectin-dependent aggregation (namely, microbial agglutination), enhanced binding of microorganisms or microbial aggregates to their 'receptors' on host cells, phagocyte activation, and opsonic enhancement of phagocytosis and killing, potentially involving one or more cellular receptors for SP-D. Binding to organisms in suspension is often -but not always -accompanied by some degree of aggregation.\nSP-D binds to purified lipopolysaccharide (LPS) isolated from a variety of Gram-negative organisms [35, 41] . In addition, LPS is the major cell wall component that is labeled on lectin blotting of outer membranes isolated from Escherichia coli [41] . The latter interactions involve the recognition of the core oligosaccharide domain, which contains glucose and heptose [41] . SP-D interacts preferentially with purified LPS molecules characterized by short or absent O-antigens and preferentially agglutinates bacterial strains expressing a predominance of rough (O-antigen-deficient) LPS [41, 44] .\nAlthough the core oligosaccharide domain of LPS constitutes the major ligand for SP-D on at least some Gram-negative bacteria, the mechanism of interaction with this group of microorganisms is probably heterogeneous. SP-D binds to some smooth, unencapsulated strains of Gram-negative bacteria by immunofluorescence. The mechanism is uncertain; the quantity or quality of binding differs from that observed for rough strains and does not necessarily result in agglutination. LPS molecules on the surfaces of bacteria show heterogeneity in the extent of maturation, so it is possible that this interaction is mediated by a subpopulation of LPS with deficient O-antigens and that the density of binding sites is too low for high-affinity binding.\nThe recognition of the surface glycoconjugates on Gramnegative bacteria by SP-D depends not only on the expression of lectin-specific residues by a given strain or species, but also on the accessibility of these residues [1, 45] . For example, SP-D binds inefficiently to the core region of LPS of encapsulated Klebsiella, but efficiently agglutinates the corresponding unencapsulated phase variants. Interactions of SP-D with the core oligosaccharides of Gram-negative organisms are also influenced by the number of repeating saccharide units associated with the terminal O-antigen of the LPS [41,44].\nOther potential ligands include the O-antigen domain of LPS, certain capsular polysaccharides, and membraneassociated glycoproteins. In this regard, SP-D can bind to di-mannose containing O-antigens expressed by a subset of Klebsiella serotypes (I Ofek, H Sahly and EC Crouch, unpublished data). Although other C-type lectins, specifically SP-A and the mannose receptor, can interact with specific capsular polysaccharides [46], a specific interaction of SP-D with capsular glycoconjugates or exopolysaccharides has not been described.\nThe mechanism of interaction with Gram-positive organisms has not been elucidated. Lipoteichoic acids, which are the major glycolipids associated with the Gram-positive cell wall, do not detectably compete with LPS for binding to SP-D (I Ofek, A Mesika, M Kalina, Y Keisari, D Chang, D McGregor and EC Crouch, manuscript submitted). In preliminary studies we observed that binding was competed only partly with maltose and/or EDTA, raising the possibility that binding might be more complex than for some Gram-negative organisms. . However, similar effects were observed when the neutrophils were preincubated with SP-D, and there was only a slight enhancement of uptake when bacteria were incubated with human SP-D and washed before their addition to neutrophils. Notably, the extent of binding and internalization was dependent on the extent of multimerization, with human SP-D multimers demonstrating the highest potency. Differences in cell type, the extent of SP-D multimerization, or differences in size or organization of bacterial aggregates could account for some of the apparent inconsistencies.\nAlthough LPS mediates the binding of SP-D to at least some Gram-negative bacteria, SP-D can also bind to spe- In the latter study the authors suggested that fungal aggregation inhibits phagocytosis. Interestingly, SP-D binding directly inhibited fungal growth and decreased the outgrowth of pseudohyphae, the invasive form of the fungus, in the absence of phagocytic cells [57] . It is possible that these effects are also secondary to agglutination, possibly as result of nutrient deprivation.\nPurified rat and human SP-D inhibit the infectivity and hemagglutination activity of influenza \nSP-D can interact with host cells, both directly and indirectly. As indicated above, SP-D can enhance the phagocytosis and killing of certain microorganisms and enhance the oxidant response to microbial binding. However, at present there is only one study that suggests that the enhancement of phagocytosis by SP-D might involve the participation of an opsonic receptor. Furthermore, the enhanced uptake of IAV seems to be mediated by viral aggregation, with enhanced interactions of the virus with its natural receptors on the host cell.\nIn any case, SP-D can interact directly with host cells, and in some cases can influence their behavior. SP-D is chemotactic and haptotactic for neutrophils and certain mononuclear phagocytes [59 • ,67-69] and can elicit directional actin polymerization in alveolar macrophages [69] . In this regard, SP-D is considerably more potent than SP-A. Early studies with natural proteins isolated from silicotic animals reported directed effects on the oxidant metabolism of isolated alveolar macrophages [70] . However, such effects can probably be attributed to endotoxin contamination and/or aggregation. Purified dodecamers do not significantly increase the production of nitric oxide [71] or of proinflammatory cytokines such as tumor necrosis factor-α (Y Kesari, H Wang, A Mesika, E Crouch and I Ofek, unpublished data). Interestingly, purified SP-D has been reported to increase the production of several metalloproteinases in the absence of a significant effect on proinflammatory cytokine production [72] .\nDespite the ability of SP-D to modulate a variety of cellular functions, little is currently known about potential cellular receptors for this protein. compartments [73] , but it is unclear whether the uptake is receptor dependent and whether SP-D is being internalized in association with specific ligands.\nThere are at least two classes of binding to host cells: CRD-dependent and CRD-independent. Some studies have demonstrated CRD-dependent binding to phagocytes that can be inhibited with EDTA or competing saccharides, both in vitro and in vivo.\nAs indicated above, the ability of SP-D to elicit the chemotaxis of neutrophilic and monocytic cells depends on the lectin activity of SP-D [68] . In addition, Kuan and coworkers reported that extracting formaldehyde-fixed alveolar macrophages with detergents largely eliminates the binding of purified SP-D, suggesting a membrane-associated ligand or glycolipid receptor [73] . Dong and Wright have extended these findings and suggest that PI can contribute to SP-D binding by alveolar macrophages [74] .\nIt is of interest that SP-D can bind to recombinant sCD14 through interactions with N-linked oligosaccharides [51 • ].\nGiven that the membrane-associated form of CD14 is widely expressed on host cells, it is possible that CD14 can serve as a binding site on macrophages and other cell types.\nThe phagocytic uptake of certain bacteria by neutrophils is also inhibited by calcium chelation or competing sugars [42]; however, this could result from the inhibition of microbial agglutination rather than lectin-dependent interactions with the phagocyte. Wang et al suggested that SP-D can bind to lymphocytic cells by a lectin-dependent mechanism [75 •• ] . In this regard, it is interesting to note that glucosylceramide, a ligand for SP-D in vitro, is one of the most abundant neutral glycolipids expressed by lymphoid cells.\nReid and co-workers were the first to present evidence for lectin-independent binding [76] . These and other studies suggested that binding does not involve known C1q or collectin receptors. The only putative receptor protein, gp-340, is a widely expressed member of the scavenger receptor superfamily [77,78 • ]. It binds to the CRD of SP-D in a calcium-dependent manner that does not require the lectin activity of SP-D. Although the protein has been immunolocalized to alveolar macrophage membranes and distributes together with SP-D in many different human tissues [10 • ,77], it has not yet been shown to mediate the binding of SP-D to these cells or to participate in signal transduction events. The cDNAs isolated from lung have not shown a membrane-spanning region [77] , and the protein is abundant as a soluble component in BAL. Given that gp-340 is a highly multimerized protein that contains numerous potential ligand binding domains (Fig. 1b) , it is possible that the protein cooperates with SP-D in the neu-tralization or clearance of certain ligands rather than specifically mediating the interactions of SP-D with host cells.\nWright and co-workers have demonstrated the binding of SP-D to isolated type II pneumocytes. The mechanism seems distinct from the binding to macrophages [79 • ]. The binding was dependent on concentration, time, and temperature and required calcium; it was not sensitive to protease treatment or to PI-phospholipase C. Although the internalized SP-D was degraded or recycled to lamellar bodies, SP-D binding did not alter the uptake of surfactant lipids.\nSP-D has demonstrated comparatively few direct effects on the metabolism of host cells, at least in situations where self-aggregation and endotoxin contamination have been excluded. One possible explanation is that modulation of cellular function requires the prior interaction of SP-D with a ligand. This would have numerous potential physiological advantages, because the presence of 'active' protein might be restricted to sites of microbial or antigenic deposition. The binding of complex, multivalent, particulate antigens to two or more CRDs could markedly alter the conformation of SP-D molecules, with respect to the spatial orientation of the arms in relation to the N-terminal crosslinking domain and/or with respect to the spatial orientation of the CRDs within a given trimeric subunit. Thus, the 'charging' of SP-D with a particulate ligand could lead to local or distant conformational changes that expose 'cryptic' binding sites for cellular receptors.\nThere is some preliminary evidence consistent with the notion that the interaction of SP-D with a ligand alters its capacity to activate host cells. Table 3 and discussed below.\nSP-D can be isolated in different multimeric forms from proteinosis lavage [32 • ] and are produced by Chinese hamster ovary K1 cells transfected with human SP-D cDNA [18] . As described previously, the effects of SP-D on the neutrophil response to influenza virus are highly dependent on the ability of SP-D to agglutinate the viral particles, and the agglutination activity is directly correlated with the extent of multimerization. Trimers can bind to the virus but have little capacity to modulate neutrophil interactions. By contrast, highly multimerized proteins show greater activity than dodecamers [81] .\nGiven these observations, factors that favor enhanced oligomerization or lead to the accumulation of trimeric subunits promote might influence SP-D function. For example, the liberation of active trimers by a hypothetical microbial protease could lead to the accumulation of molecules that might inhibit the aggregation-dependent activities of SP-D.\nIn contrast, recombinant trimeric CRDs can stimulate chemotaxis [67] and decrease viral infectivity [65 • ]. Although higher-order oligomers of SP-D can self-aggregate and precipitate in the presence of calcium in vitro, the functional consequences are not known.\nThe lectin activity of SP-A is decreased after the nitric oxide-dependent nitration of tyrosine residues [82] , and nitration decreases the ability of SP-A to enhance the adherence of Pneumocystis to alveolar macrophages [83] . However, similar findings have not yet been reported for SP-D. Conditions of mildly acidic pH, as might be found in endocytic compartments, are predicted to disrupt the lectin-dependent activities of SP-D [34]. Proteolytic degradation remains an important possibility. However, SP-D is highly resistant to degradation by a wide variety of neutral proteases in vitro, and degradation products have not yet been shown to accumulate under pathological conditions in vivo.\nGlucose concentrations at levels encountered in diabetes can interfere with SP-D's ability to interact with specific strains of IAV or other microorganisms in vitro [84 • ]. Many microorganisms release cell wall polysaccharides or glycoconjugates, which might interfere with the binding of collectins to the same or other organisms. In this regard, SP-D recovered from rats after the instillation of LPS into the airway shows decreased lectin activity, which is attributed to occupancy of the CRD with LPS [49 • ]. It seems reasonable to speculate that some organisms might compete with other organisms for binding to SP-D. Such a situation could conceivably predispose to secondary infections. Lastly, the potential inhibitory effects of competing saccharide ligands presents important methodological considerations for experiments using carbohydrate-containing cell culture medium or buffers. Non C-type lectins (such as ficolins)\nIt is difficult to predict the functions of SP-D within the airspace. Other lectins with overlapping specificity are also present. Although the levels of mannose-binding lectin are probably low in the absence of increased vascular permeability, SP-A and the macrophage mannose receptor could conceivably interact with the same ligands in the distal airways and alveoli. Such interactions could lead to antagonistic or cooperative effects. Furthermore, we have little knowledge regarding the microanatomic distribution of these molecules in specific circumstances in vivo.\nAlthough most SP-A is probably associated with the insoluble phase of the alveolar lining material, and the macrophage mannose receptor is membrane-associated, the distribution might be altered in the setting of lung injury.\nModels of SP-D deficiency show no detectable anatomical or physiological abnormalities at birth. However, the animals gradually develop a patchy, subpleural alveolar lipidosis with associated type II cell hypertrophy, the accumulation of enlarged and foamy macrophages, and an apparent expansion of peribronchial lymphoid tissue [85 • ,86 • ]. Interestingly, the mice eventually develop distal-acinar emphysema and areas of subpleural fibrosis, which could reflect a continuing inflammatory reaction associated with abnormal oxidant metabolism and metalloproteinase activity [87 • ]. By contrast, SP-A-deficient mice (-/-) show essentially normal respiratory function and surfactant lipid metabolism [88, 89] but numerous apparent host defense abnormalities [90] .\nThe capacity of SP-D to bind to specific strains of influenza A in vitro is highly correlated with the capacity of the virus to proliferate in mice in vivo [62] . Specifically, strains with more oligosaccharide attachments on the HA are preferentially neutralized by SP-D in vitro and show decreased proliferation in mice. Because the administration of mannan together with the virus increased the replication of IAV in the lung, the involvement of a mannose-type, C-type lectin was implicated.\nSP-D-sensitive IAV strains also replicate to higher titers in the lungs of diabetic mice than in nondiabetic controls [84 • ]. Replication of the virus is positively correlated with blood glucose level, and decreases in response to insulin treatment. Significantly, blood glucose levels comparable to those measured in the diabetic mice were sufficient to inhibit the interaction of SP-D with these viral strains in vitro. PR-8, a strain that does not interact with SP-D but does interact with SP-A, replicated to the same extent in diabetic and control mice.\nSP-D levels increase in association with certain infections. For example, SP-D levels, but not the levels of serum mannose-binding lectin, increase markedly after IAV infection [62] . Impressive increases in SP-D have also been observed in murine models of Pneumocystis carinii [91] and P. aeruginosa infection [92] .\nSP-D-deficient mice have not yet been extensively characterized with respect to host defense function. However, they show decreased viral clearance and enhanced inflammation after challenge with respiratory syncytial virus [93] and IAV (AM Levine, personal communication). In addition, they show increased inflammation, increased oxidant production, and decreased macrophage phagocytosis in response to intratracheally instilled group B streptococcus and Haemophilus influenzae (AM Levine, personal communication). Although the overexpression of wild-type SP-D in type II pneumocytes with the SP-D-deficient mice can prevent the lipidosis and inflammatory changes [94] , the ability of overexpressed wild-type SP-D or exogenous SP-D to ameliorate these abnormalities has not yet been described. The coexisting pulmonary abnormalities also complicate the interpretation of challenge models. For example, macrophage activation might enhance killing and offset any decrease that results more directly from SP-D deficiency. SP-D deficiency modifies the host response to instilled LPS with decreased lung injury and inflammatory cell recruitment [50].\nMolecules that can bind to potential antigens and deliver them to macrophages and other antigen-presenting cells might contribute to the development of acquired immunity. In this regard, a few published observations suggest possible roles in the development of humoral and/or cellular immunity in response to microorganisms or complex organic antigens. For example, SP-D can decrease interleukin-2dependent T-lymphocyte proliferation [95 • ]. Interestingly, single-arm mutants were at least as potent as intact dodecamers in mediating this effect. SP-D also binds to oligosaccharides associated with dust mite allergen [96 • ], and can inhibit the binding of specific IgE to these allergens, possibly through direct, CRD-dependent binding to lymphocytes [96 • ]. Thus, alterations in the level of SP-D (or the state of oligomerization) might influence the development of immunological responses and contribute to the pathogenesis of asthma and other hypersensitivity disorders.\nThere are other potential interplays between humoral immunity and collectins with regard to antimicrobial host defense. For example, increased glycosylation of IAV coat proteins, an adaptation that is believed to help the virus to evade antibody-mediated neutralization, is associated with increased reactivity with SP-D and other collectins [62]. Thus, the relative potential importance of antibody and collectin-mediated host defenses might be influenced by subtle variations in the structure of the microbial surface.\nThere is little recent information on the developmental regulation of SP-D expression. In general, SP-D increases rapidly late in gestation [97] [98] [99] [100] . The production of SP-D increases during the culture of fetal lung explants, and expression can be increased with glucocorticoids [98, 100, 101] . The exposure of fetal rats to glucocorticoids in vivo leads to precocious expression with increased numbers of SP-D-expressing cells and increased cellular levels of SP-D message [98, 101, 102] .\nAlthough SP-D is produced constitutively within the lung, protein accumulation and gene expression are inducible and increases in SP-D expression have been observed in a number of disease states or models (Tables 4 and 5 ). In general, the synthesis and secretion of SP-D increase in association with lung injury and activation of the respiratory epithelium [1] . For example, levels of SP-D mRNA and SP-D accumulation are increased within 24-72 h after intratracheal instillation of LPS [103 • ], and SP-D expression by alveolar and bronchiolar epithelial cells increases after exposure of rats to 95% O 2 for 12 h [104] . Keratinocyte growth factor (KGF) increases SP-D expression and protein production in association with pneumocyte hyperplasia and after injury caused by bleomycin [105] . In addition, the levels of SP-D can increase markedly in response to the overexpression of certain cytokines, such as interleukin-4, or in response to microbial challenge [91, 92] .\nStudies of the upstream regulatory region of the SP-D gene have demonstrated increased promoter activity in the presence of glucocorticoids, which is consistent with the findings in vivo and in lung organ culture [106] . However, no functional glucocorticoid response elements have been identified, and the effects of dexamethasone seem to be secondary and involve the effects of other transregulatory molecules.\nThe activity of the human SP-D promoter is dependent on a conserved activator protein-1 (AP-1) element (-109) that binds to members of the fos and jun families of transcriptional factors [107] . In addition, the promoter contains multiple functional binding sites for CCAAT-enhancer-binding protein (C/EBP) transcription factors. Mutagenesis experiments suggest that these are required for basal and stimulated promoter activity, and promoter activity is markedly increased in H441 cells after co-transfection with C/EBPβ cDNA (YC He and E crouch, unpublished data). The importance of the conserved AP-1 element and the presence of multiple binding sites for C/EBP transcription factors is consistent with the observed modulation of SP-D expression in the setting of tissue injury.\nSP-D promoter activity is not dependent on the binding of thyroid transcription factor 1 (TTF-1) [107] . However, promoter activity is dependent on two interacting forkhead binding sites, upstream and downstream of the AP-1 element; these sites bind to hepatic nuclear factor-3α and apparently other forkhead box proteins in H441 lung adenocarcinoma nuclear extracts [107] .\nInitial comparison of genomic and cDNA sequence suggested the existence of genetic polymorphisms in the SP-D coding sequence, including one in the N-terminal propeptide domain (Thr11 compared with Met11 in the mature protein) and three additional differences within the collagen domain at positions 102, 160, and 186 [108] . The latter substitutions are conservative to the extent that they are not expected to disrupt the collagen helix. Floros Table 5 Increased SP-D accumulation or expression in animal models\nSilicosis Rat [118] Hyperoxia Rat [104] Endotoxin (LPS) Rat [103] Challenge with P. aeruginosa Mouse [92] Challenge with IAV Mouse\n[62]\nChallenge with Pneumocystis carinii SCID mouse [91] Rat [119] Overexpression of interleukin-4 Mouse [120] SCID, severe combined immunodeficiency. and co-workers have recently confirmed the existence of polymorphisms at positions 11 and 160 of the mature protein [109] . The potential biological significance, if any, is not known. Interestingly, the 50 kDa variant of SP-D showed O-linked glycosylation of Thr11 [32 • ], suggesting that this polymorphism might be associated with altered glycosylation. Interestingly, the 50 kDa variant was recovered as trimeric subunits, raising the possibility that differences in the glycosylation of residue 11, which is immediately N-terminal to Cys15, could influence multimerization and the capacity of SP-D to participate in bridging interactions.\nThere is increasing evidence that SP-D interacts specifically with a wide variety of respiratory pathogens, modulates the leukocyte response to these organisms, and participates in aspects of pulmonary immune and inflammatory regulation (Table 6) . SP-D can influence the activity of phagocytes through CRD-dependent and CRD-independent interactions. At least some of the effects of SP-D result from aggregation with enhanced binding of the agglutinated ligand to their natural 'receptors'. Although the lung is the major site of SP-D expression, it is likely that the protein has more generalized roles in host defense and the acute response to infection and tissue injury. 16 " }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: from Xenopus laevis [16] . ETA receptors in normal lung are found in greatest abundance on vascular and airway smooth muscle, whereas ETB receptors are most often found on the endothelium. Clearance of ET-1 from the circulation is mediated by the ETB receptor primarily in the lung, but also in the kidney and liver [17] .\nActivation of both ETA and ETB receptors on smooth muscle cells leads to vasoconstriction whereas ETB receptor activation leads to bronchoconstriction. Activation of ETB receptors located on endothelial cells leads to vasodilation by increasing nitric oxide (NO) production. The mitogenic and inflammatory modulator functions of ET-1 are primarily mediated by ETA receptor activity. Binding of the ligand to its receptor results in coupling of cell-specific G proteins that activate or inhibit adenylate cyclase, stimulate phosphatidyl-inositol-specific phosholipase, open voltage gated calcium and potassium channels, and so on. The varied effects of ET-1 receptor activation thus depend on the G protein and signal transduction pathways active in the cell of interest [18] . A growing number of receptor antagonists exist with variable selectivity for one or both receptor subtypes.\nRegulation of ET-1 is at the level of transcription, with stimuli including shear stress, hypoxia, cytokines (IL-2, IL-1β, tumor necrosis factor α, IFN-β, etc), lipopolysaccharides, and many growth factors (transforming growth factor-β, platelet-derived growth factor, epidermal growth factor, etc) inducing transcription of ET-1 mRNA and secretion of protein [18] . ET-1 acting in an autocrine fashion may also increase ET-1 expression [19] . ET-1 expression is decreased by NO [20] . Some stimuli may additionally enhance preproET-1 mRNA stability, leading to increased and sustained ET-1 expression. The number of ETA and ETB receptors is also cell specific and regulated by a variety of growth factors [18] . Because ET-1 and receptor expression is influenced by many diverse physical and biochemical mechanisms, the role of ET-1 in pathologic states has been difficult to define, and these are addressed in subsequent parts of this article.\nIn the airway, ET-1 is localized primarily to the bronchial smooth muscle with low expression in the epithelium. Cellular subsets of the epithelium that secrete ET-1 include mucous cells, serous cells, and Clara cells [21] . ET binding sites are found on bronchial smooth muscle, alveolar septae, endothelial cells, and parasympathetic ganglia [22, 23] . ET-1 expression in the airways, as previously noted, is regulated by inflammatory mediators. Eosinophilic airway inflammation, as may be seen in severe asthma, is associated with increased ET-1 levels in the lung [24] . ET-1 secretion may also act in an autocrine or paracrine fashion, via the ETA receptor, leading to increased transepithelial potential difference and ciliary beat frequency, and to exerting mitogenic effects on airway epithelium and smooth muscle cells [25] [26] [27] [28] .\nAll three endothelins cause bronchoconstriction in intact airways, with ET-1 being the most potent. Denuded bronchi constrict equally to all three endothelins, suggesting considerable modulation of ET-1 effects by the epithelium [29] . The vast majority of ET-1 binding sites on bronchial smooth muscle are ETB receptors, and bronchoconstriction in human bronchi is not inhibited by ETA antagonists but augmented by ETB receptor agonists [30] [31] [32] . Since cultured airway epithelium secretes equal amounts of ET-1 and ET-3, which have equivalent affinity for the ETB receptor, bronchoconstriction could be mediated by both endothelins [33] .\nWhile ET-1 stimulates release of multiple cytokines important in airway inflammation, it does not enhance secretion of histamine or leukotrienes. ET-1 does increase prostaglandin release [32] . Inhibition of cyclo-oxygenase, however, has no effect on bronchoconstriction suggesting that, despite the release of multiple mediators, ET-1 mediated bronchoconstriction is a direct effect of activation of the ETB receptor [32] . ETA mediated bronchoconstriction may also be important following ETB receptor desensitization or denudation of the airway epithelium, as may occur during airway inflammation and during the late, sustained airway response to inhaled antigens [31, 34, 35] . Interestingly, heterozygous ET-1 knockout mice, with a 50% reduction in ET-1 peptide, have airway hyperresponsiveness but not remodeling, suggesting the decrease in ET-1 modulates bronchoconstriction activity by a functional mechanism, possibly by decreasing basal NO production [36, 37] .\nAsthma is also an inflammatory airway disease characterized by bronchoconstriction and hyperreactivity with influx of inflammatory cells, mucus production, edema, and airway thickening. ET-1 may have important roles in each of these processes. While ET-1 causes immediate bronchoconstriction [38] , it also increases bronchial reactivity to inhaled antigens [35] as well as influx of inflammatory cells [39, 40] , increased cytokine production [40] , airway edema [41] , and airway remodeling [28, 42, 43] . Airway inflammation also leads to increased ET-1 synthesis, possibly perpetuating the inflammation and bronchoconstriction [44] . ET-1 release from cultured peripheral mononuclear and bronchial epithelial cells from asthmatics is also increased [45, 46] . Inhibition of ETA or combined ETA and ETB receptors additionally leads to decreased airway inflammation in antigen-challenged animals, suggesting that the proinflammatory effects of ET-1 in the airway are mediated by ETA receptors [39, 47] .\nChildren with asthma have increased circulating levels of ET-1 [48] . Adult asthmatics have normal levels between attacks but, during acute attacks, have elevated serum ET-1 levels that correlate inversely with airflow measurements and decrease with treatment [49] . Bronchoalveolar lavage (BAL) ET-1 in asthmatics is similarly increased to concentrations that cause bronchoconstriction and inversely correlates with forced expiratory volume in 1 s (FEV 1 ) [29, 50, 51] . As in cultured epithelial cells, ET-1 and ET-3 are found in equal amounts in BAL fluid from asthmatics [33, 52] . There is also a relative increase in ETB versus ETA receptor expression in asthmatic patients, which may contribute to increased bronchoconstriction [53] . Not all asthmatics, however, have increased ET-1 as patients with nocturnal asthma have decreased BAL ET-1 levels [54] . Treatment of acute asthma exacerbations with steroids, beta-adrenergic agonists or phosphodiesterase inhibitors resulted in decreased BAL ET-1 [52, 55] . Immunostaining and in situ hybridization for ET-1 in biopsy specimens from asthmatics have shown an increase in ET-1 in the bronchial epithelium that correlates with asthma symptoms [46, 56] .\nCigarette smoking leads to increased circulating ET-1 [57] but patients with chronic obstructive pulmonary disease, in the absence of pulmonary hypertension and hypoxemia, do not have increased plasma ET-1 [58] [59] [60] . Increases in urinary ET-1 instead correlate with decreases in oxygenation, possibly through hypoxic release of ET-1 from the kidney [61, 62] . Smokers also have impaired ET-1 mediated vasodilation that correlates with bronchial hyperresponsiveness and may contribute to pulmonary hypertension [63, 64] .\nET-1 has been implicated in the pathogenesis of bronchiectasis by its ability to promote neutrophil chemotaxis, adherence, and activation [65] [66] [67] [68] [69] . Sputum ET-1 levels are increased in patients with cystic fibrosis [59] , and sputum ET-1 correlated with Pseduomonas infection in noncystic fibrosis related bronchiectasis [70] .\nET-1 has also been implicated in the pathogenesis of bronchiolitis obliterans (BO), which is characterized by injury to small conducting airways resulting in formation of proliferative, collagen rich tissue obliterating airway architecture. BO is the leading cause of late mortality from lung transplantation, and ET-1 is increased in lung allografts [71] . The pro-inflammatory and mitogenic properties of ET-1 in the airways has led to speculation that ET-1 may be involved in formation of the lesion [28] . This is further supported by the increase in BAL ET-1 in lung allografts [72, 73] . The in vivo gene transfer of ET-1 to the airway epithelium using the hemagglutinating virus of Japan in rats recently resulted in pathologic changes in the distal airways identical to those seen in human BO specimens [74] . These changes were not due to nonspecific effects of the hemagglutinating virus of Japan itself, but could be attributed to the presence of the ET-1 gene, which was localized to the airway epithelium, hyperplastic lesions, and alveolar cells.\nPulmonary hypertension is a rare and progressive disease characterized by increases in normally low pulmonary vascular tone, pulmonary vascular remodeling, and progressive right heart failure. ET-1 has been implicated as a mediator in the changes seen in pulmonary hypertension. In the pulmonary vasculature, ET-1 is found primarily in endothelial cells and to a lesser extent in the vascular smooth muscle cells. The endothelium secretes ET-1 primarily to the basolateral surface of the cell. ET-1 secretion may be increased by a variety of stimuli including cytokines, catecholamines, and physical forces such as shear stress, and decreased by NO, prostaglandins, and oxidant stress [20, [75] [76] [77] [78] . Hypoxia has been reported to increase, have no effect, or decrease ET-1 release from endothelial cells [79] [80] [81] [82] [83] .\nActivation of the receptors for ET-1 in the pulmonary vasculature leads to both vasodilation and vasoconstriction, and depends on both cell type and receptor. In the whole lung, ETA receptors are the most abundant and are localized to the medial layer of the arteries, decreasing in intensity in the peripheral circulation [84, 85] . ETB receptors are also found in the media of the pulmonary vessels, increasing in intensity in the distal circulation, while intimal ETB receptors are localized in the larger elastic arteries [85] . This distribution of receptors has important implications in understanding ET-1 regulation of vascular tone. Vascular ET-1 receptors may be increased by several factors including angiotensin and hypoxia [80, [85] [86] [87] .\nET-1 can act as both a vasodilator and vasoconstrictor in the pulmonary circulation. Generation of NO or opening of ATP-sensitive potassium channels leading to hyperpolarization results in vasodilation mediated by ETB receptors on pulmonary endothelium [88, 89] . In hypertensive, chronically hypoxic lungs with increased ETB receptor expression, augmented vasodilation is due to increased ETB mediated NO release that is inhibited by hypoxic ventilation, while inhibition of NO synthesis leads to increased ET-1 mediated vasoconstriction [85, [90] [91] [92] . Both ETA and ETB receptors, conversely, acting on vascular smooth muscle, mediate ET-1 induced vasoconstriction. In the normal lung, ET-1 causes vasoconstriction primarily by activation of the ETA receptors in the large, conducting vessels of the lung [93, 94] . In the smaller, resistance vessels of the lung, ETB receptors in the media predominate and are responsible for the ET-1 induced vasoconstriction [93] . Interestingly, preconstriction of the pulmonary circulation resulted in a shift from primarily ETA mediated to ETB mediated vasoconstriction [94] .\nThe overall effect of ET-1 on vascular tone depends on both the dose and on the pre-existing tone in the lung. ET-1 administration during acute hypoxic vasoconstriction will result in transient pulmonary vasodilation [89] . This effect is dose dependent, with lower doses leading to vasodilation while higher or repetitive doses cause vasoconstriction following an initial, brief vasodilation [89] . The role of ET-1 in the acute hypoxic vasoconstriction in the lung is not certain. ETA receptor antagonism attenuates hypoxic pulmonary vasoconstriction in several species [95] , and ET-1 may be implicated in the mechanism of acute hypoxic response by inhibition of K-ATP channels [96] .\nSeveral lines of evidence have suggested the importance of ET-1 in chronic hypoxic pulmonary hypertension. ET-1 is increased in plasma and lungs of rats following exposure to hypoxia [80, 97] . Treatment with either ETA or combined ETA and ETB receptor antagonists additionally attenuates the development of hypoxic pulmonary hypertension [98, 99] . ET-1 has also been implicated in the vascular remodeling associated with chronic hypoxia through its mitogenic effects on vascular smooth muscle cells [98, 100] .\nET-1 has also been implicated in other animal models of pulmonary hypertension. ET-1 is increased in fawn hooded rats that develop severe pulmonary hypertension when raised under conditions of mild hypoxia and in monocrotaline treated rats [101, 102] . The increase in ET-1 in both of these forms of pulmonary hypertension may be contributing to increases in vascular tone as well as in vascular remodeling [103] [104] [105] [106] 114] . Interestingly, transgenic mice overexpressing the human preproET-1 gene, with modestly increased lung ET-1 levels (35-50%), do not develop pulmonary hypertension under normoxic conditions or an exaggerated response to chronic hypoxia [107] .\nHuman pulmonary hypertension is classified as primary, or unexplained, or secondary to other cardiopulmonary diseases or connective tissue diseases (ie scleroderma). Hallmarks of the disease include progressive increases in pulmonary vascular resistance and pulmonary vascular remodeling, with thickening of the medial layer small pulmonary arterioles and formation of the complex plexiform lesion [108] . Circulating ET-1 is increased in humans with pulmonary hypertension, either primary or due to other cardiopulmonary disease [109] . Levels are highest in patients with primary pulmonary hypertension. Since the lung is the major source for clearance of ET-1 from the circulation, increased arterio-venous ratios as seen in primary pulmonary hypertension suggest either decreased clearance or increased production in the lung [17, 109] . ET-1 is also increased in lungs of patients with pulmonary hypertension, with the greatest increase seen in the small resistance arteries and the plexiform lesions [110] , and may correlate with pulmonary vascular resistance [111] . Interestingly, treatment with continuous infusion of prostacyclin resulted in clinical improvement and a decrease in the arterio-venous ratio of ET-1 [112] , possibly by decreasing ET-1 synthesis from endothelial cells [76] . Studies using ET-1 receptor antagonists in the treatment of primary pulmonary hypertension are underway and may offer hope to patients with this disease by inhibiting this pluripotent peptide's effects on vascular tone and remodeling.\nSeveral lines of evidence suggest the importance of ET-1 in lung allograft survival and rejection. The peptide has been implicated as an important factor in ischemia-reperfusion injury at the time of transplant as well as in acute and chronic rejection of the allograft.\nCirculating ET-1 is increased in humans undergoing lung transplant immediately following perfusion of the allograft. Plasma ET-1 increased threefold within minutes, remained high for 12 hours following transplantation, and declined to near normal levels within 24 hours [113] . This increase in ET-1 correlated with the increase in pulmonary vascular resistance occurring about 6 hours post-transplantation, suggesting that the release of ET-1 in the circulation may have mediated this event. ET-1 in BAL fluid from recipients of lung allografts is similarly increased several fold and remains elevated up to 2 years post-transplant [72, 73] . In recipients of single lung transplants, ET-1 was increased 10-fold in BAL fluid from the transplanted lung compared with the native lung, suggesting that the increase in ET-1 was due to the graft and not the underlying disease requiring transplant [72] . ET-1 in BAL fluid did not, however, correlate with episodes of infection or rejection.\nThe cellular source of ET-1 in lung allografts is unknown. The expression of ET-1 in nontransplanted human lungs is low and found primarily in the vascular endothelium [114] . Transbronchial biopsy specimens obtained either for surveillance or for clinical suspicion of infection or rejection following transplantation revealed the presence of ET-1 in the airway epithelium and in alveolar macrophages [115] . ET-1 was occasionally seen in lymphocytes but not in the endothelium or pneumocytes. ET-1 localization was no different in surveillance specimens compared with infected or rejecting lungs, or changed over time from transplantation. This study suggests that the source of the increased BAL ET-1 in transplanted lungs is due to the increased number of alveolar inflammatory cells and de novo expression in the airway epithelium. The biologic importance of the ET-1 from inflammatory cells is supported by the observation that peripheral mononuclear cells from dogs with mild to moderate lung allograft rejection cause vasoconstriction in pulmonary arterial rings, which is attenuated by the ETA blocker BQ123 [116] .\nAnalysis of ET-1 binding activity in failed transplanted human lungs suggested that ET-1 binding activity was not different compared with normal lung in the lung parenchyma, bronchial smooth muscle, or perivascular infiltrates. ET-1 binding was, however, decreased in small muscular arteries (pulmonary arteries and bronchial arteries) in the failed transplants, suggesting a role for ET-1 in impaired vasoregulation of transplanted lungs [117] .\nIschemia-reperfusion injury is the leading cause of early post-operative graft failure and death. In its severest manifestation, increased pulmonary vascular resistance, hypoxia, and pulmonary edema lead to cor pulmonale and death [118] . ET-1 has been implicated as a mediator of these events. The increase in pulmonary vascular resistance observed in human recipients of lung allografts follows an increase in circulating ET-1 and falls with decreases in circulating ET-1 [113] . A similar pattern is seen in dogs subjected to allotransplantation [119] . Conscious dogs with left pulmonary allografts demonstrate an increase in both resting pulmonary perfusion pressure and acute pulmonary vasoconstrictor response to hypoxia [120] . Administration of ETA selective or combined ETA and ETB receptor blockers did not change the resting tone. ETB receptor mediated hypoxic pulmonary vasoconstriction appeared, however, to be increased in allograft recipients. In another study, administration of a mixed ETA and ETB receptor antagonist (SB209670) to dogs before reperfusion of the allograft resulted in a marked increase in oxygenation, decreases in pulmonary arterial pressures and improved survival compared with control animals [121] . In a model of ischemia reperfusion, inhibitors of ECE additionally attenuated the increase in circulating ET-1 and the severity of lung injury [122] . ET-1 receptor antagonists did not, however, completely eliminate the ischemia-reperfusion injury, suggesting that changes in other vasoactive mediators, such as an increase in thromboxane, a decrease in prostaglandins, or a decrease in NO, may also contribute to the increased pulmonary vascular resistance. Administration of NO donor (FK409) to both donor and recipient dogs before lung transplantation reduced pulmonary arterial pressure, lung edema, and inflammation, and improved survival. This suggests that reductions in NO following transplantation may be partly responsible for early graft failure [123] . Treatment with NO donor was also associated with a decrease in plasma ET-1 levels.\nAcute rejection is manifested by diffuse infiltrates, hypoxia, and airflow limitation, and may lead to respiratory insufficiency and death. BAL ET-1 was increased in dogs during episodes of acute rejection that decreased with immunosuppressive treatment [124] . Acute episodes of rejection in humans, however, are not associated with further increases in BAL ET-1 [72] . Chronic rejection of allografts, manifested as BO, is the major cause of morbidity and mortality in long-term lung transplant survivors [71] . The etiology of BO following transplant is unclear but may be related to repeated episodes of acute rejection, chronic low-grade rejection, or organizing pneumonia [125] . As discussed earlier, a chronic increase in ET-1, as seen in lung allografts, may contribute to bronchospasm and proliferative bronchiolitis obliterans due to the bronchoconstrictor and smooth muscle mitogenic effects of ET-1 [28, 126] . This is further supported by the increase of BAL ET-1 in the transplanted lung, which is susceptible to BO, but not the native lung in recipients of single lung transplants [72] .\nThe mitogenic effects of ET-1 may play a role in the development of pulmonary malignancy as well as metastasis to the lung. Many human tumor cell lines, including prostate, breast, gastric, ovary, colon, etc, produce ET-1. The importance of the ET-1 may lie in its mitogenic effects on tumor growth and survival. This has been suggested by blockade of ETA receptors resulting in a decrease in mitogenic effects of ET-1 in a prostate cancer and colorectal cell lines [127, 128] . ET-1 receptors in tumor cells may also be altered with increases in the ETA receptor and downregulation of ETB receptors [129] . Other tumors may have an increase in ETB receptors, however, and blockade of ETB results in a decrease in tumor growth [130, 131] . Tumor cells may, as a result of this altered balance, lose the ability to respond to regulatory signals from their environment. ET-1 may additionally protect against Fas-ligand mediated apoptosis [132] .\nET-1 has been detected using immunohistochemistry and in situ hybridization in pulmonary adenocarcinomas and squamous cell tumors and, to a lesser extent, small cell and carcinoid tumors [133] . In situ hybridization also demonstrated a similar pattern of ET-1 mRNA expression in non-neuroendocrine tumors. ET-1 receptors have also been found in a variety of pulmonary tumor cell lines. ETA receptors were found in small cell tumors, adenocarcinomas and large cell tumors, while ETB receptors were expressed primarily in adenocarcinomas and small cell tumors [134] . ECE, which converts big ET-1 to ET-1, the committed step in ET-1 biosynthesis, was also found in human lung tumors but not in adjacent normal lung [135] . These findings, combined with the presence of ET-1 in lung tumors, suggest a possible autocrine loop that sustains and supports the growth of lung tumors. A recent study, however, suggested that, while ETA and ECE-1 were detectable in lung tumors, these genes were downregulated compared with normal bronchial epithelial cell lines [136] . It was proposed that the role of ET-1 in lung tumors is not that of an autocrine factor, but that of a paracrine growth factor to the stroma and vasculature surrounding the tumor allowing angiogenesis.\nTumor angiogenesis is necessary for continued growth of the tumor beyond the limits of oxygen diffusion. The growth of vessels into the tumor is also important to metastatic potential of the tumor. ET-1 may play an important role in angiogenesis and tumor growth and survival Available online http://respiratory-research.com/content/2/2/090 commentary review reports primary research through induction of vascular endothelial growth factor expression and sprouting of new vessels into the tumor and surrounding tissue [137, 138] . ET-1 binding activity was found in blood vessels and vascular stroma surrounding lung tumors at the time of resection, most markedly surrounding squamous cell tumors [139] . ET-1 production may be further augmented by the hypoxic environment found within large solid tumors [140] . Since metastasis is dependent on neo-vascularization, ET-1 may also be an important mediator of this phenomenon. ET-1 receptor antagonists may have a useful role in the treatment of neoplastic disease by inhibiting growth as well as metastatic potential of human tumors.\nExperimental lung injury of many different types results in increased circulating ET-1, BAL ET-1, and lung tissue ET-1 [18] . ET-1 levels in humans are also increased in sepsis, burns, disseminated intravascular coagulation, acute lung injury, and acute respiratory distress syndrome (ARDS) [141] [142] [143] [144] [145] [146] [147] . ET-1 increases also correlate with a poorer outcome with multiple organ failure, increased pulmonary arterial pressure, increased airway pressure and decreased PiO 2 /FiO 2 , while clinical improvement correlates with decreased ET-1 levels [144, 145, 147] . The arterio-venous ratio for ET-1 is increased in patients with ARDS but it is not clear whether this is due to increased secretion of ET-1 in the lungs or decreased clearance [142, 144] . In patients who succumbed to ARDS, there was also a marked increase in tissue ET-1 immunostaining in vascular endothelium, alveolar macrophages, smooth muscle, and airway epithelium compared with lungs of patients who died without ARDS. Interestingly, these same patients also had a decrease in immunostaining for both endothelial nitric oxide synthase and inducible nitric oxide synthase in the lung [148] . ARDS is also characterized by the presence of inflammatory cells in the lung. Since ET-1 may act as an immune modulator, an increase in ET-1 may contribute to lung injury by inducing expression of cytokines including tumor necrosis factor and IL-6 and IL-8 [149] . These cytokines may in turn stimulate the production of many inflammatory mediators, leading to lung injury. ET-1 additionally activated neutrophils, and increased neutrophil migration and trapping in the lung [65] [66] [67] [68] [69] .\nAnother hallmark of ARDS is disruption and dysfunction of the pulmonary vascular endothelium leading to accumulation of lung water. The role of endothelin in formation of pulmonary edema is uncertain. Infusion of ET-1 raises pulmonary vascular pressure, but it is uncertain whether ET-1 by itself increased pulmonary protein or fluid transport in the lung [150] [151] [152] . ET-1 may rather be acting synergistically with other mediators to lead to pulmonary edema [153, 154] .\nPulmonary fibrosis is the final outcome for a variety of injurious processes involving the lung parenchyma. The final common pathway in response to injury to the alveolar wall involves recruitment of inflammatory cells, release of inflammatory mediators, and resolution. The reparative phase occasionally becomes disordered, resulting in progressive fibrosis.\nET-1 in the lung may be important in the initial events in lung injury by activating neutrophils to aggregate and release elastase and oxygen radicals, increasing neutrophil adherence, activating mast cells, and inducing cytokine production from monocytes [65] [66] [67] [68] [69] 149, 155] . Among the many cytokines induced by ET-1 that are important in mediating pulmonary fibrosis are transforming growth factor-β and tumor necrosis factor α [156, 157] . ET-1 is also profibrotic by stimulating fibroblast replication, migration, contraction, and collagen synthesis and secretion while decreasing collagen degradation [158] [159] [160] [161] [162] . ET-1 additionally enhances the conversion of fibroblasts into contractile myelofibroblasts [43, 163] . ET-1 also increases fibronectin production by bronchial epithelial cells [164] . Finally, ET-1 has mitogenic effects on vascular and airway smooth muscle [126, 28] . ET-1 may thus play an important role in the initial injury and eventual fibrotic reparative process of many inflammatory events in the lung.\nSeveral lines of evidence regarding the importance of ET-1 in pulmonary fibrosis are available. Plasma and BAL ET-1 levels are increased in idiopathic pulmonary fibrosis [50, 165] . Lung biopsies from patients with idiopathic pulmonary fibrosis have additionally increased ET-1 immunostaining in airway epithelial cells and type II pneumocytes, which correlates with disease activity [166] . Scleroderma is commonly associated with pulmonary hypertension and pulmonary fibrosis. Plasma and BAL ET-1 is increased in these patients [160, 167, 168] , but it is unclear whether the presence of either pulmonary hypertension or pulmonary fibrosis increases these levels further [167] . BAL fluid from patients with scleroderma increased proliferation of cultured lung fibroblasts, which was inhibited by ETA receptor antagonist. This suggests that the ET-1 in the airspace may be contributing significantly to the fibrotic response [160] . An increase in ET-1 binding has also been reported in lung tissue from patients with scleroderma associated pulmonary fibrosis [169] . Pulmonary inflammatory cells also appear to be primed for ET-1 production because cultured alveolar macrophages from patients with scleroderma and lung involvement secrete increased amounts of ET-1 in response to stimulation with lipopolysaccharide [170] . These observations collectively suggest that augmented ET-1 release may contribute to and perpetuate the inflammatory process. Bleomycin-induced pulmonary fibrosis in animals is associated with increased ET-1 expression in alveolar macrophages and epithelium [171] . The increase in ET-1 proceeds the development of pulmonary fibrosis. The use of ET-1 receptor antagonists has produced mixed results in limiting the development of bleomycin-induced fibrosis. A decrease in fibroblast replication and secretion of extracellular matrix proteins in vitro but not a decrease in lung collagen content in vivo has been shown using ETA or combined ETA and ETB receptor antagonists after bleomycin [172] . Another group did, however, observe a decrease in fibrotic area in lungs of rats following bleomycin that were treated with a mixed ETA and ETB receptor antagonist [173] .\nWhile ET-1 seems to correlate with pulmonary fibrosis, it remains uncertain whether the increase in ET-1 is a cause or consequence of the lung disease. Pulmonary fibrosis was recently reported in mice that constitutively overexpress human ET-1 [107] . These mice were known to develop progressive nephrosclerosis in the absence of systemic hypertension [174] . The transgene was localized throughout the lung, with the strongest expression in the bronchial wall. In the lung, the mice developed age-dependent accumulation of collagen and accumulation of CD4+ lymphocytes in the perivascular space. This observation suggests that an increase in lung ET-1 alone may play a causative role in the development of pulmonary fibrosis [107, 175] .\nSince its discovery 12 years ago, much evidence has accumulated regarding the biologic activity and potential role of ET-1 in a variety of diseases of the respiratory track. As compelling as much of this evidence is, the causal relationship between ET-1 activity and disease is not complete. The increasing use of ECE and endothelin receptor antagonists in experimental and human respiratory disorders will help to clarify the role of this pluripotent peptide in health and disease." }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: RSV and PVM are viruses of the family Paramyxoviridae, subfamily pneumovirus; they are enveloped, singlestranded, nonsegmented RNA viruses that can cause intense viral bronchiolitis in humans and mice, respectively. In its most severe form, the lower respiratory tract infection caused by pneumoviruses is associated with the development of peribronchiolar infiltrates that are accompanied by submucosal edema and bronchorrhea, and ultimately leads to bronchiolar obstruction and compromised oxygen transfer. As the infection is confined to the respiratory epithelium, the responses of these cells are clearly of primary importance in determining the nature and extent of the resulting inflammatory process.\nMost of our understanding of responses to pneumovirus infection has emerged from studies of RSV infection of human epithelial target cells in vitro; a list of genes and/or gene products produced by epithelial cells in response to RSV infection in vitro is provided in Table 1 . At the cellular level, epithelial cells initially respond to RSV infection by reducing their ciliary beat frequency. Production and release of chemoattractant cytokines (chemokines) can be observed as early as 12 h after infection, leading to the recruitment of specific leukocyte subsets to the lung tissue. RSV-infected epithelial cells become resistant to tumor necrosis factor (TNF)-α-induced apoptosis, but later fuse to form giant-cell syncytia and die by cellular necrosis. We review the molecular bases (to the extent that they\nare understood) of these specific responses, and discuss several novel strategies that may permit us to study the responses to RSV and PVM infection in a more coherent and systematic manner.\nTristram et al [1] observed that explanted respiratory epithelial cells slow their ciliary beat frequency almost immediately after exposure to RSV, with complete ciliostasis seen as early as 6 h after the initial infection. The molecular basis of ciliostasis remains completely unknown.\nThe chemokines and cytokines with production and release that has been associated with RSV infection of human epithelial cells are listed in Table 1 . Much of this work was also recently reviewed elsewhere [2, 3] . We focus here on the three chemokines whose molecular mechanisms and physiologic implications are best understood.\nThe earliest reports on this subject described production of the neutrophil chemoattractant IL-8 from tissue culture supernatants from RSV-infected cells [4] [5] [6] and in nasal secretions from patients with viral rhinitis [7] . IL-8 has since been detected in lower airway secretions from patients with severe RSV bronchiolitis [8] , and the neutrophil influx observed in response to this infection is probably due, at least in part, to the activity of this chemokine.\nAt the cellular level IL-8 production can be observed in response to inactivated RSV virions, whereas IL-8 production in response to active infection was inhibited by ribavarin, amiloride, and antioxidants [9, 10] . Several groups have demonstrated activation of the transcription factor nuclear factor-κB (NF-κB) in response to RSV infection, and NF-κB is recognized for its central role in eliciting the production of IL-8 [9, 11, 12] . The transcription factor NF-IL-6 is also produced in response to RSV infection [13] , and participates in a co-operative manner with NF-κB in the regulation of IL-8 gene expression [11] , although later studies suggest that activator protein-1 may function preferentially in this role [14] . Interestingly, the NF-κB regulator IκBα, which functions by inhibiting NF-κB activation in response to TNF-α, was produced with different kinetics and does not promote a reversal of NF-κB activation in response to RSV infection as it does in response to TNF-α [15] . Most recently, Casola et al [16] demonstrated that the IL-8 promoter contains independent response elements, with nucleotides -162 to -132 representing a unique RSV response element that is distinct from elements necessary for IL-8 production in response to TNF-α. This concept of a stimulus-specific response will probably make an important contribution toward our understanding of how pneumoviruses promote transcription of unique and specific sets of independent gene products.\nThe pleiotropic chemokine regulated upon activation, normal T-cell expressed and secreted (RANTES) has also been detected in supernatants from RSV-infected epithelial cells in culture [17, 18] , as well as in upper and lower airway secretions from patients infected with this virus [7, 8] . RANTES acts as a chemoattractant for eosinophils and monocytes in vitro, although its role in vivo is somewhat less clear. Similar to IL-8, RANTES can be produced in vitro in response to inactivated virions [8] , and involves NF-κB activation, binding, and nuclear translocation [19] . However, Koga et al [20] demonstrated that stabilization of RANTES mRNA, a response to RSV infection mediated in part by nucleotides 11-389 of the RANTES gene, is probably the primary mechanism underlying increased production and secretion of RANTES protein. Further studies will determine whether a similar mechanism is also in place for IL-8 and other RSV-mediated responses. \nSeveral groups have recently shown that macrophage inflammatory protein (MIP)-1α is released from RSVinfected cells in culture [7, 21] ; MIP-1α was also detected in upper and lower airway secretions from RSV-infected patients [7, 8] . Interestingly, of the three aforementioned chemokines, MIP-1α is the one that is most closely correlated with the presence of eosinophil degranulation products; this, together with data from our PVM model of pneumovirus infection [22] , has suggested to us that MIP-1α plays a pivotal role in eosinophil recruitment in response to primary pneumovirus infection. Interestingly, production of MIP-1α in cell culture requires active viral replication [8] , which suggests that this response may proceed by a mechanism that is completely distinct from that which elicits production of RANTES and IL-8. However, no reports to date have addressed the molecular mechanism that underlies the RSV-mediated MIP-1α response.\nA list of cell-surface molecules that have been reported as expressed in response to RSV infection is shown in Table 1 . We focus here on the expression of intercellular adhesion molecule (ICAM)-1 (CD54) and the leukocyte integrin CD18.\nIncreased expression of this cell-surface adhesion protein was observed in both respiratory epithelial cell lines [23, 24] and in human nasal epithelial cells [25] in response to infection with RSV in vitro. Chini et al [26] demonstrated that the expression of ICAM-1 mRNA, similar to IL-8 and RANTES, was dependent on an intact NF-κB site in the gene promoter, and demonstrated a role for the consensus binding site for the factor CCAAT/ enhancer-binding protein. Stark et al [27] demonstrated that ICAM-1 and CD18 expressed in response to RSV serve to enhance neutrophil and eosinophil binding to epithelial cells.\nCD18 is a polypeptide of the integrin family that functions in mediating cell-cell interactions. Several groups have observed expression of CD18 on epithelial cells in response to RSV infection [27, 28] , with CD18 shown to enhance the degranulation of eosinophils in this specific setting [28] . Of particular interest are the recent findings relating expression of CD18 (along with CD14) to earlier literature on bacterial superinfections in the setting of viral infections. Earlier studies [29, 30] reported enhanced binding of bacteria to respiratory epithelial cells that were infected with RSV, findings that had clinical implications relating to acute bacterial otitis media in infants.\nTwo more recent studies addressed the question of binding sites. Saadi et al [31] determined that two strains of the pathogen Bordetella pertussis bound more efficiently to RSV-infected cells, and that the binding was reduced upon pretreatment of the cells with anti-CD14 or anti-CD18 antibodies. Similarly, Raza et al [32] reported that both CD14 and CD18 on RSV-infected epithelial cells contributed to the binding of nonpilate Neisseria meningitidis. In vivo testing is required before the clinical significance of these intriguing findings can be appreciated.\nRSV-infected epithelial cells in culture do not show features that are suggestive of apoptosis (ie no evidence of membrane blebbing, fragmentation of chromosomal DNA, or characteristic changes in nuclear morphology). Takeuchi et al [33] showed that, although RSV-infected epithelial cells express a number of apoptosis-associated genes, including interferon regulatory factor-1, IL-1β-converting enzyme and caspase 3, they do not undergo formal apoptosis.\nAs part of our attempts to understand mucosal responses in a more systematic manner (see below), we discovered that RSV-infected epithelial cells express the recently described antiapoptosis gene IEX-1L [34] . In our studies, we found that expression of IEX-1L is a response to active virus; no gene expression was observed in response to irradiated, replication-incompetent virus. Moreover, expression of IEX-1L is not observed in response to adenoviral infection, suggesting that expression of this gene is not a universal response to cellular perturbation, or indeed to all viral infections. Functionally, we also demonstrated that RSV infection protects epithelial cells from TNF-α-induced apoptosis, an effect that is temporally associated with the expression of IEX-1L.\nApoptosis is generally considered to be a highly efficient self-defense mechanism employed by host target cells, because it permits the infected host to dispose of viral proteins and nucleic acids on a single-cell basis without inducing an inflammatory response. It is thus not surprising that many viruses have evolved strategies to circumvent this response. Of interest, Krilov et al [35] demonstrated that monocytes and cord blood mononuclear cells are similarly protected from apoptosis when infected with RSV.\nAlthough virus-induced protection from apoptosis appears advantageous to the virus alone, another interpretation may be considered. Because respiratory epithelial cells are now recognized as a major source of leukocyte chemoattractants, and because leukocyte recruitment to the lung has been associated with enhanced viral clearance and prolonged survival in pneumovirus infection [22] , the ability to maintain chemoattractant production from viable cells may ultimately benefit the host organism as well.\nAvailable online http://respiratory-research.com/content/2/4/225\nIn tissue culture, RSV-infection is characterized by the formation of giant-cell syncytia. The mechanisms for the formation of these fused masses of cells depend in part on the expression of the RSV-specific fusion (F) protein on the surface of infected host cells, and in part on virusmediated changes in cytoskeletal architecture. It is important to note that RSV-induced changes in cytoskeletal architecture are not restricted to cell lines grown in vitro, as giant-cell syncytia have also been found in pathologic lung specimens obtained from both humans and animals that were infected with RSV.\nAgain, as part of our systematic study of gene expression in response to pneumovirus infection, we found that human respiratory epithelial cells respond to RSV infection with increased expression of the cytoskeletal protein cytokeratin-17 [36] . Cytokeratin-17 is a 46-kDa cytoskeletal protein that belongs to the class I acidic cytokeratin family. In the lung, expression of cytokeratin-17 is normally restricted to basal epithelial cells of the larynx, trachea, and bronchi. In RSV-infected cells, we found expression of Ck-17 predominantly at sites of syncytia formation, and thus provided the first description of a unique component of these pathognomonic structures at the molecular level. Similar to what has been reported for the production of IL-8, expression of Ck-17 is dependent on the activity of the transcription factor NF-κB, and future studies will determine the role of the NF-κB consensus site (-200 to -208 of the cytokeratin-17 promoter) in mediating this response.\nTo date, efforts to study pneumovirus-induced alterations in gene expression have relied heavily on in vitro models of virus-infected cells and cell lines. The intrinsic value of characterizing the genes identified in this artificial system is by definition limited, and the clinical and physiologic sig-nificance of any findings must ultimately be tested in vivo.\nTo some extent, the study of gene products in clinical specimens is possible, but this approach is limited, cumbersome, and dictated by sample availability. It is clear that an appropriate animal model of inflammatory bronchiolitis is required to characterize the alterations in gene expression discovered using the available in vitro models. Although RSV has been used for the study of specific allergic reactions to viral antigens, it is not a natural pathogen of mice, and intranasal inoculation of virus at high titer results in, at best, a minimal primary infection with a correspondingly minimal inflammatory response. In order to study gene expression in response to primary pneumovirus infection in vivo, we developed a novel mouse model of inflammatory bronchiolitis using the natural rodent pneumovirus pathogen and closest phylogenetic relative of RSV [37] -PVM. We presented our initial findings on PVM infection in mice in three recent publications [22, 38, 39] . A summary of these findings is presented in Table 2 and Fig. 1 .\nTo begin, we described the cellular and biochemical pathology observed in response to PVM infection in mice [38] . We found that infection could be established with as few as 30 plaque-forming units (pfu) of PVM in the inoculum, with infection resulting in significant morbidity and mortality, and viral recoveries in the order of 10 8 pfu/g lung tissue. We also noted inflammatory bronchiolitis as among the immediate responses to this infection, with bronchoalveolar lavage fluid containing virtually 100% neutrophils and eosinophils obtained as early as 3 days after inoculation. Furthermore, we found that infection was accompanied by the production of the proinflammatory chemokine MIP-1α, which was previously shown by Cook et al [40] to be an important component of the inflammatory response to the orthomyxovirus influenza virus.\nWe also described the role played by MIP-1α in the pathogenesis of PVM-induced bronchiolitis [22] . Specifi- cally, we explored the responses of gene-deleted mice to infection with PVM, and found no inflammatory response in mice deficient in MIP-1α expression (MIP-1α -/-) and only minimal virus-induced inflammation in mice that lacked the major MIP-1α receptor on granulocytes chemokine receptor (CCR)1 (CCR1 -/-). Although the inflammatory response is often considered to be unnecessary and indeed detrimental, we demonstrated that the absence of granulocytic inflammation was associated with enhanced recovery of infectious virions, as well as with accelerated mortality. These results suggest that the MIP-1α/CCR1-mediated acute inflammatory response protects mice by delaying the lethal sequelae of viral infection.\nOur most recent report on this subject [39] presents a direct comparison between the responses of mice to challenge with PVM and RSV. Although RSV is not a natural pathogen of mice, it has been used extensively in mouse models of human infection because a limited, or 'semipermissive' infection can be established via intranasal inocula-tion of virus at very high titers. In this regard, we found (as have others) that RSV infection did not result in any measurable degree of morbidity, and that viral recovery was significantly lower than that found in the inoculum; these results suggested that there was no significant viral replication in mouse lung tissue. We further demonstrated that the inflammatory response to RSV challenge was minimal, as few leukocytes were recruited to the lungs (Fig. 1) .\nTaken together, our results suggest that infection of mice with PVM provides a superior model for the study of acute inflammatory bronchiolitis in response to pneumovirus infection in vivo. The advantages of this model include the following: clinical parameters -morbidity and mortalitythat can be measured clearly and specifically; clear evidence of viral replication in lung tissue, with incremental recoveries that, at peak, are in excess of 10 8 pfu/g in response to as few as 30 pfu in the inoculum; and a dramatic granulocytic response that is modulated at least in part by the proinflammatory chemokine MIP-1α and its receptor CCR1. \nTraditionally, analysis of gene expression through measurement of steady-state levels of individual mRNAs could be conducted only one gene at a time using northern blotting, dot blots, or quantitative reverse transcription-PCR. Differential display, serial analysis of gene expression, and total gene analysis offer great promise, because they are multiplex technologies that provide simultaneous analysis of multiple mRNAs isolated under conditions of interest via PCR amplification techniques. DNA hybridization arrays are theoretically the most efficient of the gene expression analysis techniques. Although many skeptics have described these genome-based approaches as expensive, nonhypothesis-driven 'fishing expeditions', we view them as broad-based screening techniques that will enable us to identify patterns of gene expression that can then be subjected to careful characterization and analysis.\nDifferential display is a semiquantitative, reverse transcription-PCR-based technique that is used to compare mRNAs from two or more conditions of interest. Both increased and decreased expression of specific amplicons will be evident -an obvious advantage to this approach. Total RNA can be isolated from virus infected versus uninfected cells or mouse lungs both before and during infection, and differential display is performed using degenerate T11(XY) anchoring primers and random upstream oligomers, as described elsewhere [34, 36] . The resulting PCR products are separated by electrophoresis, and the gel is dried and exposed to film. An example of our results comparing cDNA amplicons from RNA extracted from RSV-infected epithelial cells daily for 4 days is shown in Fig. 2 . Differentially expressed bands are cut from the gel, eluted and reamplified using the same primers that generated the original signal, and northern blots generated from RNA extracted from pneumovirus-infected cells or tissue over time and probed with the differentially expressed amplicons serve to confirm differential expression of the identified sequence.\nThe DNA sequences of the newly identified differentially expressed amplicons are compared with sequences present in the GenBank database. Viral sequences are expected to be upregulated over time and can be identified immediately, because the entire genomes of both PVM and RSV are present in GenBank. In cases in which the amplicon represents a newly discovered gene, potential openreading frames are compared with sequences that are present in the Swiss protein database; motifs that share homologies with known proteins represent important clues to the identity of the differentially expressed gene.\nWith the help of differential display, we have identified and characterized several genes that are upregulated in RSV-infected respiratory epithelial cells. Two specific examples of genes that were found to be induced during RSV infection, and later characterized as playing independent roles in the pathophysiology of RSV infection, are the antiapoptosis gene IEX-1L [34] and the gene that encodes the cytoskeletal protein cytokeratin-17 [36] .\nUnlike DNA viruses, which are known to encode virus-specific antiapoptosis genes, RSV -an RNA virus with a small (approximately 15.2 kb) viral genome -was shown to alter host cell expression of the apoptosis inhibitor IEX-1L. After demonstrating that IEX-1L mRNA was present at sevenfold higher concentrations in RSV-infected respiratory epithelial cells when compared with uninfected cells, we concluded that this cellular response protected against TNF-α-induced programmed cell death during viral infection. Further efforts to determine which of the 11 RSV proteins participate in the trans-activation of the IEX-1L gene (either directly or indirectly) are ongoing.\nA second example of a gene that is specifically upregulated in RSV-infected respiratory epithelium, as identified by differential display, is that which encodes cytokeratin-17 [36] . Upon characterizing the molecular events that are important for cytokeratin-17 induction, we demonstrated a link to an NF-κB signaling pathway. Above, we discussed the importance of this transcription factor in the regulation of proinflammatory cytokine gene expression, and because of this involvement we were not surprised to discover its role in virus-induced cytokeratin-17 gene regulation. Perhaps the most interesting observation made during these experiments was the in situ localization of cytokeratin-17 protein to areas of cytopathic syncytia formation, suggesting a role for this cytoskeletal protein in their formation. Of note, we observed a dramatic decrease in RSV replication and in syncytia formation when we blocked cytokeratin-17 expression, suggesting that blocking syncytia formation, at least in part, impairs the direct cell-cell spread and productive replication of virus.\nAlthough there are several companies that market these systems and components, the cytokine gene macroarray systems recently developed by R&D Systems (Sigma Genosys ® ; Minneapolis, MN, USA) and Clontech (Atlas ® ; Palo Alto, CA, USA) represent some of the newer opportunities available that have a focus on gene products that are known to be involved in inflammation. These arrays consist of different cDNAs printed as PCR products onto charged nylon membranes. An example of our experience with the Sigma Genosys array is shown in Fig. 3 . For this example, total RNA was extracted from RSV-infected HEp-2 cells and uninfected controls at day 3 after infection. Three micrograms of total RNA was used in a cDNA synthesis reaction, using a proprietary mixture of 378 primer pairs and trace amounts of 32 P-radiolabeled dCTP.\nThe resulting radiolabeled products were hybridized to the macroarrays overnight at 65°C, and then washed and exposed to film. The arrow highlights one of the most obviously upregulated sequences from this experiment, which was identified as the gene encoding human MIP-1α. The physiologic importance of MIP-1α upregulation during human RSV infection and during rodent PVM bronchiolitis has already been described.\nMicroarrays can be differentiated from macroarrays in several ways. Among these differences, the microarray matrix is a glass or plastic slide, probes are labeled with fluorescent dye rather than via radioisotopes, and, most significantly, microarrays generally include a larger number and a higher density of imbedded sequences than do macroarrays. Although this may seem to be highly appealing at first, the massive amounts of data generated by microarray technology poses new challenges with respect to data normalization, management, and development of mathematical models to assist in data interpretation.\nThe pneumoviruses RSV and PVM enter respiratory epithelial cells via a receptor-mediated event. During hostcell attachment and internalization, the target cell begins to alter its gene expression, which, among other events, involves the transcriptional upregulation of cytokine and chemokine genes. As RSV replication progresses, additional changes in cellular gene expression can be observed, including induction of the potent antiapoptosis gene IEX-1L and increased expression of the otherwise quiescent gene that encodes cytokeratin-17. What we know regarding the physiologic importance of these genes and their gene products has been described, but there is more to be learned. As the available technologies evolve, we can continue to capitalize on the use of Display of amplicons generated from RNA extracted from RSV-infected cells at daily intervals following infection (days 0-4) using a single anchoring primer, T11GC (downstream primer 8) and (A-H) eight random 10mers. Two differentially expressed sequences are highlighted by arrows (the black arrow shows an upregulated amplicon, and the white arrow highlights a downregulated amplicon). Several other differentially expressed signals are also seen.\ngenomic approaches as large-scale screening tools to identify genes that play important roles in the pathophysiology of pneumovirus infection. These elegant and simple tools will provide us with the means for thorough and systematic exploration of gene expression, both in the estab- Cytokine macroarray probed with radiolabelled cDNA generated from total RNA extracted from epithelial cells 48 h after RSV infection (upper panel) or 48 h after exposure to conditioned medium (lower panel). Signal intensity of each gene under each condition is compared. The arrow highlights the signal for human MIP-1α present at 12-fold higher concentration in infected epithelial cells compared with the uninfected controls." }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: The genetic information of RNA viruses is organized very ef®ciently. Practically every nucleotide of their genome is utilized, either as protein-coding sequence or as cis-acting signals for translation, RNA synthesis or RNA encapsidation. As part of their genome expression strategy, several groups of positive-strand RNA (+RNA) viruses produce subgenomic (sg) mRNAs (reviewed by Miller and Koev, 2000) . The replication of their genomic RNA, which is also the mRNA for the viral replicase, is supplemented with the generation of sg transcripts to express structural and auxiliary proteins, which are encoded downstream of the replicase gene in the genome. Sg mRNAs of +RNA viruses are always 3¢-co-terminal with the genomic RNA, but different mechanisms are used for their synthesis.\nSome viruses, such as brome mosaic virus, initiate sg mRNA synthesis internally on the full-length minus strand RNA template (Miller et al., 1985) . Others, exempli®ed by red clover necrotic mosaic virus (RCNMV), may rely on premature termination of minus strand synthesis from the genomic RNA template, followed by the synthesis of sg plus strands from the truncated minus strand template (Sit et al., 1998) . Members of the order Nidovirales, which includes coronaviruses and arteriviruses, have evolved a third and unique mechanism, which employs discontinuous RNA synthesis for the generation of an extensive set of sg RNAs (reviewed by Brian and Spaan, 1997; Lai and Cavanagh, 1997; Snijder and Meulenberg, 1998) . Nidovirus sg mRNAs differ fundamentally from other viral sg RNAs in that they are not only 3¢-coterminal, but also 5¢-co-terminal with the genome ( Figure 1A) . A 5¢ common leader sequence of 65±221 nucleotides, derived from the 5¢ end of the genomic RNA, is attached to the 3¢ part of each sg RNA (thè mRNA body').\nVarious models have been put forward to explain the cotranscriptional fusion of non-contiguous parts of the nidovirus genome during sg RNA synthesis ( Figure 1B and C). Central to each of these models are short transcription-regulating sequences (TRSs), which are present both at the 3¢ end of the leader and at the 5¢ end of the sg RNA body regions in the genomic RNA. The TRS is copied into the mRNA and connects its leader and body part (Spaan et al., 1983; Lai et al., 1984) . Synthesis of sg mRNAs initially was proposed to be primed by free leader transcripts, which would base-pair to the complementary TRS regions in the full-length minus strand, and would be extended subsequently to make sg plus strands ( Figure 1B ; Baric et al., 1983 Baric et al., , 1985 . This model, however, was based on the report that sg minus strands were not present in coronavirus-infected cells (Lai et al., 1982) . The subsequent discovery of such molecules (Sethna et al., 1989) resulted in reconsideration of the initial`leader-primed transcription' model. Sawicki and Sawicki (1995) have proposed an alternative model ( Figure 1C ), in which the discontinuous step occurs during minus instead of plus strand RNA synthesis. In this model, minus strand synthesis would be attenuated after copying a body TRS from the plus strand template. Next, the nascent minus strand, with the TRS complement at its 3¢ end, would be transferred to the leader TRS and attach by means of TRS±TRS base pairing. RNA synthesis would be reinitiated to complete the sg minus strand by adding the complement of the genomic leader sequence. Subsequently, the sg minus strand would be used as template for sg mRNA synthesis, and the presence of the leader complement at its 3¢ end might allow the use of the same RNA signals that direct genome synthesis from the fulllength minus strand.\nSequence requirements for RNA strand transfer during nidovirus discontinuous subgenomic RNA synthesis The EMBO Journal Vol. 20 No. 24 pp. 7220±7228, 2001 Using site-directed mutagenesis of TRSs of the arterivirus equine arteritis virus (EAV), we have shown previously that base pairing between the sense leader TRS and antisense body TRSs is crucial for sg mRNA synthesis (van Marle et al., 1999a) . However, base pairing is only one step of the nascent strand transfer process and is essential in both models outlined in Figure 1 . The EAV genomic RNA contains several sequences that match the leader TRS precisely, but nevertheless are not used for sg RNA synthesis (den Boon et al., 1996; Pasternak et al., 2000) . This suggests that leader±body TRS similarity alone is, though necessary, not suf®cient for the strand transfer to occur.\nTo gain further insight into the cis-acting signals regulating sg RNA synthesis, we performed a comprehensive site-directed mutagenesis study of the EAV leader and body TRSs. Every nucleotide of the TRS (5¢-UCAACU-3¢) was substituted with each of the three alternative nucleotides. Our analysis revealed a number of striking similarities with the process of copy-choice RNA recombination, as it occurs in RNA viruses. Whereas the leader TRS plays only a targeting role in translocation of the nascent strand, body TRS nucleotides appear to ful®l diverse position-speci®c and base-speci®c functions. In addition, the sequence of the leader±body junctions of the sg mRNAs produced by these mutants provided strong evidence for the discontinuous minus strand extension model.\nEAV genome replication is not signi®cantly affected by leader TRS and body TRS mutations To dissect EAV RNA synthesis, we routinely use a fulllength cDNA clone (van Dinten et al., 1997) , from which infectious EAV RNA is in vitro transcribed. Following transfection of the RNA into baby hamster kidney (BHK-21) cells, intracellular RNA is isolated and analysed by northern blot hybridization and RT±PCR (van Marle et al., 1999a) . Due to differences in transfection ef®ciency, the total amount of virus-speci®c RNA (genomic RNA and sg mRNA) isolated from transfected cell cultures is somewhat variable. Thus, the accurate quantitation of sg mRNA synthesis by TRS mutants requires an internal standard for transfection ef®ciency. The amount of viral genomic RNA can be this standard, but only if its ampli®cation is not dramatically affected by the TRS mutations. To prove that this is the case, we used the previously described mutants L4, B4 and LB4 (van Marle et al., 1999a) , in which ®ve nucleotides of the TRS (5¢-UCAAC-3¢) were replaced by the sequence 5¢-AGUUG-3¢, either in the leader TRS (L4), RNA7 body TRS (B4) or both TRSs (LB4).\nThe three mutants were tested in three independent experiments. Intracellular RNA was isolated at 14 h posttransfection, early enough to prevent spread of the wildtype control virus to non-transfected cells (®rst cycle analysis). Transfection ef®ciencies were determined by immuno¯uorescence assays (see Materials and methods) and varied between 10 and 23% (data not shown). Prior to RNA analysis, the amount of isolated intracellular RNA was corrected for the transfection ef®ciency of the sample, so that each lane in Figure 2 represents EAV-speci®c RNA from an approximately equal number of EAV-positive cells. Phosphoimager quantitation revealed that genomic RNA replication of mutants L4, B4 and LB4 varied by not more than 30% (Table I) . These differences could re¯ect, for example, a slight in¯uence of RNA secondary structure changes in the TRS regions on genomic RNA synthesis. Remarkably, however, the genomic RNA level of the leader±body TRS double mutant LB4 was not affected by more than 10%. In view of the results obtained with these pentanucleotide TRS mutants, we assumed that the amount of genomic RNA could indeed be used as an internal standard during the analysis of mutants containing only single nucleotide replacements in leader TRS and/or RNA7 body TRS. The regions of the genome specifying the leader (L) sequence, the replicase gene (ORFs 1a and 1b) and the structural genes are indicated. The nested set of EAV mRNAs (genome and sg mRNAs 2±7) is depicted below. The black boxes in the genomic RNA indicate the position of leader and major body TRSs. (B and C) Alternative models for nidovirus discontinuous sg RNA synthesis. The discontinuous step may occur during either plus strand (B) or minus strand (C) RNA synthesis. In the latter case, sg mRNAs would be synthesized from an sg minus strand template. For details see text. Northern analysis of EAV-speci®c RNA isolated from cells transfected with RNA transcribed either from the wild-type EAV infectious cDNA clone or from TRS pentanucleotide mutants (UCAAC to AGUUG). The results of two independent experiments are shown.\nThe RNA±RNA interaction between the leader and body TRSs is not the only factor that regulates EAV sg RNA synthesis There are numerous examples of regulatory RNA±RNA interactions in both eukaryotic and prokaryotic cells, as well as in RNA viruses. Essential processes such as translation, replication and encapsidation of RNA virus genomes frequently depend on RNA±RNA interactions and higher order RNA structures. Regulation of sg RNA synthesis of +RNA viruses by RNA±RNA interactions is also not without precedent. In tomato bushy stunt virus, an RNA element located 1000 nucleotide upstream of the sg RNA2 promoter base-pairs with the promoter and is necessary for sg RNA production (Zhang et al., 1999) . Similarly, base pairing interactions between complementary sequences in the 5¢ end of the potato virus X genomic RNA and sequences upstream of two major sg RNA promoters are required for ef®cient sg RNA synthesis (Kim and Hemenway, 1999) . In RCNMV, an intermolecular RNA±RNA interaction is required for sg RNA synthesis (Sit et al., 1998) .\nRecently, we have established the pivotal role of an interaction between sense and antisense RNA sequences in the life cycle of EAV (van Marle et al., 1999a) . In that study, the role of TRS nucleotides C 2 and C 5 was tested by substituting them with G. It was concluded that base pairing between the sense leader TRS and the antisense body TRS plays a crucial role in nidovirus sg RNA synthesis. We now took a more systematic approach and performed an extensive site-directed co-variation mutagenesis study of the entire leader TRS and RNA7 body TRS, which directs the synthesis of the most abundant EAV sg RNA. Every nucleotide of the TRS (5¢-UCA-ACU-3¢) was replaced with each of the other possible nucleotides. As in the study of van Marle et al. (1999a) , every mutation was introduced into leader TRS, RNA7 body TRS and both TRSs, resulting in 54 mutant constructs. Each mutant was given a unique name: e.g. BU 1 A refers to a mutant in which a U has been changed to A at position 1 of the body TRS; LU 1 A refers to the same substitution in the leader TRS; and DU 1 A means that these two substitutions were combined in one double mutant construct. The amount of sg RNA7 was quantitated by phosphoimager scanning of hybridized gels and was corrected for the amount of genomic RNA in the same lane (as outlined above). Figure 3 shows the relative sg RNA7 level of the 54 mutants, compared with the RNA7 level of the wild-type control. For a selection of 11 interesting mutants (see below), the analysis was repeated three times (Figure 4 ), without observing signi®cant variations in sg RNA synthesis.\nThe comprehensive analysis of the effects of TRS mutations considerably expanded our understanding of van Dinten et al., 1997) was taken along as a positive control. For every mutant, the level of sg RNA7 synthesis was calculated as [(sg/g)/(sg/g) wt ] 3 100%: it was corrected for the level of genomic RNA (used as an internal standard; see text) and subsequently was related to the level of sg RNA7 produced by the wild-type control in the same experiment, which was also corrected for the corresponding genomic RNA level. The relative sg RNA7 level of the wild-type control was set at 100%.\nA.O. Pasternak et al. discontinuous sg RNA synthesis. Remarkably, the effects of single (leader or body) TRS mutations were mostly base speci®c, i.e. different nucleotide substitutions at the same position affected sg RNA7 synthesis to different extents. For example, at position 1, the BU 1 A mutant retained 44% of the wild-type RNA7 synthesis level, whereas both the BU 1 C and BU 1 G mutants lost RNA7 synthesis almost completely. Conversely, when U 1 of the leader TRS was changed to A or G, RNA7 synthesis was completely abolished, whereas 13% of the wild-type level was still maintained by LU 1 C. For position 2, only the BC 2 U mutant retained 30% of the wild-type RNA7 synthesis level, while all the other position 2 single mutants have lost 90% or more of wild-type RNA7 synthesis. Another example is position 6: BU 6 C left only 5% of wild-type RNA7 synthesis, whereas BU 6 A produced much higher RNA7 levels. This implied that for some positions (1, 2 and 6), certain mismatches in the duplex between plus leader TRS and minus body TRS, such as U±U (BU 1 A and BU 6 A) or C±A (LU 1 C and BC 2 U), are allowed to a limited extent. In contrast, no mismatches were allowed for position 5, where all single nucleotide substitutions abolished RNA7 synthesis almost completely. Surprisingly, both body TRS U to C substitutions at positions 1 and 6 (BU 1 C and BU 6 C) resulted in low levels of RNA7, despite the fact that these mutations allow the formation of a G±U base pair between the plus leader TRS, providing the U nucleotide, and the minus body TRS, providing the G. On the other hand, for positions 3 and 4, G±U base pairing was shown to be functional, because mutants LA 3 G and LA 4 G, which can form G±U base pairs between the G in the plus leader TRS and U in the minus body TRS, were the only position 3 and 4 single mutants that produced reasonable levels of RNA7. Taken together, these ®ndings suggest that other factors, besides leader± body base pairing, also play a role in sg RNA synthesis and that the primary sequence (or secondary structure) of TRSs may dictate strong base preferences at certain positions. Our analysis of the degree of complementation by the double mutants provided strong support for this assumption.\nDifferentiating between effects at the level of primary TRS sequence and the level of leader±body duplex formation For some TRS nucleotides (2, 5 and 6, except in the case of DU 6 C), the RNA7 level of double mutants was clearly higher than that of the corresponding single mutants. This means that base pairing between these leader and body TRS nucleotides is involved in sg RNA synthesis. However, none of these double mutants reached the wild-type sg RNA7 level. In the other double mutants (all position 1, 3 and 4 mutants, and DU 6 C), in clear contradiction to the predictions of the`base pairing model', RNA7 synthesis was not signi®cantly restored. Moreover, a comparison of the values for the B and D mutants in Figure 3 showed that, for almost all of these mutants (e.g. the position 1 mutants), the amount of sg RNA7 produced by the double mutant appeared to be limited by the level allowed by the body TRS mutation. Sometimes the RNA7 level of the double mutant was even less than that of the leader mutant (DU 1 C, DA 3 G, DA 4 G or DU 6 C). Clearly, for these substitutions, restoration of the possibilities for leader±body duplex formation did not restore sg RNA synthesis. Apparently this is because the effect of body TRS mutations at the level of primary sequence or secondary structure can be`dominant' over the duplex-restoring effects of the double mutations.\nBody TRS mutants thus fell into two distinct types, determined by the position and chemistry of the substitution. In mutants of the ®rst type, sg RNA synthesis was impaired mainly because of the disruption of the leader± body TRS duplex. This effect could be compensated for by introduction of the corresponding mutation in the leader TRS and, in the double mutant, sg RNA synthesis was restored compared with the corresponding single mutants. In mutants of the second type, sg RNA synthesis was down-regulated as a consequence of both TRS duplex disruption and disruption of the primary sequence (or secondary structure) of the body TRS. Obviously, the latter effect could not be compensated for by mutating the leader TRS, and the corresponding double mutants did not show restoration of sg RNA synthesis.\nIn contrast to our ®ndings with the body TRS mutants, we did not obtain leader TRS mutations that appeared to determine the level of sg RNA7 synthesis of the corresponding double mutant (Figure 3) . Thus, effects of mutations in the leader TRS were not`dominant' over the duplex-restoring effects of the double mutations, suggesting that they only affected duplex formation. This indicated that the leader TRS probably does not have an additional, sequence-speci®c function in sg RNA synthesis in addition to its participation in TRS±TRS base pairing. The fact that single leader TRS mutations at all six Nidovirus discontinuous subgenomic RNA synthesis positions severely repressed RNA7 synthesis indicated that base pairing of every TRS nucleotide contributes to sg RNA production. In this respect, it was signi®cant that the two leader TRS mutants with the highest RNA7 levels, LA 3 G and LA 4 G, can form G±U base pairs to maintain the duplex.\nThe observation that leader TRS mutations could bè rescued' by introducing complementary mutations in the body TRS, but that many body TRS mutations could not bè rescued' by corresponding changes in the leader TRS, is clearly illustrated by the U 1 A mutants. Due to the restoration of TRS base pairing possibilities, the RNA7 synthesis of double mutant DU 1 A was signi®cantly increased compared with that of LU 1 A, but not above the level of BU 1 A. Thus, restoration of the leader±body duplex in DU 1 A exerted a clear effect on sg RNA7 production compared with LU 1 A, but had no effect on sg RNA synthesis compared with BU 1 A. This exempli®ed the dominant nature of a mutation in the primary sequence of a body TRS. In contrast, for instance, the BC 2 U mutation probably affected duplex formation only, because RNA7 synthesis was restored almost to wildtype levels in the DC 2 U double mutant.\nThese results indicate that there are strong base preference constraints for some body TRS positions. To interpret these base preferences accurately, it is necessary to limit the analysis to the double mutants only, because in these mutants the down-regulation of sg RNA synthesis was only due to the sequence changes in the body TRS, and not to the disruption of the leader±body TRS duplex. There were strict preferences for positions 1, 3 and 4 of the body TRS: at position 1, only the U to A substitution allowed for a signi®cant RNA7 level (~40% of wild-type); and at positions 3 and 4, only the A to U mutants retained 15±20% of the wild-type level. For positions 2 and 5, the sequence constraints were less stringent (all substitutions allowed for >20% of wild-type level), but still only DC 2 A and DC 2 U reached >50%. At position 6 of the body TRS, only U to C was not allowed, whereas the other two double mutants still produced 50% or more of RNA7. In other words, the functional EAV RNA7 body TRS (based on the analysis of our single nucleotide substitutions) can be described as U 1 (C/u/a) 2 A 3 A 4 C 5 (U/a/g) 6 , with wild-type nucleotides shown in upper case and nucleotides that allowed for at least 50% of the wild-type RNA7 level shown in lower case. Remarkably, TRS nucleotides A 3 , A 4 and C 5 are conserved in the TRSs of all other arteriviruses (Snijder and Meulenberg, 1998) . Also the fact that DC 2 U retained 80% of RNA7 synthesis corresponded nicely to the presence of a U at this position in other arteriviruses.\nUntil recently (Almazan et al., 2000; Thiel et al., 2001) , infectious cDNA clones were lacking for coronaviruses. Consequently, most studies on coronavirus sg RNA synthesis were carried out using defective interfering (DI) RNAs. These replicons carried body TRSs from which moderate levels of sg mRNAs could be produced in the presence of helper virus. Using this system, Joo and Makino (1992) and van der Most et al. (1994) performed body TRS mutagenesis studies for the murine coronavirus (MHV). Joo and Makino systematically mutagenized the core of the MHV body TRS. In contrast to our results, they found that in only two of 21 body TRS mutants was sg RNA synthesis from the DI RNA genome abolished, whereas all others supported normal levels of sg RNA production. Thus, it is possible that the MHV TRS which was used in that study is more tolerant to single-nucleotide mismatches than the EAV sg RNA7 TRS.\nIn a similar study, van der Most et al. (1994) observed that U to C substitutions at positions 1 and 3 of the MHV body TRS, which maintained the duplex by changing a U±A base pair into a U±G base pair, reduced sg RNA levels more strongly than substitutions that disrupted the duplex (van der Most et al., 1994) . This implies that, as in the case of EAV, leader±body TRS duplex formation is not the only factor that determines coronavirus sg RNA synthesis. However, because of the limitations of the DI RNA system, the leader TRS could not be mutagenized in these studies, and body TRS-speci®c effects could not be distinguished from effects at the level of leader±body duplex formation.\nThe discontinuous step in nidovirus sg RNA synthesis occurs during minus strand RNA synthesis Due to recent studies of arterivirus and coronavirus sg RNA synthesis (van Marle et al., 1999a; Baric and Yount, 2000; Sawicki et al., 2001) , the discontinuous minus strand extension model ( Figure 1C ) has been gaining more and more ground. This model predicts that the TRSderived sequence that forms the leader±body junction in the sg mRNA is a copy of the body TRS, and not of the leader TRS. The leader-primed transcription model predicts the opposite ( Figure 1B) . Therefore, determining the origin of the leader±body junction of sg mRNAs would help to distinguish between the two models. However, in the wild-type situation, EAV leader and body TRSs are identical and consequently one cannot determine the origin of the sg mRNA leader±body junction. This problem could be overcome by tracing the mutations introduced in leader or RNA7 body TRS mutants, most of which retained part of their ability to produce mRNA7. In a previous study (van Marle et al., 1999a) , we found that nucleotides 2 and 5 of the mRNA7 leader±body junction sequence were derived exclusively from the body TRS, and not from the leader TRS. This was shown by direct sequencing of RT±PCR products obtained from the residual mRNA7 produced by mutants BC 2 G, LC 2 G, BC 5 G and LC 5 G ( van Marle et al., 1999a) .\nUsing the same approach, we analysed mRNA7 from mutants BC 2 A and BC 2 U, and these transcripts also contained the mutated nucleotide derived from the body TRS (data not shown). Assuming that only one crossover event occurs during leader±body joining, we could thus map this crossover between positions ±1 and +2 of the sg RNA junction sequence. This left the intriguing question of whether the crossover site could be mapped even more precisely. In other words, was nucleotide +1 of the junction sequence derived from the body TRS or the leader TRS?\nUsing the position 1 mutants described above, we could answer this question ( Figure 5) . The most striking result was that mRNA7 of mutants BU 1 A, BU 1 G and LU 1 C contained exclusively the body TRS-derived nucleotide at position +1. Thus, for these mutants, the crossover site could be mapped precisely between TRS nucleotide positions ±1 and +1, meaning that the complete leader± body junction sequence in an EAV sg mRNA can be body TRS derived. On the other hand, sg RNAs from mutants LU 1 A, BU 1 C and LU 1 G contained mixed populations of leader TRS-and body TRS-derived nucleotides at position +1 ( Figure 5 ): A and U for LU 1 A, C and U for BU 1 C, and G and U for LU 1 G. Remarkably, this pattern correlated with the relative amounts of sg mRNA7 produced by these mutants (Figure 3 ). Mutants that produced populations of sg RNAs that were mixed with respect to the origin of the nucleotide at position +1 of the leader±body junction had lost RNA7 synthesis almost completely. On the other hand, mutants that contained exclusively the body nucleotide at position +1 retained higher levels of RNA7 synthesis. This observation may be explained as follows: in the wild-type situation, the large majority of the crossovers probably occur between positions ±1 and +1, leading to a body TRS-derived nucleotide at position +1 in the sg RNA; however, a low number of crossovers take place between nucleotides +1 and +2, resulting in a leader TRS-derived nucleotide at position +1. Mutants in which almost all sg RNA synthesis is blocked by a substitution at position +1 may somehow be de®cient in the crossover between ±1 and +1, but may have retained the ability for crossovers between +1 and +2, which were detected by sequence analysis. Conversely, in position +1 mutants that retain reasonable sg RNA synthesis, most crossovers occur between positions ±1 and +1, and they obscure the minority of crossovers between +1 and +2 in the sequencing electropherogram. Alternatively, position +1 TRS mutations that strongly interfere with sg RNA synthesis may force a shift of the crossover site in the remaining molecules.\nWe believe that our present ®ndings strongly support the discontinuous minus strand extension model. Indeed, the fact that a complete body TRS can be copied into the sg RNA is very dif®cult to reconcile with the alternative model, in which sg RNA synthesis from the genomic minus strand template is primed by free plus strand leader transcripts that contain the leader TRS at their 3¢ end ( Figure 1B) . To explain the presence of a complete copy of the body TRS in the sg mRNA in this model, one would have to assume that a 3¢±5¢ exonuclease activity trims back the free leader transcript prior to its extension into an sg mRNA (Baker and Lai, 1990) . Note that there would not be a single base pair left to hold these`trimmed' leader molecules on the template. Such an enzymatic activity, which is unprecedented in +RNA viruses, exists in yeast retrotransposon Ty5 (Ke et al., 1999) , in which reverse transcription is primed by an internal region in a tRNA. However, in this system, it is not a part of the duplex that is removed, but the single-stranded 3¢ tail of the tRNA, which cannot base-pair with the Ty5 RNA.\nRemoval of the TRS at the 3¢ end of the nidovirus leader, which has already base paired with the template, would be very energetically unfavourable for the RdRp. Instead of starting elongation using the intact and properly positioned leader as a primer, it would have to disrupt the newly formed duplex, degrade part of the leader RNA and then reinitiate polymerization, without any base pairing between primer and template. It has been shown that in¯uenza virus transcription does not require a sequence match between the (cellular) RNA primer and the (viral) template (Plotch et al., 1981) . However, if in the nidovirus system the`trimmed' leader RNA could also be ®xed on the template solely by RNA±protein interactions, the targeting of the nascent strand by TRS base pairing would be extremely puzzling.\nSequence data of sg RNA leader±body junctions from other arteriviruses are also dif®cult to reconcile with the leader-primed transcription model. For the porcine and simian arteriviruses (Meulenberg et al., 1993; Godeny et al., 1998) , the leader±body junctions of some sg RNAs mapped two nucleotides upstream of the body TRS, which again would not leave a single nucleotide to hold the putative free leader on the template after the hypothetical back trimming'. On the other hand, these ®ndings and our data can be explained readily by the discontinuous minus strand extension model ( Figure 1C ). The six-nucleotide Fig. 5 . Sequence analysis of mRNA7 leader±body junctions from position 1 TRS mutants. Sequences were determined directly from sg mRNA7-speci®c RT±PCR products. For the U 1 A and U 1 C mutants, the sequence shown corresponds to the plus strand of sg RNA7. For sequencing-related technical reasons, the minus strand sequence was determined for the U 1 G mutants; a mirror image of the electropherogram is shown with the corresponding plus strand sequence listed at the top of the panel. For every mutant, a sequence alignment of the leader (red) and body (blue) TRSs and surrounding sequences is shown (TRSs are boxed). The mRNA7 leader±body junctions detected by our sequence analysis are shown in yellow. duplex formed between the body TRS complement at the 3¢ end of the leaderless sg minus strand and the leader TRS in the genomic RNA template should suf®ce to position the nascent minus strand properly for subsequent elongation to add the complement of the leader sequence. In most cases, the nascent minus strand contains the entire body TRS complement at its 3¢ end at the moment of strand transfer, leading to a body TRS-derived leader±body junction sequence in the sg mRNA molecule. In a small number of transcripts, however, minus strand synthesis appears to be interrupted before nucleotide +1 of the body TRS is copied and, after strand transfer, resumes by incorporating the complement of the +1 nucleotide of the leader TRS. As stated above, we postulate that the detection of this phenomenon is determined by the level of crossovers between the ±1 and +1 position that is allowed by the mutations introduced at the +1 position of body TRS or leader TRS. We cannot, however, formally exclude that a`back trimming' activity degrades the 3¢-terminal nucleotide of the minus strand before or after strand transfer. However, note that in the discontinuous minus strand extension model ( Figure 1C ), such an activity would not disturb the proper positioning of the nascent minus strand on the leader template, because the TRS± TRS duplex would be shortened by one nucleotide only.\nNidovirus discontinuous minus strand extension resembles similarity-assisted, copy-choice RNA recombination Due to their discontinuous sg RNA synthesis, nidoviruses occupy a special`niche' in the +RNA virus world. Their mode of sg RNA production is clearly different from that of other +RNA viruses and resembles another welldocumented +RNA virus feature: RNA recombination (for recent reviews see Nagy and Simon, 1997; Aaziz and Tepfer, 1999; Worobey and Holmes, 1999) . Most of the experimental evidence supports an RdRp template switch (Kirkegaard and Baltimore, 1986) as the main mechanism of RNA recombination. Mechanistically, such a template switch involves the transfer of a nascent strand from one RNA template (donor) to the other (acceptor). Also, nidovirus discontinuous sg RNA synthesis involves transfer of a nascent RNA strand, the sg RNA, but now from one site to another in the same template.\nBased on the data currently available, we refer to the discontinuous minus strand extension model as our working model for nidovirus sg RNA synthesis. If one applies the`recombination terms' to this model (Chang et al., 1996; Brian and Spaan, 1997; van Marle et al., 1999a) , the donor strand would be the body part of the genomic RNA template, the acceptor strand would be the leader part of the genomic RNA template and the nascent strand would be the discontinuously synthesized minus strand. Nagy and Simon (1997) have de®ned three main classes of RNA recombination: similarity-essential, similarity-non-essential and similarity-assisted recombination. The latter is de®ned as a mechanism in which strand transfer is determined by both sequence similarity between the parental RNAs and additional RNA determinants, present in only one of the parental RNAs.\nThe results of our present study strongly suggest that nidovirus discontinuous sg RNA synthesis can be considered a special case of high-frequency similarity-assisted RNA recombination. While the only obvious function of the leader TRS is to ensure the ®delity of the strand transfer by base pairing with the 3¢ end of the nascent strand, the body TRS in the donor template indeed has additional, sequence-speci®c functions. One of these functions apparently is to pause (or terminate) nascent strand synthesis and thereby provide the opportunity for strand transfer. In addition, body TRS-derived nucleotides may play a role in the reinitiation of nascent strand synthesis on the acceptor template. Given the compact nature of the EAV TRS, it is quite possible that some nucleotides ful®l multiple tasks.\nRNA secondary structure of the body TRS may regulate sg RNA synthesis The sequence-speci®c function of the body TRS, revealed in this study, may be exerted at the level of either primary sequence or secondary structure. For a number of +RNA viruses, RNA secondary structure motifs located in the (proximity of) sg RNA promoters are vital for sg RNA synthesis. In alfalfa mosaic virus (Haasnoot et al., 2000) , turnip crinkle virus (TCV) (Wang et al., 1999) and barley yellow dwarf virus (Koev et al., 1999) , stem±loop structures in sg RNA promoter regions of the template strand are required for sg RNA synthesis. The sg RNA1 promoter of the latter virus is especially interesting, since it contains two stem±loop domains. For one of them, secondary structure, but not the primary sequence, is important for sg RNA synthesis, whereas the other domain acts through primary sequence, and not secondary structure (Koev et al., 1999) . Similarly, RNA secondary structure may play only a minor role in the sequence-speci®c recognition of the BMV sg RNA promoter by the RdRp Siegel et al., 1997) .\nWe have suggested previously that RNA secondary structure of body TRS regions contributes to their attenuating potential and thereby determines the relative portion of the nascent minus strands that is transferred to the leader TRS in the template (Pasternak et al., 2000) . At present, it is unknown whether EAV body TRSs are part of an RNA structural motif that is essential for body TRS function, or whether they are recognized by a protein factor in a sequence-speci®c manner. However, the latter seems less likely than the former, since even LB4 (Figure 2 ), in which ®ve TRS nucleotides were substituted, still produced some sg RNA7, although~30-fold less than the wild-type control. The fact that some sequences in the EAV genome match the leader TRS perfectly, but are not used for sg mRNA synthesis, also argues against the recognition of a speci®c sequence (Pasternak et al., 2000) . More probably, mutagenesis of the RNA7 body TRS disturbed an RNA structure that is necessary for its function. This could, for example, explain the fact that the BU 6 C substitution reduced the amount of RNA7 by 20-fold (and could not be rescued by the same mutation in the leader TRS), whereas the wild-type RNA6 body TRS contains a C at the same position. If a protein factor were involved in sequence-speci®c TRS recognition, then one would expect it to recognize all TRSs similarly. If RNA structure is important for recognition by such a protein, then the BU 6 C substitution probably disturbs a structural motif of the RNA7 TRS, which is not present in the RNA6 TRS. On the other hand, conservation of part of the TRS in other arteriviruses suggests a sequence-speci®c recognition. Further studies are required to distinguish between these possibilities.\nIn the TCV satellite RNA recombination system, the hairpin structure in the acceptor strand, as well as the donor±acceptor homology region, are necessary for the template switch . The hairpin has been postulated to bind the RdRp, whereas the homology region targets the nascent strand to the crossover site. The TCV RdRp probably recognizes the secondary and/or tertiary structure of the hairpin, while individual nucleotides play a less important role . In EAV, the leader TRS in the acceptor template is predicted to reside in the loop of an extensive hairpin, and its base pairing interaction with the body TRS complement at the 3¢ end of the nascent minus strand would resemble certain antisense RNA-regulated control mechanisms that are based on interactions between single-stranded tails and hairpin loops (van Marle et al., 1999a, and references therein) . It is possible that the EAV RdRp, or its accessory proteins, also binds to the stem of the long hairpin that presents the leader TRS. In any case, the leader TRS itself does not seem to be recognized by a protein in a sequence-speci®c manner.\nThe body TRS is a better candidate to serve as a protein recognition site. This protein would then mediate the pausing of the nascent strand synthesis and/or nascent strand transfer. This would resemble the DNA-dependent RNA polymerase I termination system, in which speci®c DNA-binding terminator proteins bind to termination sequences (Reeder and Lang, 1997) , or a function of the HIV nucleocapsid protein, which promotes the minus strand strong-stop DNA transfer (Guo et al., 1997) . The EAV replicase component nsp1, which recently was shown to possess an sg RNA synthesis-speci®c activity (Tijms et al., 2001) , may be a good candidate for such a regulatory role. Residues predicted to form a zinc ®nger structure in nsp1 were shown to be necessary for sg RNA synthesis. Interestingly, zinc ®nger structures in the HIV nucleocapsid protein facilitate strand transfer (Guo et al., 2000) . Finally, it should be noted that the RNA structure of the nascent strand may also in¯uence pausing, strand transfer or reinitiation, as illustrated by the fact that stable hairpin structures in the nascent strand promote termination of transcription by Escherichia coli RNA polymerase (Wilson and von Hippel, 1995) .\nSite-directed mutagenesis, RNA transfections and immuno¯uorescence analysis Site-directed mutagenesis of EAV leader and body TRSs was carried out as described by van Marle et al. (1999a) , and all mutant constructs were sequenced. Following in vitro transcription from infectious cDNA clones, full-length EAV RNA was introduced into BHK-21 cells by electroporation, as described by van Dinten et al. (1997) . Immuno¯uorescence assays with EAV-speci®c antisera were performed at 14 h posttransfection as described by van der Meer et al. (1998) . To visualize the nuclei for cell counting, nuclear DNA was stained with 5 mg/ml Hoechst B2883 (Sigma). Cells were counted using the Scion Image software (Scion Corporation) and the percentage of transfected cells was calculated on the basis of the number of cells positive for the EAV replicase component nsp3 (Pedersen et al., 1999) .\nFor RNA analyses, cells were lysed at 14 h post-transfection. Intracellular RNA isolation was performed using the acidic phenol method as described by Pasternak et al. (2000) . Total intracellular RNA was resolved in denaturing agarose±formaldehyde gels. Hybridization of dried gels with the radioactively labelled oligonucleotide probe E154, which is complementary to the 3¢ end of the EAV genome and recognizes all viral mRNA molecules (genomic and subgenomic), and phosphoimager quantitation of individual bands were performed as described by Pasternak et al. (2000) . To determine the leader±body junction sequence of sg mRNA7, mRNA7-speci®c RT±PCRs were carried out as described by van Marle et al. (1999b) using an antisense (RT and PCR) primer from the RNA7 body region and a sense PCR primer matching a part of the leader sequence. RT±PCR products were sequenced directly as described by Pasternak et al. (2000) using the leader-derived primer, an ABI PRISMÔ sequencing kit (Perkin Elmer) and an ABI PRISMÔ 310 Genetic Analyser (Perkin Elmer)." }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: Anaemia is a common condition in critically ill patients, and RBC transfusions are often used in the treatment and management of this patient population. In fact, one study [1] reported that 25% of all critically ill patients received RBC transfusions. Many laboratory studies [2] [3] [4] [5] [6] [7] [8] have examined the physiological responses (ie compensatory mechanisms) of the body to anaemia, which include the following [9] : increased cardiac output, decreased blood viscosity, capillary changes, increased oxygen extraction, and other tissue adaptations to meet oxygen requirements. Although critically ill patients are affected by a number of factors that predispose them to the adverse consequences of anaemia, persistence of this condition is of particular concern because it may cause the compensatory mechanisms in these patients to become impaired, risking oxygen deprivation in vital organs [9] . However, critically ill patients may also be at increased risk from the adverse effects of RBC transfusions, such as pulmonary oedema from volume overload, immune suppression resulting in increased risk of infection, and microcirculatory injury from poorly deformable RBCs.\nIt is the aim of the present commentary to justify the statement 'Transfusing to normal haemoglobin concentration will not improve outcome.' If we define normal haemoglobin as being greater than 115 g/l for women and greater than 125 g/l for men, then there is no evidence in the literature to justify maintaining such high concentrations by the use of RBC transfusions in any anaemic patient. There may, however, be some debate about adopting a transfusion threshold of 100 g/l, which is well below 'normal'. transfusion threshold would, obviously, reduce the number of allogeneic RBCs transfused. It is our goal to impress upon the reader that transfusing to a level equal to or greater than 100 g/l does not improve mortality and other clinically important outcomes in a critical care setting. We first explore the reasons why a reduction in the total number of allogeneic blood transfusions would be beneficial. Second, we examine the current evidence for using a lower transfusion strategy, specifically that employed in the Transfusion Requirements In Critical Care (TRICC) trial.\nRBC transfusions have inherent risks that may be categorized as follows [11] [12] [13] [14] [15] : transfusion-transmitted infections; immune-related reactions (acute or delayed haemolytic reactions, febrile, allergic, anaphylactic reactions and graft-versus-host disease); and nonimmunerelated reactions (fluid overload, hypothermia, electrolyte toxicity and iron overload).\nMajor improvements in donor screening procedures and laboratory testing have dramatically improved the safety of the blood supply [16] . Currently, the risk of transmitting an infectious agent through blood transfusion ranges from 1:100,000 for hepatitis B virus to 1:1,000,000 for HIV (Canadian Blood Services, personal communication, 2000). The most important threats to the blood supply remain new and unknown pathogens. More recently, concern has focused on the potential transmission of prions through RBCs. Also, infectious agents with long latency periods pose particular risks to young individuals who require RBCs, such as multiple trauma victims. The risk : benefit ratio for a 24-year-old trauma victim with a 50-year life expectancy differs markedly from that for a person aged 80 years who is undergoing coronary artery bypass surgery. In summary, because there is a risk of transmitting diseases through the blood supply, we should always strive to use RBCs according to the best available evidence in order to ensure that we do more good than harm to our patients.\nIt is a long-standing observation [17] [18] [19] [20] [21] that blood transfusions are associated with immune suppression. This clinical phenomenon was first observed in renal transplant patients who had received blood transfusions while on dialysis before the transplant [22] , and has been observed repeatedly in transplant centres around the world [23, 24] . Recently, Opelz et al [25] reported a multicentre clinical trial in which all renal allograft recipients received modern immunosuppressive regimens. Those patients who were allocated to receive three allogeneic RBC units before renal transplant had a 1-year graft survival rate of 90%, as compared with 82% for patients who were not transfused (P = 0.02). These data suggest that there are long-term immunosuppressive effects following transfusion of nonleukocyte-reduced allogeneic RBCs.\nA large number of studies [26] [27] [28] [29] [30] [31] [32] [33] [34] have also suggested that allogeneic transfusions accelerate cancer growth, perhaps due to altered immune surveillance. These altered immune responses after allogeneic RBC transfusions may also predispose critically ill transfusion recipients to nosocomial infections [35] [36] [37] [38] [39] [40] and increased rates of multiplesystem organ failure [41] , which may ultimately result in higher mortality rates. However, most studies that examined the association between cancer recurrence and postoperative infection after transfusion [42, 43] only provided weak causal inferences because of poor study design and the lack of independence between allogeneic RBC transfusions and the potential complication.\nA recent meta-analysis [44] combined the results from seven RCTs, and was unable to detect clinically important decreases in mortality and postoperative infections. We added the results of a new RCT by van de Watering et al [45] to the above meta-analysis. The relative risk for allcause mortality was 1.05 (95% confidence interval 0.88-1.25), and was 1.10 (95% confidence interval 0.85-1.43) for postoperative infections. However, this meta-analysis excluded two positive RCTs [40, 46] because of the significant statistical heterogeneity introduced by these two studies. If all available RCTs are combined, ignoring heterogeneity, then the relative risk difference for postoperative infections across all studies is 1.60 (95% confidence interval 1.00-2.56; P = 0.05). Thus, the available evidence suggests that universal prestorage leukoreduction could have clinical effects that range from none to decreasing rates of infection by as much as 50% in high-risk patients. In summary, despite convincing laboratory evidence and some supportive clinical studies, the clinical significance of the immunosuppressive effects of allogeneic RBC transfusions have not been clearly established [47] . More importantly, the impact of a leukoreduction programme has not been studied in a large population of patients who are expected to have significant exposure to allogeneic RBCs.\nThe majority of complications from allogeneic RBC transfusion, however, are no more frequent in the intensive care setting than in other patient populations, with the possible exception of pulmonary oedema, hypothermia and electrolyte disturbance. Hypothermia and electrolyte disturbances occur most frequently with massive transfusions. In critically ill patients, the optimal effective circulatory volume may be difficult to determine, and as a consequence pulmonary oedema may be a much more frequent complication of RBC transfusion than in other patient populations. This may explain the significantly higher rate of pulmonary oedema in patients transfused using a threshold of 100 g/l (5.3% in the restrictive transfusion group versus 10.7% in the liberal transfusion group; P < 0.01), as reported in the TRICC trial [10] . As an alternative explanation, the more frequent use of RBCs might have resulted in more frequent episodes of transfusion-related acute lung injury in the liberal strategy group (7.7% in the restrictive strategy versus 11.4% in the liberal strategy; P = 0.06), as reported in the TRICC trial.\nClinical evidence is also insufficient to definitively establish a correlation between the age of RBCs being transfused and patient mortality; however, laboratory evidence has shown many storage-related changes that may result in impairment of blood flow and oxygen delivery at the microcirculatory level. Marik et al [48] demonstrated an association between a fall in gastric intramucosal pH and transfusion of RBCs stored for longer than 15 days. In addition, there is ample laboratory evidence that prolonged RBC storage adversely affects RBCs, potentially results in the generation of cytokines, and alters host immune function. In another study, Fitzgerald et al [49] , using an animal model of transfusion, consistently observed a lack of efficacy of transfused, stored rat blood to improve tissue oxygen consumption as compared with fresh cells or other blood substitutes.\nThree retrospective clinical studies tested the association between the age of transfused blood and duration of stay in the intensive care unit (ICU) [50] and mortality [51, 52] . Martin et al [50] observed a statistically significant association between the transfusion of aged blood (>14 days old) and increased duration of ICU stay (P = 0.003) in 698 critically ill patients. In patients who received a transfusion, aged RBCs was the only predictor of duration of stay (P < 0.0001). In survivors, only median age of blood was predictive of duration of stay (P < 0.0001). Purdy et al [51] demonstrated a negative correlation (r = -0.73) between the proportion of RBC units of a given age transfused to survivors and increasing age of RBCs in patients admitted to the ICU with a diagnosis of severe sepsis (n = 31). Those investigators also noted that these latter units were more likely to be older. A recently reported study by Vamvakas and Carven [52] evaluated the effect of duration of RBC storage on postoperative pneumonia in 416 consecutive patients undergoing coronary artery bypass grafting. Those investigators noted an adjusted increase of 1% in the risk of postoperative pneumonia per day of average increase in the duration of RBC storage (P < 0.005) in transfused patients. Each of these three studies also noted that patients who received a large number of RBC units had a higher mortality. Although these risks are relatively small when viewed collectively, they become significant when one considers that 25% of all critically ill patients in Canada are transfused during their ICU stay [1] .\nUntil recently, physicians have depended on clinical judgement when deciding at what haemoglobin level to transfuse a critically ill patient. As a result, significant variation has been shown to exist in transfusion practice among Canadian critical care physicians [53] , which is due largely to a lack of published data on the subject. An arbitrary haemoglobin level of 100 g/l has historically been used as a threshold to transfuse critically ill patients.\nSix observational studies investigated the importance of anaemia on transfusion practices in various settings. Of these, three large cohort studies, which were performed in different patient populations (intensive care [1] , coronary artery bypass surgery [54] and hip fractures [55]), reached different conclusions. RBC transfusions in particular improved outcome in critically ill patients with cardiovascular disease, but increased the risk of myocardial infarction in coronary artery bypass surgery patients. Transfusion had no impact on short-term or long-term mortality in hip-fracture patients. Three smaller studies [56] [57] [58] evaluated the relationship between anaemia and adverse outcomes in vascular disease patients. Although increased numbers of ischaemic events were observed in anaemic patients, the validity of these studies is uncertain, given that the decision to transfuse a patient was often correlated with illness burden of the patient. It is also possible that comorbidity was not adequately adjusted for in those studies.\nTransfusion thresholds were compared in 10 randomized clinical trials [10, [59] [60] [61] [62] [63] [64] [65] [66] [67] . Although the clinical settings varied, each trial randomized patients to be transfused on the basis of a 'conservative' or a 'liberal' strategy. The definitions of conservative and liberal strategies varied, and actually overlapped between studies. Of these 10 trials, only three included more than 100 patients and only one trial evaluated the impact of transfusion on symptoms. In the first trial of patients undergoing elective coronary artery bypass surgery [65] , the differences between perioperative haemoglobin levels were small, event rates were very low, and there were no differences in any outcome. In the second trial [67] , patients undergoing knee arthroplasty were randomly assigned to receive autologous blood transfusion immediately after surgery or to receive autologous blood if haemoglobin level fell below 9 g/dl [67] . Again, no differences in outcome were observed. The third trial of hip fracture patients undergoing surgical repair [64] found no differences in outcomes; however, five deaths were recorded at 60 days after surgery in the symptomatic group, and two deaths occurred in the 10 g/dl group. The numbers of patients in these trials were too small to evaluate the effect of lower transfusion triggers on clinically important outcomes such as mortality, morbidity and functional status.\nIn 1999, Hebert et al [10] reported the results of the TRICC trial. Patients (n = 838) were randomized either to a restrictive strategy (haemoglobin concentration maintained between 70 and 90 g/l, with a trigger set at 70 g/l) or to a liberal strategy (haemoglobin concentration maintained between 100 and 120 g/l, with a trigger at 100 g/l). To date, the TRICC trial is the only large study that has investigated these parameters. The groups were comparable at baseline. The average daily haemoglobin concentration ranged from 85 ± 7.2 g/l in the restrictive group to 107 ± 7.3 g/l in the liberal group (P < 0.01). The average number of transfusions was reduced by 52% in the restrictive group (2.6 ± 4.1 versus 5.6 ± 5.3 RBCs/patient; P < 0.01). Cardiac events, primarily pulmonary oedema and myocardial infarction, were more frequent in the liberal strategy (P < 0.01; Table 1 ). On examination of composite outcomes, the number of patients with multiorgan failure was found to be substantially increased in both groups, with the results being marginally better in the restrictive strategy group (20.6% versus 26.0%; P = 0.07; Table 2 ). Overall, the restrictive transfusion group showed a lower 30-day mortality (18.7% versus 23.3%; P = 0.11; Fig. 1 ). Kaplan-Meier survival curves, however, were significantly different in the subgroup of patients with an Acute Physiology and Chronic Health Evaluation II score of 20 or less (P = 0.02; Fig. 2 ). In addition, although 60-day mortality (22.8% versus 26.5%; P = 0.23) and ICU mortality (13.9% versus 16.2%; P = 0.29) were not statistically significant, they did show a consistent trend in terms of absolute values that favoured the restrictive strategy. The key observation from the TRICC trial is not that the restrictive strategy is better, but rather that it is at worst equivalent to the liberal strategy and at best superior to the liberal strategy.\nAt this juncture, preclinical and clinical evidence support the adoption of a more restrictive transfusion strategy in most critically ill patients. However, there remain divergent views regarding the risks and benefits of treating anaemia in patients with cardiovascular disease. Laboratory-based studies [68, 69] suggest that patients with cardiovascular disease may require higher haemoglobin concentrations to maintain oxygen delivery in partially occluded or diseased coronary arteries. Studies to demonstrate how anaemia affects contractile function of the left ventricle have rarely shown important effects above haemoglobin concentrations of 70 g/l. Indeed, it is more important to address the underlying pathophysiological causes of the acute coronary syndrome with proven therapy such as aspirin and β-blockers, rather than treating mild-to-moderate anaemia as an initial step. If the effects of RBC transfusion were either limited or increased then there would be no debate; however, the use of allogeneic RBCs has been shown to be associated with immunomodulation [12, 47] and/or alteration in the delivery of oxygen in the microcirculation [70, 71] , resulting in increased rates of infections and organ failure.\nFew clinical studies have attempted to elucidate the risk : benefit ratio of anaemia and transfusion in cardiac patients. Two small RCTs [62, 72] examined transfusion practice in patients undergoing coronary artery bypass grafting, and concluded that a conservative approach to the administration of RBCs may be safe. However, two recent cohort studies suggested that anaemia may increase the risk of mortality in critical illness [73] and following surgery in patients with cardiovascular disease [74] . There were 418 and 420 patients in the restrictive and liberal transfusion groups, respectively. *Difference calculated by subtracting mean values of restrictive group from those of liberal group. † Three patients were lost to 60-day mortality rate; therefore n = 835. ‡ Nonsurvivors are considered to have all organs failing on date of death. § Changes in MOD score from baseline, while also incorporating adjustment for death. Data from Hébert et al [10] .\nIn a study of Jehovah's Witnesses (a group that refuses RBC transfusion on religious grounds) undergoing surgical procedures [74] , it was noted that mortality was significantly increased in patients with cardiac disease after a decrease in haemoglobin levels from 100-110 g/l to 60-69 g/l. In that study, patients with no cardiac disease and similar changes in haemoglobin levels showed no increase in mortality, which is in accordance with the results of the TRICC trial [10] . In the study by Hébert et al. [73] of 4470 critically ill patients, a correlation between Critical Care Vol 5 No 2 Alvarez et al [10] .\nKaplan-Meier estimates of survival in the 30 days after admission to the ICU in the restrictive and liberal transfusion strategy groups (all patients). Data from Hébert et al [10] .\nKaplan-Meier estimates of survival in the 30 days after admission to the ICU in the restrictive and liberal transfusion strategy groups (patients with APACHE II score ≤20). Data from Hébert et al [10] .\nanaemia and mortality rates was observed. Those investigators also found that the risk of anaemia appeared to decrease with RBC transfusion in patients with cardiac disease. In patients with cardiac disease, mortality increased when haemoglobin concentrations were below 95 g/l, as compared with anaemic patients with other diagnoses (55% versus 42%; P = 0.09). In the subgroup of patients with cardiac disease, increasing haemoglobin values in anaemic patients was associated with improved survival (odds ratio 0.80 for each 10 g/l increase; P = 0.012). One possible explanation for the discrepancy between the TRICC trial and this observational study may be that the attending physicians who recruited patients into the study did not enter those patients who were considered to have severe cardiac disease.\nHébert et al. [73] sought to examine further whether a restrictive transfusion strategy was at least as effective as a liberal strategy in critically ill patients with cardiac disease. In the subgroup of patients with cardiovascular disease from the TRICC trial, those investigators suggested that most haemodynamically stable critically ill patients with cardiovascular disease may be transfused when haemoglobin concentrations fall below 70 g/l, and that the hemoglobin concentration should be maintained between 70 and 90 g/l. Average daily haemoglobin concentrations were 85 ± 6.2 g/l in the restrictive transfusion group and 103 ± 6.7 g/l in the liberal transfusion group (P < 0.01). In the 357 patients with cardiovascular disease, the 30-day mortality rate was 23% in the restrictive transfusion group versus 23% in the liberal group (95% confidence interval of the difference -8.4% to 9.1%; P = 1.00). Other mortality rates, including 60-day (26% versus 27%; P = 0.90), ICU (19% versus 16%; P = 0.49) and hospital mortality (27% versus 28%; P = 0.81), were not significantly different between groups. Kaplan-Meier survival curves comparing time to death demonstrated similar trends in the two groups ( Fig. 3 ; P = 0.98). The multiple organ dysfunction (MOD) scores, during the entire study period, were also not significantly different between groups (8.6 ± 4.9 versus 9.0 ± 4.4; P = 0.40), but the change in MOD score from baseline values was significantly lower in the restrictive group than in the liberal group (0.2 ± 4.2 versus 1.3 ± 4.4; P = 0.02).\nCombined measures of morbidity and mortality, or composite outcomes, were also examined. When all patients who died were given a score of 24, the total MOD score between groups was not different (P = 0.39), or were the changes in MOD scores significantly different from baseline (2.7 ± 6.9 versus 4.0 ± 7.3; P = 0.08). Among the specific subset of cardiac patients with ischaemic heart disease (n = 257), there were no discernible differences in 30-day and 60-day as well as ICU mortality rates. However, a nonsignificant (P = 0.3) decrease in overall survival rate in the restrictive group was noted in those patients with confirmed ischaemic heart disease, severe peripheral vascular disease or severe comorbid cardiac disease (Fig. 4) .\nIn conclusion, a restrictive RBC transfusion strategy generally appears to be safe in most critically ill patients with cardiovascular disease, with the possible exception of patients experiencing acute myocardial infarction or unstable angina. \nSurvival over 30 days in patients with ischemic heart disease in the restrictive and liberal allogeneic RBC transfusion strategy groups. This graph illustrates Kaplan-Meier survival curves for all patients with ischemic heart disease in both study groups. There is no difference in mortality in patients in the restrictive group (dashed line) as compared to the liberal group (solid line) (P = 0.30).\nThe need to reduce the amount of allogeneic blood transfusions in order to reduce the associated risks has been firmly established. RBCs are associated with clinically important immune suppression, and stored RBCs have been shown to cause adverse microcirculatory effects that result in increased organ failure.\nThe question for some time has been whether critically ill patients are able to tolerate lower levels of haemoglobin without deleterious effects, thus reducing the amount of exposure to allogeneic transfusions. In the only large RCT, Hébert et al [10] established that there was no difference in mortality rates between restrictive and liberal transfusion strategies in noncardiac, critically ill patients. Although those investigators were able to show convincing trends that the liberal strategy may in fact be deleterious in terms of absolute values, statistical significance was not achieved. However, the fact that no difference between the two strategies was achieved is of great importance, because this means that the total number of transfusions can be reduced by approximately half without any impact on mortality. In addition, these findings are easily put into clinical practice. Although many questions remain, the TRICC trial and many laboratory and clinical studies have established that transfusing to normal haemoglobin concentrations does not improve organ failure and mortality in the critically ill patient. As such, a restrictive transfusion strategy will reduce exposure to allogeneic transfusions, result in more efficient use of RBCs, save blood overall, and decrease health care costs." }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: This year's symposium was dominated by the results of recent clinical trials. After 10 years of 'magic bullet' trials in sepsis, a number of successful therapeutic options are now emerging. In addition, recent advances in our understanding of the soup of mediators observed in sepsis offer yet more tantalizing targets for new therapies.\nIn contrast, the eagerly awaited results from Italy of the prone positioning trial in ARDS were disheartening. The epidemiology of both sepsis and ARDS, and their impact on clinical studies and the future provision of critical care were also hot topics. The era of extracorporeal liver replacement therapy is upon us, with considerable early promise and the probability of wide availability. Finally, as always, ethics remained an area of interest.\nThis report summarizes and discusses the presentations on the above topics.\nAngus (Pittsburgh, PA, USA) presented his group's work on the epidemiology of sepsis in the USA (accepted for publication in Critical Care Medicine). They developed a method for identifying hospitalized patients with sepsis based on ICD9 criteria, the most widely recorded coding system used in US hospitals. Prospective testing of the method found it to be both sensitive and reliable. They then applied it to a representative selection of US hospitals. Their results indicated that about 50% of intensive care unit (ICU) patients have systemic inflammatory response syndrome, and that approximately 20% of these progress to severe sepsis. Mortality for severe sepsis was greater than 30%. Demographically, those at the extremes of age represent the most at-risk groups, in whom the mortality is also the highest. These data provides yet another reminder that the increasing demands on health care resources caused by the ageing population is predicted to exceed intensive care provision within the next The 21st International Symposium on Intensive Care and Emergency Medicine, Brussels, Belgium, 20-23 March 2001 10-20 years. Finally, those investigators found a striking demographic peak in patients aged 20-30 years, which they attributed largely to human immunodeficiency virus.\nThe long-standing debate between the two schools of sepsis theory -microcirculatory dys-autoregulation versus cellular dysfunction -shows signs of resolution. New techniques for studying tissue oxygen tension, presented by Ince (Amsterdam, The Netherlands), provide more evidence that microcirculatory dys-autoregulation results in significant shunting. This occurs predominantly in the submucosal and serosal portions of organs, and is an early event. These studies show that the macroscopic restoration of global oxygen delivery fails to improve oxygen consumption as the mucosa becomes hyperoxic, whereas the submucosa and serosa remain hypoxic. Somewhat counterintuitively, this can be reversed in the face of resistant hypotension with vasodilators, at least in animal models.\nThe cellular dysfunction camp, although still somewhat doubtful as to the importance of these microcirculatory findings, have now clearly established that their championed mechanism of mitochondrial failure is a late but crucial event in the evolution of sepsis. Fink (Pittsburgh, PA, USA) presented evidence that mitochondrial failure in septic cells is triggered by the activation of the enzyme poly-adenosine diphosphate ribose polymerase [1] . This enzyme represents a significant target for novel therapies, which are apparently already in development. The debate regarding the toxicity of oxygen and the formation of free radicals continues despite the absence of demonstrated effectiveness of scavenging therapies, and is a testament to the incomplete understanding of this area.\nThe round-table conference preceding this year's symposium concentrated on distilling current knowledge on the microscopic events in critically ill patients into an explanation of the macroscopic multiorgan failure that is so commonly encountered. The conclusions of the conference appeared to relate mostly to future directions for research, in particular the study of organ-organ interactions. Marshall (Toronto, Canada) proposed the development of an alternative to the much-maligned physiological scoring systems, based on the staging systems widely used in the field of oncology. He proposed that mediator levels, in addition to physiological variables, will soon be used usefully to characterize septic patients. He also suggested that, in the light of the recent successful mediator trials in sepsis, future therapies will be directed in a manner analogous to the control of glucose in diabetic patients.\nThe natural anticoagulants antithrombin III (AT III), tissue factor pathway inhibitor (TFPI) and activated protein C (APC), and the cytokine tumour necrosis factor (TNF)-α are the latest inflammatory mediators to be targeted in large multicentre clinical trials in an attempt to improve the current dismal outcome for patients with severe sepsis.\nThe KyberSept AT III study recruited over 2300 patients from 200 centres, with high Simplified Acute Physiology Scale scores (median 50), and a mortality of nearly 40% [2] . Unfortunately, no overall benefit was shown between AT III and placebo, although results were more encouraging in an analysis of the subgroup of patients who received AT III but no heparin, which is known to inhibit AT III. Interestingly, improvements in quality of life scores were seen in survivors who received AT III in comparison to those who received placebo, suggesting that morbidity may be reduced, although again this was an analysis of a subgroup. Patients in the AT III group who received concomitant heparin had a significantly higher incidence of bleeding events, and outcome worsened as the dose of heparin increased. Explanations for the failure of this study included the inhibitory effects of heparin and the failure to achieve AT III activity levels of greater that 200% from baseline in the treatment population, a level established as required for therapeutic benefit in phase II trials.\nPhase II clinical trial results using TFPI (TFPI n = 141, placebo n = 69; unpublished data) show a mortality benefit in the sicker sepsis patients who already have coagulation problems. Results of the phase III multicentre study are expected to be presented at the 22nd International Symposium on Intensive Care and Emergency Medicine, in Brussels in 2002.\nHuman trials of various anti-TNF-α formulations have been variable to date, and include North American sepsis trial (NORASEPT) I [3] , International sepsis trial (INTERSEPT) [4] and NORASEPT II [5] . Possible reasons have included a lack of biological activity of the anti-TNF-α formulation studied, inappropriate timing of therapy, redundancy of proinflammatory mediators and hetereogeneity of patient populations. The Monoclonal Anti-TNF, A Randomized controlled Sepsis Trial (MONARCS) study used a different anti-TNF-α formulation (F[ab′]2 fragment of a murine monoclonal antibody to human TNF-α), and stratified patients based on demonstrable abnormalities in immunological pathways (highly elevated interleukin-6 levels -a circulating cytokine that is induced by TNF-α). Unpublished results revealed 28-day mortality rates of 44 and 48% in the anti-TNF-α and placebo groups, respectively, in those patients who had high interleukin-6 levels on recruitment to the study (n = 488 anti-TNF-α, n = 510 placebo). This represented a relative mortality reduction of 14%. Relative mortality reduction in all patients (n = 1305 anti-TNF-α, n = 1329 placebo), independent of baseline interleukin-6 levels, was only 10%.\nThe Recombinant Human Activated Protein C Worldwide Evaluation in Severe Sepsis (PROWESS) study is hot off the press [6] , and presentation of the results at the congress allowed those of us who still carry the unopened New England Journal of Medicine issue in our briefcases to catch up! A total of 164 sites from 11 countries recruited 1690 patients with severe sepsis, before the trial was prematurely stopped following the second safety analysis. Twenty-eight-day all-cause mortality rates for placebo and APC were 31 and 25% respectively, with a relative risk reduction of 19%. Resolution of cardiovascular and respiratory function was more rapid in survivors who received APC, although ICU and hospital stay did not differ. There was a trend towards an increase in serious bleeding events in the APC group (3% APC versus 2% placebo), but these events were primarily due to trauma or instrumentation. Although this is an exciting breakthrough, we all recognize that when APC reaches the market place it will seriously stretch ICU finances, especially because there appear to be other mediators on the horizon that we will be encouraged to use, in combination, to fight the inflammatory 'soup'.\nTwo opposing epidemiological views of ARDS were presented by Lemaire (Créteil, France) and Evans (London, UK). Broad agreement does seem to exist as to the incidence of this condition, which is in the order of 10/100,000, although there is significant variation between countries. It was argued that this variation results from the availability of ventilated beds, with higher incidences apparent in countries with greater provision, emphasizing that this condition can be considered the result of intensive care intervention or, as one speaker put it, 'physician-induced lung injury'.\nEarly results from the Acute Lung Injury Verification of European Epidemiology (ALIVE) study (unpublished data), sponsored by the European Society of Intensive Care Medicine, are at odds with recent trial findings. The ALIVE study, which included over 6000 patients surveyed in 1998, found a 50-60% 28-day mortality, which compares to only 20-30% in the control groups of recent trials. Pneumonia was the commonest cause, responsible for 50% of cases, with sepsis identified as the cause in a further 20-30%. Astonishingly, this study found the ratio of arterial oxygen tension to fractional inspired oxygen at ICU admission was highly predictive of mortality, despite continuing controversy regarding this measurement.\nA diverse range of views were presented from the Third International Consensus Conference on ARDS (unpublished data), held in Barcelona late last year. The decision as to how to change the defining criteria for this condition remains unresolved. The debates surrounding chest X-ray criteria, the use of the ratio of arterial oxygen tension to fractional inspired oxygen, and the level/utility of pulmonary artery wedge pressure measurements continue.\nIn addition, a debate has arisen as to whether ARDS can be a unilateral process, and whether it can coexist with cardiac failure. There appears to be increasing recognition that ARDS represents only a small subset of patients with acute lung failure (approximately 30%). Surprisingly little is known about the remainder of this larger group. In contrast to the ALIVE study, several centres have reported their 28-day mortality at 40%, which represents an improvement from the 60% of 10 years ago. However, it was argued that a 28-day follow-up period is too short for clinical trials, as the long-term quality of life for patients with ARDS is poor compared with that of critically ill patients without this condition. Results suggest that the recovery of lung function is good overall, but is dependent on severity. Treatment recommendations include the universal adoption of the US National Institutes of Health protective lung ventilation strategy [7] . There was general agreement that recruitment manoeuvres are beneficial, but how and when to employ them remains controversial.\nRouby (Paris, France) put forward a new classification for ARDS based on computed tomography findings. He observed that patients can be split into three groups, depending on the appearance of the upper lobes. In group 1 the upper lobes are normal, and positive end-expiratory pressure (PEEP) is of little benefit and results in significant over-distension. Survival in this group is approximately 60%. In group 2 the upper lobes are abnormal, PEEP is of dramatic benefit, but survival is only approximately 25%. In group 3 there are mixed/patchy abnormalities, the effects of PEEP are less predictable, but, as in group 1, survival is approximately 60%.\nGattinoni (Milan, Italy) presented the results of the longawaited Italian multicentre randomized controlled trial of prone positioning in ARDS (unpublished data). This trial was terminated after 3 years despite having only recruited 304 patients; enrollment of 750 patients was originally planned, in order to achieve sufficient statistical power. At trial outset, recruitment was encumbered by the lack of familiarity with and scepticism regarding this procedure in many of the centres. However, by the end of the study many participants were unwilling to enter patients into the trial, because they felt it unethical to deny them this intervention. The trial protocol resulted in patients in the treatment group being prone for an average of only 7 out of 24 h for a 10-day period. Overall there was no difference in mortality between the control and treatment groups at day 10, time of ICU discharge, or at 6 months. Interestingly, analysis of subgroups revealed a significant difference in the outcome at 10 days for patients with the most severe disease, although this disappeared by ICU discharge. In retrospect, the design of this ambitious trial was flawed by its failure to establish the optimal utilization of this manoeuvre. \nThe opening session reported that we are making progress in supporting the failing liver (Wendon, London, UK). Current optimism should probably be limited to extracorporeal methods, because the molecular adsorbent recirculating system (essentially extracorporeal albumin dialysis) has been shown to have beneficial clinical effects as well as improved survival in two small randomized controlled trials [8, 9] . The equipment is familiar to all those who use dialytic therapies, and we will undoubtedly hear more about this system in the next few years.\nThe slide of a patient reading the newspaper through a transparent helmet, while receiving noninvasive ventilation (NIV) resembled pictures of a NASA astronaut! However, it was reported to be well tolerated for prolonged periods, and significantly reduces the complications associated with NIV (pressure areas, tolerance of mask). The recent Consensus Conference [10] examined weaning aspects of NIV and emphasized the reduced weaning time and avoidance of reintubation, but called for more randomized controlled trials. Finally, although continuous positive airway pressure has been shown to be beneficial in pulmonary oedema, caution is still advised with the use of bilevel positive airway pressure because of the reporting of myocardial infarction in several studies. However, the groups studied were unmatched and starting points were different, so conclusions should not be drawn until randomized controlled trial results are available in this area.\nThis was a well-attended session, which, according to Levy (Providence, RI, USA), was in complete contrast to the interest shown in the USA for the subject. Although there were few new data in the session, the emphasis on a strategy for lawsuits was welcome. Suggestions included statements from scientific societies at a national and international level, open reporting in medical files of decisions to withdraw or withhold treatment, and family involvement in decision making that will ultimately involve better media education.\nThe last day of this year's symposium was sadly abandoned by many due to the Belgian rail strike. Despite this, the usual convivial atmosphere, both in and around the congress, was as abundant as ever. Overall, the 'state of the art' lectures, pro/con debates, seminars and tutorials were of the usual high standard, although, yet again, access to many of the symposium's venues was limited by the lack of capacity of the secondary rooms. The 21st International Symposium was marked by a sense of renewed enthusiasm that real positive progress is occurring at the coal face of intensive care." }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: The heme oxygenase-1/carbon monoxide (HO-1/CO) system has recently seen an explosion of research interest due to its newly discovered physiological effects. This metabolic pathway, first characterized by Tenhunen et al. [1, 2] , has only recently revealed its surprising cytoprotective properties [3, 4] . Research in HO-1/CO now embraces the entire field of medicine where reactive oxygen/nitrogen species, inflammation, growth control, and apoptosis represent important pathophysiological mechanisms [3] [4] [5] [6] . Indeed, the number of publications in recent years concerning HO-1 has increased exponentially, while the list of diseases and physiological responses associated with changes in HO-1 continues to expand [5] .\nUntil now, relatively few studies have addressed the role of HO-1/CO in pulmonary medicine. Several investigators have focused on the diagnostic application of the HO-1/CO system, by measuring exhaled CO (E-CO) in various pathological pulmonary conditions, such as asthma or chronic obstructive pulmonary disease (COPD) [7] . In another experimental approach, investigators have examined the expression of HO-1 in lung tissue from healthy or diseased subjects [8, 9] . This review will highlight the actions of HO-1/CO in the context of heme degradation have antioxidant properties [18, 19] . The liberated heme iron undergoes detoxification either by extracellular efflux or by sequestration into ferritin, an intracellular iron-storage molecule with potential cytoprotective function [20] [21] [22] [23] . Of the three known isoforms of HO (HO-1, HO-2, and HO-3), only HO-1 responds to xenobiotic induction [24] [25] [26] [27] . Constitutively expressed in many tissues, HO-2 occurs at high levels in nervous and vascular tissues, and may respond to regulation by glucocorticoids [25, 28, 29] . HO-1 and HO-2 differ in genetic origin, in primary structure, in molecular weight, and in their substrate and kinetic parameters [25, 26] . HO-3 displays a high sequence homology with HO-2 but has little enzymatic activity [27] . This review will focus on the inducible, HO-1, form.\nIn addition to the physiological substrate heme, HO-1 responds to induction by a wide variety of stimuli associated with oxidative stress. Such inducing agents include hypoxia, hyperoxia, cytokines, nitric oxide (NO), heavy metals, ultraviolet-A (320-380 nm) radiation, heat shock, shear stress, hydrogen peroxide, and thiol (-SH)-reactive substances [3] . The multiplicity of toxic inducers suggest that HO-1 may function as a critical cytoprotective molecule [3, 4] . Many studies have suggested that HO-1 acts as an inducible defense against oxidative stress, in models of inflammation, ischemia-reperfusion, hypoxia, and hyperoxia-mediated injury (reviewed in [3] ). The mechanisms by which HO-1 can mediate cytoprotection are still poorly understood. All three products of the HO reaction potentially participate in cellular defense, of which the gaseous molecule CO has recently received the most attention [30, 31] . The administration of CO at low concentrations can compensate for the protective effects of HO-1 in the presence of competitive inhibitors of HO-1 activity [32] [33] [34] . While HO-1 gene transfer confers protection against oxidative stress in a number of systems, clearly not all studies support a beneficial role for HO-1 expression. Cell-culture studies have suggested that the protective effects of HO-1 overexpression fall within a critical range, such that the excess production of HO-1 or HO-2 may be counterprotective due to a transient excess of reactive iron generated during active heme metabolism [35, 36] . Thus, an important caveat of comparative studies on the therapeutic effects of CO administration versus HO-1 gene delivery arises from the fact that the latter approach, in addition to producing CO, may have profound effects on intracellular iron metabolism.\nHO-1 expression is primarily regulated at the transcriptional level. Genetic analyses have revealed two enhancer sequences (E1, E2) in the murine HO-1 gene located at -4 kb (E1) and -10 kbp (E2) of the transcriptional start site [37, 38] . These enhancers mediate the induction of HO-1 by many agents, including heavy metals, phorbol esters, endotoxin, oxidants, and heme. E1 and E2 contain repeated stress-responsive elements, which consist of overlapping binding sites for transcription factors including activator protein-1 (AP-1), v-Maf oncoprotein, and the cap'n'collar/basic-leucine zipper family of proteins (CNC-bZIP), of which Nrf2 (NF-E2-related factor) may play a critical role in HO-1 transcription [39] . The promoter region of HO-1 also contains potential binding sites for nuclear factor κB (NF-κB), though the functional significance of these are not clear [40] . Both NF-κB and AP-1 have been identified as regulatory elements responsive to oxidative cellular stress [40, 41] . In response to hyperoxic stress, AP-1 factors mediated the induction of HO-1 in cooperation with signal-transducer and activator of transcription (STAT) proteins [41] . Furthermore, a distinct hypoxia-response element (HRE), which mediates the HO-1 response to hypoxia, represents a binding site for the hypoxia-inducible factor-1 (HIF-1) [42] .\nThe toxic properties of CO are well known in the field of pulmonary medicine. This invisible, odorless gas still claims many victims each year by accidental exposure. CO evolves from the combustion of organic materials and is present in smoke and automobile exhaust. The toxic actions of CO relate to its high affinity for hemoglobin (240-fold greater than that of O 2 ). CO replaces O 2 rapidly from hemoglobin, causing tissue hypoxia [43] [44] [45] . At high concentrations, other mechanisms of CO-induced toxicity may include apoptosis, lipid peroxidation, and inhibition of drug metabolism and respiratory enzyme functions [44] .\nOnly recently has it become known that, at very low concentrations, CO participates in many physiological reactions. Where a CO exposure of 10,000 parts per million (ppm) (1% by volume CO in air) is toxic, 100-250 ppm (one hundredth to one fortieth as much) will stimulate the physiological effects without apparent toxicity [4] . The majority of endogenous CO production originates from active heme metabolism (>86%), though a portion may be produced in lipid peroxidation and drug metabolism reactions [46] . Cigarette smoking, still practiced by many lung patients, represents a major source of chronic lowlevel exposure to CO. Inhaled CO initially targets alveolar macrophages and respiratory epithelial cells.\nThe exact mechanisms by which CO acts at the molecular level remain incompletely understood. CO potentially exerts its physiological effects by influencing at least three known pathways (Fig. 2 ). By complexation with the heme moiety of the enzyme, CO activates soluble guanylate cyclase (sGC), stimulating the production of cyclic 3':5'guanosine monophosphate (cGMP) [47] . The sGC/cGMP pathway mediates the effects of CO on vascular relaxation, smooth muscle cell relaxation, bronchodilation, neurotransmission, and the inhibition of platelet aggregation, coagulation, and smooth muscle proliferation [48] [49] [50] [51] . Furthermore, CO may cause vascular relaxation by directly activating calcium-dependent potassium channels [52] [53] [54] . CO potentially influences other intracellular signal transduction pathways. The mitogen-activated protein kinase (MAPK) pathways, which transduce oxidative stress and inflammatory signaling (i.e. response to lipopolysaccharide), may represent an important target Possible mechanism(s) of carbon monoxide action Figure 2 Possible mechanism(s) of carbon monoxide action. Endogenous carbon monoxide (CO) arises principally as a product of heme metabolism, from the action of heme oxygenase enzymes, although a portion may arise from environmental sources such as pharmacological administration or accidental exposure, or other endogenous processes such as drug and lipid metabolism. The vasoregulatory properties of CO, including its effects on cellular proliferation, platelet aggregation, and vasodilation, have been largely ascribed to the stimulation of guanylate cyclase by direct heme binding, leading to the generation of cyclic GMP. The anti-inflammatory properties of CO are associated with the downregulation of proinflammatory cytokine production, dependent on the selective modulation of mitogen-activated protein kinase (MAPK), such as the 38 kilodalton protein (p38MAPK). In addition to these two mechanisms, CO may potentially interact with any hemoprotein target, though the functional consequences of these interactions with respect to cellular signaling remain poorly understood.\nAnti-Platelet Aggregation Anti-Proliferation \n?\nInhibition of pro-inflammatory cytokine production\nModulation of hemoprotein function of CO action [32, 34, 55, 56 ]. An anti-apoptotic effect of CO and its relation to MAPK has recently been described. The overexpression of HO-1 or the exogenous administration of CO prevented tumor necrosis factor α (TNF-α)induced apoptosis in murine fibroblasts [57] . In endothelial cells, the anti-apoptotic effect of CO depended on the modulation of the p38 (38 kilodalton protein) MAPK pathway [34] . The role of the remaining heme metabolites, (i.e. Fe and biliverdin IXα) in the modulation of apoptosis is currently being investigated and is beyond the scope of this review. Recent studies have reported a potent anti-inflammatory effect of CO, involving the inhibition of proinflammatory cytokine production after endotoxin stimulation, dependent on the modulation of p38 MAPK [32] . The clinical relevance of p38 MAPK lies in the possibility of modulating this pathway in various clinical conditions to downregulate the inflammatory response [58] .\nOxidative stress arising from an imbalance between oxidants and antioxidants plays a central role in the pathogenesis of airway disease [59] . In lung tissue, HO-1 expression may occur in respiratory epithelial cells, fibroblasts, endothelial cells, and to a large extent in alveolar macrophages [41, 60, 61] . HO-1 induction in these tissues, in vitro and in vivo, responds to common causes of oxidative stress to the airways, including hyperoxia, hypoxia, endotoxemia, heavy metal exposure, bleomycin, diesel exhaust particles, and allergen exposure [4, 41, 61] . Induction of HO-1 or administration of CO can protect cells from these stressful stimuli [10, 41] . In one of the experiments that best illustrate the protective role of CO in vivo, rats were exposed to hyperoxia (>98% O 2 ) in the absence or presence of CO at low concentration (250 ppm). The CO-treated rats showed increased survival and a diminished inflammatory response to the hyperoxia [11] . As demonstrated in a model of endotoxin-induced inflammation, the protection afforded by CO most likely resulted from the downregulated synthesis of proinflammatory cytokines (i.e. TNF-α, IL-1β) and the upregulation of the anti-inflammatory cytokine interleukin-10 (IL-10) [32] . Furthermore, increases in exhaled CO (E-CO) have been reported in a number of pathological pulmonary conditions, such as unstable asthma, COPD, and infectious lung disease; these increases may reflect increased endogenous HO-1 activity [7] . Elevated carboxyhemoglobin (Hb-CO) levels have also been reported in these same diseases in nonsmoking subjects, where both the E-CO and Hb-CO levels decrease to normal levels in response to therapy [62] .\nE-CO in humans originates primarily from both systemic heme metabolism, which produces CO in various tissues, and localized (lung) heme metabolism, as a result of the combined action of inducible HO-1 and constitutive HO-2 enzymatic activity. Endogenously produced or inspired CO is eliminated exclusively by respiration [63] . Elevation of E-CO may also reflect an increase in exogenous sources such as smoking or air pollution. In addition to changes in environmental factors, elevations of E-CO in lung diseases may reflect an increase in blood Hb-CO levels in response to systemic inflammation, as well as an increase in pulmonary HO-1 expression in response to local inflammation [9, 62, 64] .\nThe diagnostic value of measuring E-CO remains controversial due to many conflicting reports (i.e. some reports indicate differences in E-CO measurements between disease activity and controls, and some reports do not). The possible explanations for these discrepancies include large differences in patient populations and in the methods used for measuring E-CO, and undefined corrections for background levels of CO. Furthermore, remarkable differences arise between studies in the magnitude of the E-CO levels in the control groups as well as in treated or untreated asthma patients. When active or passive smoking occurs, or in the presence of high background levels of CO, the measurement of E-CO is not particularly useful for monitoring airway inflammation. In patients who smoke, E-CO can be used only to confirm the smoking habit [65, 66] . Comparable to the beginning era of measurements of exhaled NO, a standardization in techniques and agreement on background correction should be reached for E-CO measurements, to allow proper conclusions to be drawn in this area of investigation.\nAsthma, a form of allergic lung disease, features an accumulation of inflammatory cells and mucus in the airways, associated with bronchoconstriction and a generalized airflow limitation. Inflammation, a key component of asthma, involves multiple cells and mediators where an imbalance in oxidants/antioxidants contributes to cell damage. Several pathways associated with oxidative stress may participate in asthma. For example, the redox-sensitive transcription factors NF-κB and AP-1 control the expression of proinflammatory mediators [59, [67] [68] [69] .\nIn light of the potential protective effects of HO-1/CO on inflammatory processes, the study of HO-1 in asthma has gained popularity. In a mouse model of asthma, HO-1 expression increased in lung tissue in response to ovalbumin aerosol challenge, indicating a role for HO-1 in asthma [70] . In a similar model of aeroallergen-induced asthma in ovalbumin-sensitized mice, exposure to a CO atmosphere resulted in a marked attenuation of eosinophil content in bronchoalveolar lavage fluid (BALF) and downregulation of the proinflammatory cytokine IL-5 [10] . This experiment showed that exogenous CO can inhibit asthmatic responses to allergens in mice.\nRecent human studies have revealed higher HO-1 expression in the alveolar macrophages and higher E-CO in untreated asthmatic patients than in healthy nonsmoking controls [71, 72] . Patients with exacerbations of asthma and patients who were withdrawn from inhaled steroids showed higher E-CO levels than steroid-treated asthmatics or healthy controls [73] . Higher levels of E-CO may also occur in children with persistent asthma than in healthy controls [74] . E-CO levels may correlate with functional parameters such as peak expiratory flow rate. A low rate in asthma exacerbations correlated with high E-CO, whereas normalization of the rate with oral glucocorticoid treatment resulted in a reduction of E-CO [75] . Furthermore, increased E-CO was associated with greater expression of HO-1 in airway alveolar macrophages obtained by induced sputum in untreated asthmatic patients than in controls. These asthma patients also showed higher bilirubin levels in the induced sputum, indicating higher HO activity [71] . Furthermore, patients with asthma show an increased Hb-CO level at the time of exacerbation, with values decreasing to control levels after oral glucocorticoid treatment [62] . In human asthmatics, E-CO and airway eosinophil counts decreased in response to a one-month treatment with inhaled corticosteroids [73] . In direct contrast to such studies promoting E-CO as a useful noninvasive tool for monitoring airway inflammation, other studies reported no difference in E-CO levels of asthma patients versus healthy controls, or between patients with stable and unstable asthma. In one such report, no further change in E-CO occurred in asthma patients after a one-month treatment of inhaled corticosteroids, despite observed decreases in airway eosinophil content and bronchial responsiveness to metacholine [76] . A recent study accentuates this finding in asthma excerbations, where no decrease in E-CO of children with asthma could be detected after oral prednisolone treatment [77] . In human allergic responses, results on elevation of E-CO are also inconclusive. A clear elevation of E-CO after allergen exposure occurred in patients with asthma during the late response, and during the early response immediately after the inhalation [78] . However, another report showed that no elevation of E-CO occurred in allergen-induced asthma within 48 hours after allergen challenge [79] . Finally, increases in E-CO were measured in allergic rhinitis, correlating with seasonal changes in exposure to allergen (pollen) [80] .\nAirway inflammation plays an important role in the development of COPD, characterized by the presence of macrophages, neutrophils, and inflammatory mediators such as proteinases, oxidants, and cytokines. Further-more, the inflammatory consequences of chronic microbiological infections may contribute to the progression of the disease. The current paradigm for the pathogenesis of COPD involves imbalances in protease/antiprotease activities and antioxidant/pro-oxidant status. Proteases with tissue-degrading capacity, (i.e. elastases and matrix metalloproteinases), when insufficiently inhibited by antiproteases, can induce tissue damage leading to emphysema. Oxidants that supersede cellular antioxidant defenses can furthermore inactivate antiproteases, cause direct injury to lung tissue, and interfere with the repair of the extracellular matrix. Smoking plays an important role in both hypotheses. Cigarette smoke will act primarily on alveolar macrophages and epithelial cells, which react to this oxidative stress by producing proinflammatory cytokines and chemokines and releasing growth factors. Nevertheless, smoking cannot be the only factor in the development of COPD, since only 15-20% of smokers develop the disease [81, 82] .\nExposure to reactive oxygen species (from cigarette smoke or chronic infections) and an imbalance in oxidant/antioxidant status are the main risk factors for the development of COPD. To defend against oxidative stress, cells and tissues contain endogenous antioxidant defense systems, which include millimolar concentrations of the tripeptide glutathione (GSH). A close relation exists between GSH concentration and HO-1, whereby depletion of GSH augments the transcriptional regulation of HO-1 by oxidants, suggesting that the HO-1/CO system acts as a secondary defense against oxidative stress [83] [84] [85] [86] . Accumulating clinical evidence suggests that HO-1/ CO may also play an important part in COPD. Alveolar macrophages, which produce a strong HO-1 response to stimuli, may represent the main source of CO production in the airways [60, 64] . Patients with COPD have displayed higher E-CO than healthy nonsmoking controls [87] . Furthermore, much higher levels of HO-1 have been observed in the airways of smokers than in nonsmokers [64] . Among subjects who formerly smoked, patients with COPD have lower HO-1 expression in alveolar macrophages than healthy subjects [88] . A microsatellite polymorphism that is linked with the development of COPD may occur in the promoter region of HO-1, resulting in a lower production of HO-1 in people who have the polymorphism. Thus, a genetically dependent downregulation of HO-1 expression may arise in subpopulations, possibly linked to increased susceptibility to oxidative stress [89] [90] [91] . Future studies on both genetic predisposition and possible therapeutic modalities will reveal the involvement of the HO-1/CO system in COPD.\nCystic fibrosis (CF) involves a deposition of hyperviscous mucus in the airways associated with pulmonary dysfunc-tion and pancreatic insufficiency, which may be accompanied by chronic microbiological infections. E-CO readings were higher in untreated versus oral-steroidtreated CF patients [92] . Furthermore, E-CO increased in patients during exacerbations of CF, correlating to deterioration of the forced expiratory volume in one second (FEV 1 ), with normalization of the E-CO levels after treatment [93] . E-CO levels may correlate with exhaled ethane, a product of lipid peroxidation that serves as an indirect marker of oxidative stress. Both E-CO and exhaled ethane were higher in steroid-treated and untreated CF patients than in healthy controls [94] . E-CO was higher in children with CF than in control patients. In addition to the inflammatory and oxidative stress responses to continuous infectious pressure in these patients, E-CO may possibly respond to hypoxia. E-CO increased further in CF children following an exercise test, and correlated with the degree of oxyhemoglobin desaturation, a finding suggestive of an increased HO-1 expression in CF patients during hypoxic states induced by exercise [95] .\nIn patients with pneumonia, higher Hb-CO levels can be measured at the onset of illness, with values decreasing to control levels after antibiotic treatment [62] . E-CO levels were reported to be higher in lower-respiratory-tract infections and bronchiectasis, with normalization after antibiotic treatment [96, 97] . Furthermore, E-CO levels in upper-respiratory-tract infections were higher than in healthy controls [74, 80] . The relationship between higher measured E-CO in these infectious states and higher Hb-CO levels cannot be concluded from these studies.\nThe role of HO-1 in the development of interstitial lung disease remains undetermined. Comparative immunohistochemical analysis has revealed that lung tissue of control subjects, patients with sarcoidosis, usual interstitial pneumonia, and desquamative interstitial pneumonia, all showed a high expression of HO-1 in the alveolar macrophages but a weak expression in the fibrotic areas [98] . The antiproliferative properties of HO-1 suggest a possible beneficial role in limiting fibrosis; however, this hypothesis is complicated by a newly discovered relation between IL-10 and HO-1. IL-10 produced by bronchial epithelial cells promotes the growth and proliferation of lung fibroblasts [99] . HO-1 expression and CO treatment have been shown to increase the production of IL-10 in macrophages following proinflammatory stimuli [32] . Conversely, IL-10 induces HO-1 production, which is apparently required for the anti-inflammatory action of IL-10 [100] .\nA recent report clearly shows the suppression of bleomycin-induced pulmonary fibrosis by adenovirus-mediated HO-1 gene transfer and overexpression in C57BL/6 mice, involving the inhibition of apoptotic cell death [101] .\nOverall, more research is needed to elucidate the mechanisms of HO-1 in interstitial lung disease and its possible therapeutic implications.\nHO-1 action may be of great importance in solid tumors, an environment that fosters hypoxia, oxidative stress, and neovascularization. HO-1 may have both pro-and antagonistic effects on tumor growth and survival. HO-1 and CO cause growth arrest in cell-culture systems and thus may represent a potential therapeutic modality in modulating tumor growth [16] . The overexpression of HO-1 or administration of CO in mesothelioma and adenocarcinoma mouse models resulted in improved survival (>90%) as well as reduction in tumor size (>50%) [17] . Furthermore, HO-1 expression in oral squamous cell carcinomas can be useful in identifying patients at low risk of lymph node metastasis. High expression of HO-1 was detected in groups without lymph node metastasis in this report [102] . In contrast to growth arrest, HO-1 may protect solid tumors from oxidative stress and hypoxia, possibly by promoting neovascularization. In one study, zinc protoporphyrin, a competitive inhibitor of HO-1 enzyme activity, suppressed tumor growth [103] .\nCO may represent a critical mediator of the body's adaptive response to hypoxia, a common feature in pulmonary vascular disease [104] . Since CO can modulate vascular tone by inducing cGMP and large, calcium-dependent potassium channels, HO-1 and CO probably play important roles in pulmonary vascular diseases [54] . A NOmediated HO-1 induction occurred in the hepatopulmonary syndrome during cirrhosis, associated with enhancement of vascular relaxation [105] . In portopulmonary hypertension, elevated levels of cGMP and inducible nitric oxide synthase (iNOS) expression in the vascular endothelium, and HO-1 expression in macrophages and bronchial epithelium have been described [106] . In transgenic mice models, ho-1 -/and ho-1 +/+ mice did not differ in their development of pulmonary hypertension following chronic hypoxia treatment, despite the development of right ventricular dilation and right myocardial infarction in ho-1 -/mice [107] . The preinduction of HO-1 protein with chemical inducers, however, prevented the development of pulmonary hypertension in the rat lung as a consequence of chronic hypoxia treatment [108] . Transgenic mice overexpressing HO-1 in the lung were resistant to hypoxia-induced inflammation and hypertension [109] . Further research is needed to elucidate the potential role of HO-1 and CO in primary human lung vascular diseases such as primary pulmonary hypertension.\nSupplemental oxygen therapy is often used clinically in the treatment of respiratory failure. Exposure to high oxygen tension (hyperoxia) may cause acute and chronic lung injury, by inducing an extensive inflammatory response in the lung that degrades the alveolar-capillary barrier, leading to impaired gas exchange and pulmonary edema [110, 111] . Hyperoxia-induced lung injury causes symptoms in rodents that resemble human acute respiratory distress syndrome [112] .\nHyperoxia induced HO-1 expression in adult rats but apparently not in neonatal rats, in which the expression and activities of HO-1 and HO-2 are developmentally upregulated during the prenatal and early postnatal period [113] .\nBoth HO-1 and HO-2 potentially influence pulmonary adaptation to high O 2 levels. In one example, the adenoviral-mediated gene transfer of HO-1 into rat lungs protected against the development of lung apoptosis and inflammation during hyperoxia [114] . In vitro studies showed that the overexpression of HO-1 in lung epithelial cells or rat fetal lung cells caused growth arrest and conferred resistance against hyperoxia-induced cell death [15, 16] . An oxygen-tolerant variant of hamster fibroblasts that moderately overexpressed HO-1 in comparison with the parent line resisted oxygen toxicity in vitro. The treatment of this oxygen-tolerant strain with HO-1 antisense oligonucleotides reduced the resistance to hyperoxia. In contrast, additional, vector-mediated, HO-1 expression did not further increase oxygen tolerance in this model [115] .\nIn vivo studies with gene-deleted mouse strains have provided much information on the roles of HO-1 and HO-2 in oxygen tolerance. Dennery et al. demonstrated that heme oxygenase-2 knockout mice (ho-2 -/-) were more sensitive to the lethal effects of hyperoxia than wild-type mice [116] . In addition to the absence of HO-2 expression, however, the mice displayed a compensatory increase in HO-1 protein expression, and higher total lung HO activity. Thus, in this model, the combination of HO-2 deletion and HO-1 overexpression resulted in a hyperoxiasensitive phenotype. Recent studies of Dennery et al. have shown that HO-1-deleted (ho-1 -/-) mice were more resistant to the lethal effects of hyperoxia than the corresponding wild type [117] . The hyperoxia resistance observed in the ho-1 -/strain could be reversed by the reintroduction of HO-1 by adenoviral-mediated gene transfer [117] . In contrast, mouse embryo fibroblasts derived from ho-1 -/mice showed increased sensitivity to the toxic effects of hemin and H 2 O 2 and generated more intracellular reactive oxygen species in response to these agents [118] . Both ho-1 -/-and ho-2 -/strains were anemic, yet displayed abnormal accumulations of tissue iron. Specifically, ho-1 -/accumulated nonheme iron in the kidney and liver and had decreased total iron content in the lung, while ho-2 -/mice accumulated total lung iron in the absence of a compensatory increase in ferritin levels [116, 119] . The mechanism(s) by which HO-1 or HO-2 deletions result in accumulation of tissue iron remain unclear. These studies, taken together, have indicated that animals deficient in either HO-1 and HO-2 display altered sensitivity to oxidative stress conditions. Aberrations in the distribution of intra-and extra-cellular iron, may underlie in part, the differential sensitivity observed [116, 117] .\nOtterbein et al. have shown that exogenous CO, through anti-inflammatory action, may protect the lung in a rat model of hyperoxia-induced lung injury. The presence of CO (250 ppm) prolonged the survival of rats in a hyperoxic (>95% O 2 ) environment, and inhibited the appearance of markers of hyperoxia-induced lung injury (i.e. hemorrhage, fibrin deposition, edema, airway protein accumulation, and BALF neutrophil influx) [11] . Furthermore, in a mouse model, CO inhibited the expression of proinflammatory cytokines (TNF-α, IL-1β, and IL-6) in mice induced by the hyperoxia treatment. Using genedeleted mice, Otterbein and colleagues also observed that the protection afforded by CO in this model, similar to a lipopolysaccharide-induced model of lung injury, depended on the p38 MAPK pathway (Otterbein et al., unpublished observation, as reviewed in [3] ).\nIn direct contrast to these studies, the group of Piantadosi and colleagues reported no significant difference in the hyperoxia tolerance of rats at CO doses between 50 and 500 ppm [120] . In their model, CO did not alter the accumulation of fluid in the airway. Furthermore, CO, when applied in combination with hyperoxia, increased the activity of myeloperoxidase, a marker of airway neutrophil influx. This study also suggested that inhalation of CO (50-500 ppm) did not alter the expression of HO-1 or other antioxidant enzymes such as Manganese superoxide dismutase (MnSOD) in vivo [120] . Furthermore, Piantadosi and colleagues were able to induce oxygen tolerance in rats and HO-1 expression with hemoglobin treatment, but this tolerance also occurred in the presence of HO inhibitors, thereby not supporting a role for HO activity in oxygen tolerance [121] . Although no consensus has been reached as to the protective role of CO inhalation and/or HO-1 induction in hyperoxic lung injury, human studies will be required to show if CO will supersede NO in providing a significant therapeutic benefit in the context of severe lung diseases [122] . While antioxidant therapies have been examined, until now no human studies exist on the role of HO-1 and CO in acute respiratory distress syndrome (ARDS) and bronchopulmonary dysplasia [123] .\nLung transplantation is the ultimate and often last therapeutic option for several end-stage lung diseases. After lung transplantation, there remains an ongoing hazardous situation in which both acute and chronic graft failure, as well as complications of the toxic immunosuppressive regimen used (i.e. severe bacterial, fungal, and viral infections; renal failure; and Epstein-Barr-virus-related lymphomas), determine the outcome [124] . The development of chronic graft failure, obliterative bronchiolitis (OB), determines the overall outcome after lung transplantation. OB, which may develop during the first months after transplantation, is the main cause of morbidity and death following the first half-year after transplantation, despite therapeutic intervention. Once OB has developed, retransplantation remains the only therapeutic option available [124, 125] . Little is known about the pathophysiological background of OB. The possible determinants of developing OB include ongoing immunological allograft response, HLADR mismatch, cytomegalovirus infection, acute rejection episodes, organ-ischemia time, and recipient age [125] . OB patients displayed elevated neutrophil counts in the BALF, and evidence of increased oxidant activity, such as increased methionine oxidation in BALF protein and decreases in the ratio of GSH to oxidized glutathione (GSSG) in epithelial lining fluid. [126, 127] .\nSo far, only very limited research data are available on the possible role for HO-1 in allograft rejection after lung transplantation. Higher HO-1 expression has been detected in alveolar macrophages from lung tissue in lung transplant recipients with either acute or chronic graft failure than in stable recipients [128] . The protective role of HO-1 against allograft rejection has been shown in other transplantation models, in which solid organ transplantation typically benefits from HO-1 modulation. A higher expression of protective genes such as HO-1 has been observed in episodes of acute renal allograft rejection [129] . Furthermore, the induction of HO-1 alleviates graft-versus-host disease [130] . Adenoviral-HO-1 gene therapy resulted in remarkable protection against rejection in rat liver transplants [131] . The upregulation of HO-1 protected pancreatic islet cells from Fas-mediated apoptosis in a dose-dependent fashion, supporting an anti-apoptotic function of HO-1 [132, 133] . HO-1 may confer protection in the early phase after transplantation by inducing Th2-dependent cytokines such as IL-4 and IL-10, while suppressing interferon-γ and IL-2 production, as demonstrated in a rat liver allograft model [134] .\nBeneficial effects of HO-1 modulation have also been described in xenotransplantation models, in which HO-1 gene expression appears functionally associated with xenograft survival [135] . In a mouse-to-rat heart trans-plant model, the effects of HO-1 upregulation could be mimicked by CO administration, suggesting that HOderived CO suppressed the graft rejection [136] . The authors proposed that CO suppressed graft rejection by inhibition of platelet aggregation, a process that facilitates vascular thrombosis and myocardial infarction.\nHO-1 may also contribute to ischemic preconditioning, a process of acquired cellular protection against ischemia/ reperfusion injury, as observed in guinea pig transplanted lungs [137] . HO-1 overexpression provided potent protection against cold ischemia/reperfusion injury in a rat model through an anti-apoptotic pathway [138, 139] . The induction of HO-1 in rats undergoing liver transplantation with cobalt-protoporphyrin or adenoviral-HO-1 gene therapy resulted in protection against ischemia/ reperfusion injury and improved survival after transplantation, possibly by suppression of Th1-cytokine production and decreased apoptosis after reperfusion [140, 141] . Until now, no reports have addressed E-CO measurements in lung transplantation, where it is possible that differences in E-CO will be found in patients with acute and chronic allograft rejection.\nThe evolution of CO in exhaled breath may serve as a general marker and diagnostic indicator of inflammatory disease states of the lung, though more research will be required to verify its reliability. Increases in exhaled CO presumably reflect changes in systemic and airway heme metabolic activity from the action of HO enzymes. Evidence from numerous in vitro and animal studies indicates that HO-1 provides a protective function in many, if not all, diseases that involve inflammation and oxidative stress. Thus, the exploitation of HO-1 for therapeutic gain could be achieved through the modulation of HO-1 enzyme activity or its up-and downstream regulatory factors, either by gene transfer, pharmacological inducers, or direct application of CO by gas administration or chemical delivery [142] [143] [144] [145] . The CO-releasing molecules (transition metal carbonyls) developed by Motterlini et al. [144] show promise in the pharmacological delivery of CO for therapeutic applications in vascular and immune regulation. The CO-releasing molecules have been shown to limit hypertension in vivo and promote vasorelaxation in isolated heart and aortic rings [144] .\nUltimately, the challenge remains in applying the therapeutic potentials of HO-1 to the treatment of human diseases. In vivo models of transplantation have shown that HO-1 gene therapy protects against allograft rejection [129, 134] . Given the toxic therapy that every transplant patient receives, especially after lung transplantation, the field of transplantation medicine may bring the first frontier for human applications of HO-1 gene therapy or exogenous CO administration. The potential use of inhalation CO as a clinical therapeutic in inflammatory lung diseases has also appeared on the horizon. In one promising study, an inhalation dose of 1500 ppm CO at the rate of 20 times per day for a week produced no cardiovascular side effects [146] . Cigarette smoking and CO inhalation at identical intervals produced comparable Hb-CO levels of approximately 5%. The question of whether or not CO can be used as an inhalation therapy will soon be replaced by questions of \"how much, how long, and how often?\" The fear of administering CO must be weighed against the severe toxicity of the immunosuppressive agents in current use, and the often negative outcome of solid organ transplantation. " }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: Unfortunately, conventional public health disease surveillance-which relies on physician and laboratory reporting and manual analysis of surveillance data-is ill equipped for timely detection of such threats. 3 The reportable disease system relies on health care professionals to recognize, diagnose, and report cases and suspected outbreaks to public health officials 4, 5 ; however, it is unlikely that without an event or alert to raise his or her index of suspicion, a physician will attribute the early symptoms and signs of disease in a bioattack victim appropriately and report the case. 6 A key limitation of the current system is that the lone physician is blind to the cases his or her colleagues in a nearby hospital are seeing-knowledge that might lead the physician to consider uncommon diseases more strongly in his or her diagnostic reasoning. Mandatory laboratory reporting 4 is also illequipped for early detection, because it takes time before tests are ordered and specimens are obtained, transported, processed, and resulted.\nSufficiently early detection of a biological attack may be accomplished through surveillance schemes that can detect infected individuals earlier in the disease process. For completeness, we note that biosensors are being developed (and deployed) that detect organisms in the air and that this type of detection, if feasible, occurs fundamentally much earlier, because the delay introduced by the incubation period of the disease is eliminated from the surveillance system. 7 However, such approaches face unsolved technical problems in the analysis of contaminated specimens (the norm in air sampling). Biosensors also need to be in the right place-on every person's lapel or every street corner and hallway-to provide complete surveillance coverage.\nSurveillance methods that can detect disease at an earlier stage are an important research direction for public health surveillance. These methods are generally referred to as syndromic surveillance because they have the goal of recognition of outbreaks based on the symptoms and signs of infection and even its effects on human behavior prior to first contact with the health care system. 8 Because the data used by syndromic surveillance systems cannot be used to establish a specific diagnosis in any particular individual, syndromic surveillance systems must be designed to detect signature patterns of disease in a population to achieve sufficient specificity. For example, it would be absurd to use only the symptom of fever to attempt to establish a working diagnosis of inhalational anthrax in an individual, but it would be very reasonable to establish a working diagnosis of anthrax release in a community if we were to observe a pattern of 1,000 individuals with fever distributed in a linear streak across an urban region consistent with the prevailing wind direction two days earlier. It would be beyond reasonable and, in fact, imperative to establish a working diagnosis of public health emergency if presented with such information.\nOne recent example of a form of syndromic surveillance is drop-in surveillance-the stationing of public health workers in emergency departments (EDs) and special clinics during high-profile events such as the Super Bowl to capture data on patients presenting with symptoms potentially indicative of bioterrorism. The major disadvantage of this approach is the cost of round-the-clock staffing for manual data collection.\nA less expensive approach-and the one taken in the Realtime Outbreak and Disease Surveillance (RODS) system-is detection based on data collected routinely for other purposes. Examples of such data include absenteeism data, sales of over-the-counter (OTC) health care products, and chief complaints from EDs. 9 The expenses of manual data collection are avoided; however, the data obtained typically are noisy approximations of what could be obtained by direct interviewing of the patient (in the case of individual level data). Both approaches may play complementary roles with current methods of public health surveillance 10-12 by assisting the physician and public health official with a continuously updated picture of the ''health status'' of a population. 13, 14 A focus of our research has been syndromic surveillance from free-text chief complaints routinely collected by triage nurses in EDs and acute care clinics during patient registration. We have deployed this type of surveillance at the 2002 Winter Olympics and in the States of Pennsylvania and Utah. We described a previous version of the RODS system, 12 but the system has undergone considerable subsequent development both architecturally and functionally. This report provides a detailed description of the current version of RODS, an example of a computer-based public health surveillance system that adheres to the National Electronic Disease Surveillance System (NEDSS) specifications of the Centers for Disease Control and Prevention (CDC). 15, 16 Background\nThe role of public health surveillance is to collect, analyze, and interpret data about biological agents, diseases, risk factors, and other health events and to provide timely dissemination of collected information to decision makers. 17 Conventionally, public health surveillance relies on manual operations and off-line analysis.\nExisting syndromic surveillance systems include the CDC's drop-in surveillance systems, 8 Early Notification of Community-based Epidemics (ESSENCE), 10,18 the Lightweight Epidemiology Advanced Detection and Emergency Response System (LEADERS), 19 the Rapid Syndrome Validation Project (RSVP), 20 and the eight systems discussed by Lober et al. 11 Lober et al. summarized desirable characteristics of syndromic surveillance systems and analyzed the extent to which systems that were in existence in 2001 had those characteristics. 11 A limitation of most systems (e.g., ESSENCE, 10 Children's Hospital in Boston, 11 University of Washington 11 ) was batch transfer of data, which may delay detection by as long as the time interval (periodicity) between batch transfers. For example, a surveillance system with daily batch transfer may delay by one day the detection of an outbreak.\nSome systems required manual data input (e.g., CDC's dropin surveillance systems, RSVP, 20 and LEADERS 19 ), which is labor-intensive and, in the worst case, requires round-theclock staffing. Manual data input is not a feasible mid-or long-term solution even if the approach is to add items to existing encounter forms (where the items still may be ignored by busy clinicians).\nA third limitation for existing surveillance systems is that the systems may not exploit existing standards or communication protocols like Heath Level 7 (HL7) even when they are available.\nThe data type most commonly used among surveillance systems is symptoms or diagnoses of patients from ED and/or physician office visits. Other types of data identified in that study include emergency call center and nurse advice lines. Other types of data being used include sales of over-thecounter health care products, prescriptions, telephone call volumes to health care providers and drug stores, and absenteeism. We have conducted studies demonstrating that the free-text chief complaint data that we use correlate with outbreaks. 21, 22 Design Objectives\nThe overall design objective for RODS is similar to that of an early warning system for missile defense; namely, to collect whatever data are required to achieve early detection from as wide an area as necessary and to analyze the data in a way that they can be used effectively by decision makers. It is required that this analysis be done in close to real time. This design objective is complex and difficult to operationalize because of the large number of organisms and the even larger number of possible routes of dissemination all requiring potentially different types of data for their detection, different algorithms, and different time urgencies. For this reason, our focus since beginning the project in 1999 has been on the specific problem of detecting a large-scale outbreak due to an outdoor (outside buildings) aerosol release of anthrax. Additional design objectives were adherence to NEDSS standards to ensure future interoperability with other types of public health surveillance systems, scalability, and that the system could not rely on manual data entry, except when it was done in a focused way in response to the system's own analysis of passively collected data.\nThis report describes RODS 1.5, which was completely rewritten as a Java 2 Enterprise Edition (J2EE) application since the previous publication describing it. RODS 1.5 is multidata type enabled, which means that any time series data can be incorporated into the databases and user interfaces. The deployed RODS system currently displays and analyzes health care delivery site registrations and separately monitors sales of OTC health care products.\nOverview RODS uses clinical data that are already being collected by health care providers and systems during the registration process. When a patient arrives at an ED (or an InstaCare in Utah), the registration clerk or triage nurse elicits the patient's reason for visit (i.e., the chief complaint), age, gender, home zip code, and other data and enter the data in a registration computer. The registration computer then generates an HL7 ADT (admission, discharge, and transfer) message and transmits it to the health system's HL7 message router (also called an integration engine). There usually is only one message router per health system even if there are many hospitals and facilities. These processes are all routine existing business activities and do not need to be created de novo for public health surveillance. Figure 1 shows the flow of clinical data to and within RODS. The hospital's HL7 message router, upon receipt of an HL7 message from a registration computer, deletes identifiable information from the message and then transmits it to RODS over a secure virtual private network (VPN), or a leased line, or both (during the 2002 Winter Olympics we utilized both types of connections to each facility for fault tolerance). The RODS HL7 listener maintains the connection with the health system's message router and parses the HL7 message as described in more detail below. It then passes the chief complaint portion of the message to a Bayesian text classifier that assigns each free-text chief complaint to one of seven syndromic categories (or to an eighth category, other). The database stores the category data, which then are used by applications such as detection algorithms and user interfaces.\nData about sales of OTC health care products are processed separately by the National Retail Data Monitor, which is discussed in detail in another article in this issue of JAMIA. 23 The processing was kept separate intentionally because, in the future, the servers for the National Retail Data Monitor may operate in different physical locations than RODS. The RODS user interfaces can and do display sales of OTC health care products as will be discussed, but other user interfaces can be connected to the National Retail Data Monitor as well.\nPrior to September 2001, RODS received data only from hospitals associated with the UPMC Health System, and efforts to recruit other hospitals met with resistance. After the terrorist attacks (including anthrax) in the Fall of 2001, other hospitals agreed to participate. Although data in this project are de-identified, certain information such as the number of ED visits by zip code were considered proprietary information by some health systems. Health Insurance Portability and Accountability Act (HIPAA) concerns also were very prominent in the discussions. Data-sharing agreements were executed with every participating health system that addressed these concerns. As an additional precaution, all RODS project members meet annually with University of Pittsburgh council to review obligations and are required to sign an agreement every year stating that they understand the terms of the data-sharing agreements and agree to abide by the terms. RODS began as a research project at the University of Pittsburgh in 1999 and has functioned with IRB approvals since that time.\nHealth care facilities send admission, discharge, and transfer (ADT) HL7 messages to RODS for patient visits in EDs and walk-in clinics. A minimal data set is sent, as shown in Figure 2 , which qualifies as a HIPAA Limited Data Set. 24 Currently the data elements are age (without date of birth), gender, home zip code, and free-text chief complaint.\nThe HL7 listener receives HL7 messages from the message routers located in each health system. The HL7 listener then passes the received HL7 message to the HL7 parser bean, an Enterprise JavaBean (EJB) in the RODS business logic tier. The HL7 parser bean uses regular expressions to parse the fields in an HL7 message. The HL7 parser bean then stores the parsed elements into a database through a managed database connection pool.\nAlthough nearly all health systems utilize the HL7 messaging standard, the location of individual data elements in an HL7 message may differ from health system to health system. For example, some care providers' systems record free-text chief complaint in the DG1 segment instead of the PV2 segment of an HL7 message. To resolve this mapping problem, a configuration file written in eXtensible Markup Language (XML), a standard protocol often used to define hierarchical data elements, defines where each of the data elements can be found in the HL7 message. When an HL7 listener starts up, it reads the hospital-dependent configuration file and passes the configuration information to the parser bean.\nWe also use this configuration file to define the database table and field in which the HL7 parser bean should store each data element. This approach is useful because it allows the HL7 data to be stored to an external database. We anticipate that health departments with existing NEDSS or other public health surveillance databases may wish to use just this component of RODS for real-time collection of clinical data.\nFor hospitals that do not have HL7 message routers (two of approximately 60 in our experience to date), RODS accepts ED registration data files through either a secure Web-based data upload interface or a secure file transfer protocol. In general, these types of data transfers are technically trivial and for that reason are used by many groups but do not have the reliability of a HL7 connection (and have very undesirable time latencies).\nRODS checks the integrity of the data in the HL7 messages that it receives. This processing is necessary because hospital data flows may have undesirable characteristics such as duplicates. RODS identifies and deletes duplicates by using a database trigger that creates a composite primary key before inserting the data. RODS also filters out scheduling messages, which are identified by the fact that they have future admitted date and time.\nRODS monitors all data feeds to ensure continuous connections with health systems. If RODS does not receive data for six hours, it sends an alert to the RODS administrator and the sending health system's administrator. Because the commercial message routers that hospitals use queue up HL7 messages when encountering networking or system problems, data integrity is preserved.\nRODS uses an Oracle8i database to store ED registration data.\n(Oracle, Redwood Shores, CA). To ensure fast response for an online query (e.g., the daily counts of respiratory syndrome in a county for the past six months), we developed a cache \nFor connectivity with the HL7 message routers, we utilize hardware-based routers. The VPN router is a Cisco PIX 501 and the leased-line routers are a pair of Cisco 2600s (Cisco Systems, Inc., San Jose, CA).\nAll of the RODS processes can be run on a single computer, but in our current implementation-serving Pennsylvania F i g u r e 2. Sample HL7 admission, discharge, and transfer (ADT) message from an emergency department. The circled fields are age, gender, home zip code, admitted date and time, and free-text chief complaint, respectively.\nand Utah as an application service provider-we use five dedicated servers: firewall, database, Web server, a geographic information system (GIS) server, and computation. The processes are written in Java code and can run on most platforms, but here we describe the specific platforms we use to indicate approximate sizing and processing requirements. \nWe developed RODS applications using the Java 2 Enterprise Edition Software Toolkit (J2EE SDK) from Sun Microsystems for cross-platform Java application development and deployment. 26 We followed contemporary application programming practices-a multitiered application consisting of a client tier (custom applications such as HL7 listeners and detection algorithms), business logic tier, database tier, and Web tier.\nBusiness logic such as the HL7 parser bean was implemented as Enterprise JavaBeans (EJBs). NEDSS specifies EJB as the standard for application logic. RODS uses Jboss, an opensource J2EE application server, to run all EJBs. 10 The Web tier comprises the graphical user-interface to RODS and uses Java Server Pages (JSP), Java Servlets, and ArcIMS. The database tier was implemented in Oracle 8i.\nRODS uses a naive Bayesian classifier called Complaint Coder (CoCo) to classify free-text chief complaints into one of the following syndromic categories: constitutional, respiratory, gastrointestinal, neurological, botulinic, rash, hemorrhagic, and other. CoCo computes the probability of each category, conditioned on each word in a free-text chief complaint and assigns a patient to the category with the highest probability. 27 The probability distributions used by CoCo are learned from a manually created training set. CoCo can be retrained with local data, and it can be trained to detect a different set of syndromes than we currently use. CoCo runs as a local process on the RODS database server. CoCo was developed at the University of Pittsburgh and is available for free download at <http://health.pitt.edu/rods/sw>.\nOver the course of the project, RODS has used two detection algorithms. These algorithms have not been formally field tested because the emphasis of the project to date has been on developing the data collection infrastructure more than field testing of algorithms.\nThe Recursive-Least-Square (RLS) adaptive filter 28 currently runs every four hours, and alerts are sent to public health officials in Utah and Pennsylvania. RLS, a dynamic autoregressive linear model, computes an expected count for each syndrome category for seven counties in Utah and 16 counties in Pennsylvania as well as for the combined counts for each state. We use RLS because it has a minimal reliance on historical data for setting model parameters and a high sensitivity to rapid increases in a time series e.g., a sudden increase in daily counts. RLS triggers an alert when the current actual count exceeds the 95% confidence interval for the predicted count.\nDuring the 2002 Olympics we also used the What's Strange About Recent Events (WSARE 1.0) algorithm. 29 WSARE performs a heuristic search over combinations of temporal and spatial features to detect anomalous densities of cases in space and time. Such features include all aspects of recent patient records, including syndromal categories, age, gender, and geographical information about patients. The criteria used in the past for sending a WSARE 1.0 alert was that there has been an increase in the number of patients with specific characteristics relative to the counts on the same day of the week during recent weeks and the p-value after careful adjustment for multiple testing for the increase was #0.05. Version 3.0 of WSARE, which will incorporate a Bayesian model for computing expected counts rather than using unadjusted historical counts currently, is under development.\nWhen an algorithm triggers an alert based on the above criteria, RODS sends e-mail and/or page alerts to its users. RODS uses an XML-based configuration file to define users' e-mail and pager addresses. The e-mail version of the alert includes a URL link to a graph of the time series that triggered the alarm with two comparison time series: total visits for the same time period and normalized counts.\nRODS has a password-protected, encrypted Web site at which users can review health care registration and sales of OTC health care products on epidemic plots and maps. When a user logs in, RODS will check the user's profile and will display data only for his or her health department's jurisdiction. The interface comprises three screens-Main, Epiplot, and Mapplot.\nThe main screen alternates views automatically among each of the available data sources (currently health care registrations and OTC products in Pennsylvania and Utah and OTC sales only for other states). The view alternates every two minutes as shown in Figure 3 . The clinic visits view shows daily total visits and seven daily syndromes for the past week. The OTC data view shows daily sales for five product categories and the total, also for the past week. Users also can set the view to a specific county in a state. If the normalize control box is checked, the counts in the time series being displayed will be divided by (normalized by) the total daily sales of OTC health care products or ED visits for the region.\nThe Epiplot screen provides a general epidemic plotting capability. The user can simultaneously view a mixture of different syndromes and OTC product categories for any geographic region (state, county, or zip code), and for any time interval. The user also can retrieve case details as shown in Figure 4 . The Get Cases button queries the database for the admission date, age, zip code, and chief complaint (verbatim, not classified into syndrome category) of all patients in the time interval and typically is used to examine an anomalous density (spike) of cases. The Download Data button will download data as a compressed comma separated file for further analyses.\nThe Mapplot screen is an interface to ArcIMS, an Internetenabled GIS product developed by Environmental Systems Research Institute, Inc. Mapplot colors zip code regions to indicate the proportion of patients presenting with a particular syndrome. The GIS server also can overlay state boundaries, county boundaries, water bodies, hospital locations, landmarks, streets, and highways on the public health data as shown in Figure 5 . Similar to Epiplot, Mapplot also can display case details for a user-selected zip code.\nRODS has been in operation for four years and, like most production systems, has acquired many fault-tolerant features. For example, at the software level, HL7 listeners continue to receive messages and temporarily store the messages when the database is off-line. A data manager program runs every ten minutes and, on finding such a cache, it loads the unstored messages to the database when the database is back on-line. In addition, the data manager program monitors and restarts HL7 listeners as necessary.\nThe database uses ''archive log'' mode to log every transaction to ensure that the database can recover from a system failure.\nThe hardware architecture also is fault tolerant. All servers have dual power supplies and dual network cards. All hard drives use Redundant Arrays of Inexpensive Disk configurations. In addition to dual power supplies, all machines are connected to an uninterrupted power supply that is capable of sending an e-mail alert to the RODS administrator when the main power is down.\nAn important component of RODS that currently is used only at the UPMC Health System in Pittsburgh is the Health System Resident Component (HSRC). The HSRC is located within the firewall of a health system and connects directly to the HL7 message router. The HSRC currently receives a diverse set of clinical data from the HL7 message router including culture results, radiology reports, and dictated F i g u r e 3. Health care registrations view in the Main screen of RODS. The Main screen alternates views every 2 minutes among data types available in the public health jurisdiction. The figure shows eight plots of health care registration data-total visits, botulinic, constitutional, gastrointestinal (GI), hemorrhagic, neurological, rash, and respiratory. After 2 minutes, over-the-counter data will be displayed. The Main screen can be used as a ''situation room'' display. emergency room notes. Its purpose is to provide additional public health surveillance functions that would not be possible if it were located outside of the firewall due to restrictions on the release of identifiable clinical data. The HSRC uses patient identifiers to link laboratory and radiology information to perform case detection. In the past, we have used HSRC to monitor for patients with both a gram-positive rod in a preliminary microbiology culture report and ''mediastinal widening'' in a radiology report. The HSRC is a case detector in a distributed outbreak detection system that is capable of achieving much higher specificity of patient diagnostic categorization through access to more information.\nHSRC also removes identifiable information before transmitting data to the RODS system, a function provided by the health system's message router in other hospitals that connect to RODS.\nThe HSRC at UPMC Health System functions as an electronic laboratory reporting system, although the state and local health departments are not yet ready to receive real-time messaging from the system. Currently, it sends email alerts to the director of the laboratory and hospital infection control group about positive cultures for organisms that are required to be reported to public health in the state of Pennsylvania. 30 It also sends messages to hospital infection control when it detects organisms that cause nosocomial infections. These organisms include Clostridium difficile, methicillin-resistant Staphylococcus aureus, and vancomycin-resistant Enterococcus.\nWe have been able in HSRC to prototype one additional feature, which is a ''look-back'' function that facilitates very rapid outbreak investigations by providing access to electronic medical records to public health investigators as shown in Figure 6 . This feature requires a token that can be passed to a hospital information system that can uniquely identify a patient, and the reason we have prototyped this feature in the HSRC and not in RODS is simply that HSRC runs within the firewall so an unencrypted token can be used. The lookback is accomplished as follows: when a public health user identifies an anonymous patient record of interest (e.g., one of 20 patients with diarrhea today from one zip code), HSRC calls the UPMC Health System Web-based electronic medical record system and passes it the patient identifier. UPMC Health System then requests the user to log in using the UPMC-issued password before providing access to the record directly from its own secure Web site. This approach is not intended to be implemented in HSRC, but rather in the RODS system outside of the firewall of a health system. It is intended to use encrypted identifiers that the health system would decrypt to retrieve the correct record. The HSRC could provide the encryption-decryption service or it could be provided by another data system in the hospital. We estimate that the prevalence of health systems that have Web-based results review in the United States is 30% to 50% and growing so that this approach could very quickly improve the efficiency of outbreak investigations. For these reasons, we have moved to an application service provider model for dissemination in which we encourage state and local health departments to form coalitions to support shared services. We also have been fortunate to have sufficient grant funding from the Commonwealth of Pennsylvania to be able to support these services on an interim basis while sustainable funding models evolve.\nOur original design objectives for RODS were real-time collection of data with sufficient geographic coverage and sampling density to provide early syndromic warning of a large-scale aerosol release of anthrax. Although we have not achieved all of our initial design objectives, progress has been substantial. The research identified two types of data-freetext chief complaints and sales of OTC health care prod- ucts-that can be obtained in real time or near real time at sampling levels of 70% or higher for most of the United States. These results were obtained through large-scale deployments of RODS in Pennsylvania and Utah and through building the National Retail Data Monitor described in the accompanying article in this issue of JAMIA. The deployments also provided insights about organizational and technical success factors that would inform an effort to scale the project nationally.\nThe project established the importance of HL7 message routers (also known as integration engines) for public health surveillance. HL7 message routers are a mature, highly prevalent technology in health care. We demonstrated that free-text triage chief complaints can be obtained in real time from most U.S. hospitals through message routers and that these data represent early syndromal information about disease. Many other clinical data of value to public health are transmitted using the HL7 standard (e.g., orders for diagnostic tests, especially microbiology tests, reports of chest radiographs, medications, and test results) and can be integrated into RODS or other surveillance systems capable of receiving HL7 messages.\nAs a result of our efforts to disseminate this technology by giving it away, we have learned that most health departments do not have the technical resources to build and maintain real-time electronic disease surveillance systems. Our application service provider model has been much more success-ful, and we now recommend that states form coalitions to share the costs of such services.\nThe project very early identified the need for a computing component to reside within the firewall of a health system, connected to the hospital's HL7 message router. This component would function as a case detector in a distributed public health surveillance scheme linking laboratory and radiology data to increase the specificity of case detection. It has proven very difficult to disseminate this technology, perhaps due to the complexity of the idea. Nevertheless, the threat of bioterrorism has created a need for such technology, and this approach, or something with equivalent function, must be deployed.\nAdherence to NEDSS architectural standards was an early design objective that we have met. RODS 1.5 closely follows NEDSS architectural, software, messaging, and data specifications. Our success is a strong validation of those standards. We will gain further understanding of the standards as we attempt to use RODS components including HL7 listeners, natural language parsers, message parsers, databases, user interfaces, notification subsystems, and detection algorithms with other NEDSS compliant systems. An ongoing project will use RODS to collect chief complaints and integrate them into the Utah Department of Health's planned NEDSS system.\nWe have demonstrated the ability to rapidly deploy RODS in a special event with the added advantage that the system F i g u r e 6. Look-back function of RODS. The user has selected one patient to investigate using the screen that is in the background and partly hidden by overlap. RODS has logged the user into the results-review function of an electronic medical record and requested that patient's chart, which is shown on the screen in the foreground.\npersisted after the event. This experience suggests strongly that RODS or similar systems be considered an alternative to drop-in surveillance.\nOur future plans are to meet our initial design objective to develop early-warning capability for a large, outdoor release of anthrax, especially ensuring that the data and analysis produced by RODS are reviewed by public health. This goal will require improvements in the interfaces and the detection algorithms to reduce false alarms and to vastly improve the efficiency with which anomalies are evaluated by use of multiple types of data, better interfaces, and implementation of the look-back function. We would like to enlarge as quickly as possible the application service provider to include more states and more types of clinical data so that states will be in a position to prospectively evaluate the detection performance from different types of data on naturally occurring outbreaks.\nOur long-term goals are to add additional disease scenarios to the design objectives such as detection of in-building anthrax release, vector-borne disease, food-borne disease, and a communicable disease such as severe acute respiratory syndrome (SARS).\nRODS is a NEDSS-compliant public health surveillance system that focuses on real-time collection and analysis of data routinely collected for other purposes. RODS is deployed in two states and was installed quickly in seven weeks for the 2002 Olympics. Our experience demonstrates the feasibility of such a surveillance system and the challenges involved.\nOutbreaks, emerging infections, and bioterrorism have become serious threats. It is our hope that the front-line of public health workers, astute citizens, and health care workers will detect outbreaks early enough so that systems such as RODS are not needed. However, timely outbreak detection is too important to be left to human detection alone. The notion that public health can operate optimally without timely electronic information is as unwise as having commercial airline pilots taking off without weather forecasts and radar." }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: The ef®ciency of +1 ribosomal frameshifting at a speci®c codon is used as a sensor to regulate polyamine levels in mammalian cells. The frameshifting occurs in decoding the gene antizyme 1, which has two partially overlapping open reading frames (ORFs). Protein sequencing showed that the reading-frame shift occurs at the last codon of ORF1, causing a proportion of ribosomes to enter ORF2 to synthesize a transframe protein (Matsufuji et al., 1995) . ORF2 encodes the main functional domains (Matsufuji et al., 1990; Miyazaki et al., 1992) of antizyme but has no ribosome initiation site of its own. The antizyme 1 protein binds to ornithine decarboxylase (ODC) (Murakami et al., 1992a; Cof®no, 1993, 1994) , inhibits it (Heller et al., 1976) and targets it for degradation by the 26S proteosome without ubiquitylation (Murakami et al., 1992b (Murakami et al., , 1999 . ODC catalyzes the ®rst and usually ratelimiting step in the synthesis of polyamines, conversion of ornithine to putrescine. Putrescine is a substrate for the synthesis of spermidine and spermine. Because of its inhibition of ODC, antizyme 1 is a negative regulator of the synthesis of polyamines. In addition, antizyme 1 is a negative regulator of the polyamine transporter (Mitchell et al., 1994; Suzuki et al., 1994; Sakata et al., 1997) . As discovered by Matsufuji and colleagues (Gesteland et al., 1992) and Rom and Kahana (1994) , increasing polyamine levels elevate frameshifting in decoding antizyme 1 mRNA and so increase the level of antizyme 1. Since antizyme 1 negatively regulates the synthesis and uptake of polyamines, the frameshifting is the sensor for an autoregulatory circuit. A second mammalian paralog of antizyme, antizyme 2, has very similar properties to antizyme 1, including the regulatory frameshifting, but does not stimulate degradation of ODC under certain conditions where antizyme 1 is active (Ivanov et al., 1998a; Zhu et al., 1999; Y.Murakami, S.Matsufuji, I.P.Ivanov, R.F.Gesteland and J.F.Atkins, in preparation) . Just like antizyme 1, antizyme 2 mRNA is ubiquitously expressed in the body but is 16 times less abundant than mRNA of antizyme 1 (Ivanov et al., 1998a) . In addition to antizyme 1 and 2, mammals have a third paralog of the gene, antizyme 3 (also encoded by two ORFs), which is expressed only during spermatogenesis (Ivanov et al., 2000) . Zebra®sh also have multiple antizyme genes, which differ in their expression patterns and activities (Saito et al., 2000) .\nNumerous studies have addressed the regulation of fungal ODC in response to exogenously added polyamines. In the cases examined, Physarum polycephalum (Mitchell and Wilson, 1983) , Saccharomyces cerevisiae (Fonzi, 1989; Toth and Cof®no, 1999) and Neurospora crassa (Barnett et al., 1988; Williams et al., 1992) , added polyamines, especially spermidine, result in signi®cant repression of ODC activity. The mechanisms of repression seem to vary from fungus to fungus and are apparently different from the mechanism of polyamine-dependent regulation of ODC in higher eukaryotes. In some cases, the existence of an antizyme-like protein has been suggested but has either been disproved, as in the case of N.crassa (Barnett et al., 1988) , or has never been substantiated, as is the case with S.cerevisiae.\nAs expected from their small cationic nature and ability to neutralize negative charges locally, polyamines play key roles in processes ranging from the functioning of certain ion channels (Williams, 1997) , nucleic acid packaging, DNA replication, apoptosis, transcription and translation. The role of polyamines can be complex as illustrated by the transfer of the butylamine moiety of spermidine to a lysine residue to form hypusine in mammalian translation initiation factor eIF-5A, the only known substrate for this reaction (Tome et al., 1997; Lee et al., 1999) . Spermine negatively regulates the growth of prostatic carcinoma cells at their primary site (Smith et al., 1995) , but at later stages of tumor progression it fails to induce antizyme, which correlates with cells becoming refractory to spermine (Koike et al., 1999) . Lack of antizyme function is also important in the early deregulation of cellular proliferation in oral tumors (Tsuji Conservation of polyamine regulation by translational frameshifting from yeast to mammals The EMBO Journal Vol. 19 No. 8 pp. 1907±1917, 2000 ã European Molecular Biology Organization et al., 1998) and probably others. The levels of polyamines are altered in many tumors, and inhibitors of polyamine synthesis are being tested for antiproliferative and cell death effects. The synthesis of ODC varies during the cell cycle in normal cells (Linden et al., 1985; Fredlund et al., 1995) . It is induced by many growth stimuli and is constitutively elevated in transformed cells (Pegg, 1988; Auvinen et al., 1992) with some phosphorylated ODC being translocated to the surface membrane where it is important for mitotic cytoskeleton rearrangement events (Heiskala et al., 1999) .\nAntizyme is one example of certain mRNA-contained signals that can elevate speci®c frameshifting >1000-fold above the background level of normal translational errors. In addition to antizyme, frameshifting is also involved in the decoding of some bacterial and yeast genes and especially in many mammalian Retroviruses and Coronaviruses, plant viruses and bacterial insertion sequences (Atkins et al., 1999) . The site of frameshifting in both mammalian antizyme 1 and 2 mRNAs is UCC UGA, where quadruplet translocation occurs at UCCU (underlined) to shift reading to the +1 frame, immediately before the UGA stop codon of the initiating frame (Matsufuji et al., 1995; Ivanov et al., 1998a) . For the frameshifting to occur with an ef®ciency of 20% or more, it is important that the 3¢ base of the quadruplet is the ®rst base of a stop codon. Other important features are a pseudoknot just 3¢ of the shift site and a speci®c sequence 5¢ of the shift site (Matsufuji et al., 1995; Ivanov et al., 1998a) . A pseudoknot 3¢ of the shift site is a common stimulator for eukaryotic ±1 frameshifting, but the synthesis of antizyme is the only known case utilizing +1 frameshifting.\nComparative analysis of RNA sequences from different organisms is informative about important features and the different options selected by evolution. Since most of the known examples of programmed frameshifting are in viruses or chromosomal mobile elements, the opportunity for comparison of frameshift cassettes in divergent organisms where the time of divergence can be approximated is limited. A start has been made with the frameshifting required for bacterial release factor 2 expression (Persson and Atkins, 1998) , but antizyme provides the ®rst opportunity for such a comparison in eukaryotes. Antizyme genes in genetically tractable lower eukaryotes would be helpful for understanding the functionally important interactions responsible for autoregulatory programmed frameshifting.\nIdenti®cation of an antizyme gene in Schizosaccharomyces pombe A search for DNA sequences encoding protein sequences homologous to Drosophila melanogaster antizyme (Ivanov et al., 1998b) and Homo sapiens antizyme 1 identi®ed the same S.pombe anonymous cDNA clone (DDBJ/EMBL/GenBank accession No. D89228). The similarity is limited (~10% identity, 24% similarity to both human antizyme 1 and D.melanogaster antizyme); however, it is highest in regions that are most highly conserved among the previously identi®ed antizymes ( Figure 1A ). Closer examination of the cDNA nucleotide sequence provided further evidence that it encodes an S.pombe homolog of antizyme. The initiating AUG codon for the ORF that is similar to higher eukaryotic antizymes (ORF2 of those genes) is not the 5¢-most AUG in this cDNA. In fact, there are eight AUGs closer to the 5¢ end. The ®rst or the second AUGs would initiate translation of an ORF (ORF1) that overlaps the longer downstream ORF (ORF2) such that a +1 translational frameshifting event in the overlap would generate a protein product analogous to the products of antizyme genes from higher eukaryotes. Furthermore, the last 12 nucleotides of ORF1 (UGG-UGC-UCC-UGA) are identical to the last 12 nucleotides of mammalian antizyme 1 ORF1s, including the frameshift site. Eleven of these 12 nucleotides are identical to the corresponding regions of all previously identi®ed antizyme genes ( Figure 1B ). Previous experiments with the mammalian frameshift sequence tested in S.pombe have shown that this short 12 nucleotide sequence, by itself, is suf®cient to stimulate measurable levels (up to 0.5%) of +1 frameshifting (Ivanov et al., 1998c) . To con®rm the ORF con®guration of the putative S.pombe antizyme gene, a region corresponding to the two overlapping ORFs plus~80 nucleotides of the 5¢ UTR and 370 nucleotides of the 3¢ UTR, was ampli®ed from both S.pombe genomic DNA and a cDNA library. The sequence of the ampli®ed DNA con®rmed that there are indeed two overlapping ORFs with the deduced con®guration. This sequence (DDBJ/EMBL/GenBank accession No. AF217277) differs from the previously sequenced cDNA clone by three nucleotides (two in the coding region and one in the 3¢ UTR); one changes an alanine codon to proline, another is a silent mutation within a proline codon. Since the sequences from the cDNA library and genomic DNA are identical, we conclude that the differences with clone No. D89228 are most likely due to strain variation. This gene contains no introns within the ampli®ed region.\nThe S.pombe protein was tested for antizyme activity using a gene fusion with glutathione S-transferase (GST). In this construct, ORF1 and ORF2 of antizyme are fused in-frame by deleting the T nucleotide that encodes U of the stop codon of ORF1. This GST±antizyme fusion gene was expressed in Escherichia coli and the protein was puri®ed by af®nity chromatography. ODC inhibitory activity was tested by incubating the recombinant antizyme protein with an S.pombe crude extract and then assaying the mixture for ODC activity. The results ( Figure 2) show that the recombinant protein can inhibit S.pombe ODC. GST alone (1 mg) does not inhibit S.pombe ODC (data not shown). In light of these results, the S.pombe gene will be called S.pombe ODC antizyme (SPA). Interestingly, the S.pombe ODC was also inhibited by mouse antizyme 1 and antizyme 2 (both expressed as GST fusions); however, the yeast fusion protein did not inhibit mouse ODC (data not shown).\nDeletion and overexpression of SPA Although the effects of overexpression of antizyme on cellular physiology have been tested previously in mammalian cells, the physiological changes associated with complete absence of antizyme activity have not yet been investigated because of the complication of multiple antizymes. The single S.pombe antizyme provides the chance to explore a knockout. SPA deletion strains were I.P. Ivanov et al. generated by replacing the two ORFs of the gene with the ORFs of either URA4 or LEU2 (see Materials and methods). Complete deletion of SPA (both ORFs) did not affect the viability of S.pombe cells in rich (YE) or minimal (MM) media. Temperature had no differential effect on mutant and wild-type cell growth. Similarly, the growth rates, mating ef®ciencies and overall morphology of the knockout strains are apparently indistinguishable from those of wild-type cells (results not shown).\nIn wild-type S.pombe cells the most abundant polyamine is spermidine followed by putrescine ( Figure 3 ). Spermine and cadaverine are found in much smaller amounts. This distribution of polyamine content is very similar to that in other fungi for which polyamine concentrations have been measured (for references, see review by Tabor and Tabor, 1985) . The effect of SPA deletion on cellular polyamine contents was examined in both exponentially growing and stationary phase cells ( Figure 3 ). The cellular concentrations of putrescine, spermidine and cadaverine (but not spermine) were higher in the knockout strains than in wild-type cells. The greatest effect was seen on putrescine and cadaverine content, with smaller effects on spermidine, presumably because eukaryotic ODC activity directly catalyzes decarboxylation of both ornithine and lysine to produce putrescine and cadaverine, respectively (Pegg and McGill, 1979) , but subsequent regulatory events affect homeostasis of spermidine and spermine. The effect of inactivating antizyme on the polyamine contents in exponentially growing cells is modest (<2-fold in all cases). The effect becomes very pronounced in cells in stationary phase with up to 40-and 10-fold increases of putrescine and cadaverine contents, respectively, in the knockout strains.\nTo test overexpression of SPA, two versions of the gene were cloned into pREP3 expression vector behind a strong, thiamine-repressible promoter (nmt1). One had the wild- type SPA sequence while in the second, ORF1 and ORF2 are fused in-frame. SPA wild type and an SPA deletion strain were transformed with each of the overexpression constructs. Derepression of the nmt1 promoter is a gradual process since it requires dilution of the intracellular pool of thiamine (the repressor) through cell division. After 2.5 days of exponential growth under derepressed conditions, yeast strains transformed with either SPA overexpression construct show signi®cant increases in doubling time ( Figure 4A ). The growth inhibition is greater with the construct expressing the in-frame version of SPA and after prolonged incubation (5±7 days); these cells cease growth and accumulate in G 1 as determined bȳ ow cytometry (data not shown). The fact that the inframe overexpression construct, which differs by a single nucleotide from the wild-type construct, confers a more severe phenotype is consistent with the hypothesis that translational frameshifting is required for expression of SPA. The growth phenotype associated with SPA overexpression is only partially relieved by adding 100 mM putrescine to the media (1 mM had no further effect) (data not shown). To see whether the slower growth is correlated with aberrant polyamine levels the polyamine contents of the deletion strain carrying in-frame SPA overexpression vector were measured under derepressed and repressed conditions, in both cases after 2 days of exponential growth ( Figure 4B ). As expected, overexpression of SPA results in signi®cant reduction in the intracellular levels of all four polyamines. After longer (4±5 days) incubation under derepressed conditions, no putrescine and cadaverine can be detected (data not shown).\nTranslational frameshifting during expression of SPA Previously, we developed an assay for measuring antizyme translational frameshifting in both S.cerevisiae (Matsufuji et al., 1996) and S.pombe (Ivanov et al., 1998c) . Brie¯y, the nucleotide sequence to be assayed is inserted between GST and lacZ, such that ORF1 of the assayed sequence is fused in-frame to GST, while ORF2 is fused in-frame to lacZ. b-galactosidase activity provides a measure of frameshifting ef®ciency. To determine whether translational frameshifting occurs in the overlap of ORF1 and ORF2 of SPA, a region of SPA including all but the ®rst codon of ORF1 plus 180 nucleotides downstream of the ORF1 stop codon was tested. +1 frameshifting occurred at 2.2% compared with a construct in which ORF1 and ORF2 are fused in-frame. This result is consistent with +1 frameshifting being crucial for expression of SPA.\nPrevious experiments have shown that the frameshift cassette of mammalian antizyme 1 can direct ef®cient +1 frameshifting when tested in S.pombe. The reverse experiment was conducted here. The SPA gene was translated in vitro in rabbit reticulocyte lysate and its resulting frameshift ef®ciency measured. With no addition of polyamines, frameshifting ef®ciency is~1.5%. Addition of spermidine to the translation mixture to a ®nal concentration of 1 mM results in a 3.7-fold increase in frameshifting to~5.5%, a level even higher than that observed in the endogenous system in vivo (autoradiogram not shown). The observed ef®ciency of frameshifting with the SPA frameshifting cassette in vivo in S.pombe is signi®cantly more than that expected from its limited nucleotide similarity to the antizyme frameshift sites of higher eukaryotes. This prompted a search for additional stimulatory elements within the SPA frameshift cassette.\nThe following experiments were done in a strain carrying deletion of SPA (high polyamines) because it gives higher frameshifting and higher b-galactosidase activity in general; however, we obtained similar ratios for mutant to wild-type frameshifting ef®ciency in a strain with the intact SPA gene. Deleting 5¢ sequences up to the third to last sense codon of ORF1 has little or no effect on frameshifting ef®ciency. Deleting all but the last sense codon (UCC) of ORF1 leads to a 4-to 5-fold reduction in frameshifting ef®ciency ( Figure 5A ). This implies that the conservation of the six nucleotides 5¢ of the UCC-UGA frameshift site is due to their importance for stimulating +1 frameshifting. It also suggests that no additional ORF1 sequences of SPA stimulate the +1 recoding event. The 180 nucleotide 3¢ region was searched for possible structure by computer RNA folding algorithms plus visual inspection. The algorithms predicted several minimal structures in that region. 3¢ deletion constructs (constructs del.3,3¢±81,3¢) tested the importance of any putative structure on the frameshifting ef®ciency. The results ( Figure 5B and C) show that all of these deletions lead to a signi®cant (~10-fold) reduction in +1 frameshifting, indicating the presence of a major 3¢ stimulatory element in the 180 nucleotide region immediately following the frameshift site of SPA. However, the results indicate that none of the putative RNA structures in this region are suf®cient for the activity of this element. Several additional 3¢ deletions delineated the boundaries of this stimulatory element from the frameshift site to 150 and 180 nucleotides downstream (since construct del.150,3¢ stimulates 5.5-fold more +1 frameshifting than del.129,3¢, 150 nucleotides downstream probably contain most of the 3¢ stimulator).\nIn the experiments described above, two of the characteristics of the autoregulatory circuit of mammalian antizyme 1 were con®rmed: SPA inhibition of ODC and the +1 translational frameshifting. The key question left is whether the recoding event is responsive to polyamine levels in cells. As shown above, overexpression of SPA leads to signi®cant reduction of polyamine levels in S.pombe. An SPA + strain was co-transformed with an SPA wild-type overexpressing plasmid (cells overexpressing wild-type SPA grow slowly but continuously) and a construct that monitors the +1 frameshifting from an SPA frameshift sequence. The +1 frameshifting was compared with that in SPA non-overexpressing cells (in both cases frameshifting was measured relative to in-frame control). The results ( Figure 6 ) show a signi®cant reduction (6.5-fold) in frameshifting ef®ciency in SPA-overproducing cells that correlates with a decrease of polyamine content (4.5-fold for putrescine and 3.9-fold for spermidine). This indicates that polyamines modulate the frameshifting ef®ciency of SPA. An alternative but less likely possibility is that SPA overexpression reduces frameshifting because high levels of SPA transcript titrate some factor limiting for frameshifting.\nThe SPA frameshift signals direct 2-fold more frameshifting in Dspa::LEU2 cells (4.4%) than in SPA + cells (in both cases the measurement is done during stationary phase); however, the relatively high standard deviations for both measurements make it dif®cult to draw ®rm conclusions from this particular result.\nA search of Caenorhabditis elegans expressed sequence tag (EST) sequences with mammalian antizyme 1 sequence identi®ed 20 clones. These sequences could be deconvoluted into a contiguous cDNA sequence. Primers designed on the basis of this sequence were used to PCR amplify and subclone this cDNA from a C.elegans cDNA library. The sequence of the subcloned cDNA was con®rmed (DDBJ/EMBL/GenBank accession No. AF217278); the subsequently released genomic sequence of this C.elegans gene (DDBJ/EMBL/GenBank accession No. AF040659) con®rms our cDNA data. The amino acid sequence deduced from the cDNA sequence revealed that the longer ORF has similarity to previously reported antizyme sequences (overall 27% identity, 39% similarity to human antizyme 1; 19% identity, 34% similarity to Drosophila antizyme). These similarities are higher than that of SPA to these two antizyme genes and again are concentrated in the regions most highly conserved among previously identi®ed antizymes ( Figure 1A ). Just like mammalian antizymes, the longer ORF (ORF2) lacks an appropriate in-frame initiation codon, and expression could be provided by initiation in a short upstream overlapping ORF (ORF1) leading to +1 ribosomal frameshifting in the overlap. The putative C.elegans antizyme frameshift site (the nucleotides proximal to the end of ORF1) has 18 of 26 nucleotides identical to the consensus sequence for antizyme frameshift sites ( Figure 1B) .\nFrameshifting for expression of C.elegans antizyme was investigated in heterologous systems. Two constructs containing the entire antizyme cDNA, one with the wildtype sequence and one with a single nucleotide deletion that fuses ORF1 to ORF2 in-frame (in-frame control), were transcribed in vitro and the RNA was translated in rabbit reticulocyte lysate. The products were examined by SDS±PAGE (Figure 7) . The main product from both constructs has an apparent M r of 21 kDa, slightly greater than the predicted M r of 17.7 kDa [aberrant, slower than expected, mobility is observed with antizyme proteins from other species (Ivanov et al., 1998a) ]. From the ratio of wild-type to in-frame product, we estimate that the ef®ciency of frameshifting of C.elegans antizyme in reticulocyte lysate is~0.8%, which is somewhat lower than SPA frameshifting in the same system. Addition of spermidine to the translation reactions almost doubles the ef®ciency of frameshifting to~1.5% (the exact numbers are not easy to determine because of dif®culty in de®ning background values). The frameshifting properties of C.elegans antizyme mRNA were also tested in vivo in S.pombe cells. A sequence including all but the ®rst codon of ORF1 plus 180 nucleotides downstream was inserted between GST and lacZ of the PIU-LAC plasmid. Comparison of the b-galactosidase activity of cells (Dspa::LEU2 strain) transformed with the wild-type construct and the in-frame control constructs indicated 3.5% +1 frameshifting. From the frameshifting observed in the heterologous systems, as well as the sequence considerations discussed above, we conclude that expression of this C.elegans gene requires ribosomal frameshifting.\nSearching the EST database with the newly discovered C.elegans antizyme identi®ed antizyme orthologs in four other nematode species. In two cases (Necator americanus and Haemonchus contortus), the cDNA sequences in the database were suf®cient to make contigs of the complete coding regions. In the other two cases [Onchocerca volvulus (DDBJ/EMBL/GenBank accession No. AF217279) and Pristioncus paci®cus (DDBJ/EMBL/ GenBank accession No. AF217280)] the complete cDNA sequences were obtained by PCR amplifying and sequencing the full genes from cDNA libraries. As with the previously identi®ed eukaryotic antizyme genes, the ORF con®guration of the newly found nematode orthologs implies the necessity for +1 frameshifting for synthesis of full-length protein.\nThe C.elegans antizyme mRNA frameshift site UUU-UGA is unique, differing from the UCC-UGA of previously known antizyme mRNAs. The C.elegans antizyme gene shares this feature with N.americanus and H.contortus but not with P.paci®cus and O.volvulus antizymes. The phylogenetic tree of nematode antizyme protein sequences matches exactly the phylogenetic relationship (Blaxter, 1998) of the nematodes expressing them, indicating that these gene sequences are the result of divergent evolution within the nematode lineage (data not shown). These results also show that the UUU-UGA frameshift site evolved after the last common ancestor of P.paci®cus and C.elegans but before the divergence of C.elegans, N.americanus and H.contortus (probably 450± 500 million years ago).\nThe ability of UUU-UGA sequence to direct +1 frameshifting was further tested in a mammalian system in the context of the mammalian antizyme mRNA (i.e. in the presence of the 3¢ RNA pseudoknot and 5¢ stimulator). A BMV-coat-protein±antizyme 1 gene fusion construct, which has a TCC-TGA to TTT-TGA substitution, was transcribed and then translated in a rabbit reticulocyte lysate. Eleven percent frameshift ef®ciency was seen in the absence of exogenously added polyamines, 2.2 times the ef®ciency seen with the UCC-UGA transcript. The frameshift ef®ciency becomes 18% when 0.6 mM spermidine is added, which is 1.3 times that with the wild type (Matsufuji et al., 1995) . Similar results were obtained in cultured mammalian (Cos7) cells transfected with TTT-TGA mutant construct, the frameshift being higher than that of wild-type construct in both high-and lowpolyamine conditions (our unpublished results). These results demonstrate that the putative C.elegans frameshift site (UUU-UGA) is, if anything, shiftier than UCC-UGA in the antizyme 1 context and is subject to polyamine stimulation.\nThe results presented show that the yeast S.pombe has a homolog of mammalian antizyme. This is the ®rst documented example of antizyme-type regulation of ODC in a lower eukaryote.\nDeleting SPA from the yeast genome has no detectable effect on viability or any other overt phenotypic effect but, as expected, it results in altered accumulation of polyamines in the cell. Interestingly, the effect is most pronounced in cells in stationary phase, where the knockout cells accumulate up to 40 times more putrescine than wild-type counterparts. This compares with a <2-fold increase of putrescine in exponentially growing cells. A likely explanation for this observation is that the usual rate of ornithine decarboxylation in exponentially growing cells is close to capacity given`normal' concentrations of substrate, enzyme and product. At the same time, all newly synthesized polyamines are continuously diluted through Fig. 6 . Effect of polyamine depletion on SPA +1 frameshifting. Polyamine depletion is achieved by overexpression of the wild-type version of SPA. The same cultures were assayed both for frameshifting and polyamine content. Numbers above columns indicate fold reduction of frameshifting and polyamine content compared with cells that do not overexpress SPA. Antizyme genes in S.pombe and C.elegans cell growth and division at a rate that is almost identical to the rate of maximum capacity synthesis. Cells in stationary phase can no longer dilute newly synthesized polyamines, and more importantly lack an effective antizymeindependent mechanism of shutting off ODC. This suggests that SPA is the primary regulator of ODC activity in S.pombe, not only during cell growth (short term regulation) but also in non-dividing cells (longer term regulation).\nOverexpression of SPA (5±7 days derepression) leads to complete depletion of intracellular putrescine. This result implies that in S.pombe ornithine decarboxylation is the only source of putrescine synthesis (the pathway from arginine via agmatine is not utilized). The complete depletion of cadaverine in SPA overexpressing cells suggests that ODC is the only enzyme in S.pombe that can decarboxylate lysine, which is also the case in rat tissues (Pegg and McGill, 1979) .\nIt is somewhat perplexing that addition of putrescine to the media leads to only partial relief of the growth phenotype associated with SPA overexpression. There are two likely explanations. (i) Perhaps S.pombe imports putrescine poorly. (ii) Alternatively, like the mammalian system, maybe SPA inhibits not only ODC but also the polyamine transporter. Further experiments will help to distinguish between these two models.\nIt is unclear how widespread the antizyme gene is within the fungal kingdom. We have identi®ed and cloned antizyme homologs from two other ®ssion yeasts (Schizosaccharomyces octosporus and Schizosaccharomyces japonicus) and from two distantly related fungi (Botryotinia fuckeliana and Emericella nidulans) (our unpublished results). The antizyme frameshift site of the latter two fungi has evolved in a unique way different from all other known antizymes, but nevertheless even these two distantly related fungi have conserved the autoregulatory +1 frameshifting. The fact that the yeast S.pombe has an antizyme gene suggests the possibility that the higher eukaryotic metazoans may all have an antizyme gene.\nThe only previously reported antizyme activity in unicellular organisms is from E.coli, but recent analyses suggest that E.coli does not have a true antizyme (Ivanov et al., 1998d) . This makes SPA the ®rst bona ®de antizyme in a unicellular organism.\nThe remarkable similarity of the core sequence important for antizyme frameshifting from S.pombe to humans could be due to convergent or divergent evolution. The near identity of this sequence in worms, Drosophila, Xenopus, zebra®sh and humans argues against convergent evolution, as if antizyme frameshifting arose in a common ancestor perhaps more than one billion years ago.\nThree cis-acting RNA elements are known to stimulate mammalian antizyme 1 frameshifting. One is a 50 nucleotide sequence immediately 5¢ of the shift site (Matsufuji et al., 1995; our unpublished results) . A second stimulator is the UGA stop codon of ORF1 and the third is an RNA pseudoknot starting 3 nucleotides 3¢ of the UGA stop codon. Among frameshift sites of the previously identi®ed antizymes from mammals all the way to Drosophila, there is substantial similarity in the sequences immediately 5¢ of the shift site. Sixteen of the last 18 nucleotides of ORF1 are completely conserved in these genes. Schizosaccharomyces pombe and C.elegans antizymes have 9 of 9 and 6 of 9 (14 out of 19 in O.volvulus) nucleotides identical to the consensus, respectively. For the 5¢ sequences, generally, the more distantly related two antizymes are, the more the similarity is con®ned to the 3¢ end of that region. Our SPA ORF1 deletion data show that mutation of nucleotides that are part of the 5¢ consensus sequence leads to reduced frameshifting ef®ciency. This is another indication that conservation of nucleotide sequence in this region is because of its importance for stimulating ef®cient +1 frameshifting. It is quite striking that in all antizyme gene sequences identi®ed so far, including a number of unpublished ones, ORF1 ends with a UGA stop codon. This is particularly surprising since any of the other two stop codons can substitute for UGA to stimulate antizyme 1 frameshifting, although slightly less ef®ciently, in vitro (Matsufuji et al., 1995) and in vivo (our unpublished results).\nThe 3¢ pseudoknot that stimulates frameshifting in antizyme 1 is highly conserved in all known vertebrate antizymes, including mammalian antizyme 2 ( Figure 1B) . None of the invertebrate antizyme mRNAs identi®ed so far, including those presented here, has a sequence in the equivalent region that can be simply folded to a comparable RNA structure. However, sequences immediately 3¢ of the frameshift site are conserved between invertebrates and vertebrates. The conservation of this region between Drosophila and the vertebrate counterparts has already been noted (Ivanov et al., 1998b) . The C.elegans antizyme gene contains the sequence YGYCCCYCA (Y = pyrimidine) in this region, which is identical to the consensus. The antizyme genes from the other four nematodes also have a similar sequence ( Figure 1B) . The signi®cance of this similarity is not clear [in fact, sequences in this region appear to play no role in antizyme 1 in vitro frameshifting outside of the RNA pseudoknot context (Matsufuji et al., 1995) ].\nOnly two examples are known where RNA elements 3¢ of the frameshift site stimulate +1 frameshifting. One is the RNA pseudoknot of mammalian antizyme 1 and the second is a short RNA sequence immediately following the frameshift site of Ty3 (Farabaugh et al., 1993) . Additional examples would be very helpful in deciphering the role such elements play in the mechanism of +1 frameshifting. It is currently not known how many and which of the invertebrate antizyme genes contain 3¢ frameshift stimulators. The results presented here show that an S.pombe 3¢ stimulator enhances frameshifting 10-fold. This stimulator appears completely different from the 3¢ RNA pseudoknot in vertebrates. Our deletion experiments indicate that none of the predicted RNA structures contained within the minimally required 3¢ region [up to 150±180 nucleotides downstream of the frameshift site ( Figure 5C )] are suf®cient to confer the stimulatory effect. The SPA 3¢ stimulator may act directly through sequence or may have an unusual RNA structure involving non-Watson±Crick base pairing. More detailed mutagenesis combined with phylogenetic analysis would be required to discern the nature of the 3¢ stimulator of SPA.\nThe nematode antizymes were analyzed for the presence of possible 5¢ or 3¢ stimulators¯anking the core frameshift site. Computer RNA folding programs did not identify any potentially interesting structure. More importantly, phylogenetic analysis with the ®ve identi®ed nematode antizymes failed to identify any conservation of primary RNA sequence (or for that matter potential secondary structure) outside of the core region that is shared between two or more members. This could indicate that no such extra cis-acting stimulators exist in nematode antizymes or that they are located in a very different place within the mRNA, for example the 3¢ untranslated region (the latter suggestion is not supported by our sequence analysis).\nA common mechanism for frameshifting is re-pairing of the peptidyl tRNA in the new reading frame. However, an alternative mechanism whereby the peptidyl tRNA merely occludes the ®rst base of the next codon, has been documented for yeast Ty3 frameshifting (Farabaugh et al., 1993) . Results of experiments with some mutants of the mammalian antizyme 1 shift site pointed to an occlusion mechanism (Matsufuji et al., 1995) . However, the mechanism with the wild-type, UCC-UGA, shift site is not clear. For C.elegans antizyme the UUU-UGA sequence would be an obvious candidate for a re-pairing since Phe-tRNA could pair perfectly with UUU in both frames. But with UCC-UGA the Ser-tRNA ®rst reading UCC could at best pair two out of three with CCU. This important problem warrants further investigation.\nThe frameshift ef®ciency of SPA frameshift site is lower than that observed with mammalian antizyme 1 even when both are tested in the same organism (S.pombe) [for the frameshift ef®ciency of antizyme 1 cassette in S.pombe, see Ivanov et al. (1998c) ]. It is possible that the observed ef®ciencies for S.pombe antizyme are arti®cially low because the constructs do not include all the cis-acting stimulatory elements. On the other hand there is no reason why a lower level of frameshifting does not correctly re¯ect the evolved balance with the other characteristics of the complex system such as relative protein stabilities.\nLike other core cellular processes, the antizyme polyamine regulatory scheme is conserved from yeast S.pombe to human. It is not obvious why this very special mechanism is so exquisitely preserved over vast evolutionary time. Perhaps there is another whole aspect to the system that our experiments do not yet detect. From this viewpoint it would seem very important to exploit the genetics systems of S.pombe and C.elegans to understand more thoroughly the physiological effects of perturbing the antizyme system.\nThe SPA gene was ampli®ed using the following primers: 5¢-CAAAACAAGTTTTCATTATTGGTTTTTTTTAAATCAATCCCC (sense) and 5¢-CGTAAATCCAATCTAAATTTAATCTTCAACTAA-ATCATGAAAAGCCTC (antisense). The S.pombe cDNA library used as a template in the ampli®cation was kindly provided by R.Rowley (University of Utah). The C.elegans antizyme gene was ampli®ed using the following primers: 5¢-CCCAGGAATTCCTCGAGTATTTTGA-GTATAATTTTAC (sense) and 5¢-CGGCCGCTCGAGTTAGACCTT-GTAGCTCATGATG (antisense). This same ampli®ed DNA was used to make the constructs for in vitro transcription and translation of C.elegans antizyme by cloning it into pTZ18U plasmid using the SacI and HindIII sites incorporated in the two primers. The in-frame construct was made using a two-step PCR. The cDNA sequences of O.volvulus and P.paci®cus antizyme genes were obtained by performing 5¢ and 3¢ RACE PCR with cDNA libraries, which were kindly provided by Ralf Sommer (P.paci®cus) and Susan Haynes (O.volvulus). The SPA overexpression constructs were made by amplifying the gene with the primers 5¢-GCATCCGAATTCCCAAATCCAAGCATCATACGCC (sense) and 5¢-GCATCCGGATCCGCCAGTGTTCTTACTTTGAGA-TGC (antisense), and then inserting BamHI-digested product between the MscI and BamHI sites of pREP3 plasmid. The in-frame construct was made by two-step PCR and subsequently all in-frame SPA constructs described below were made by one-step PCR using this plasmid's DNA as a template. To make the constructs for frameshift assays in S.pombe, DNA fragments with a given nucleotide length (as described in the main text), were ampli®ed from both the SPA and C.elegans antizyme constructs described above. These fragments were then cloned between the KpnI and BstEII sites of PIU-LAC plasmid (Ivanov et al., 1998c) . The PCR primers included an`AC' spacer between the 5¢ cloning site (BstEII) and the antizyme sequences in order to correct the reading frame. The in vivo frameshifting assays in S.pombe (strains ura4-D18 leu1-32 ade6-M216 h ± and Dspa::LEU2 ura4-D18 leu1-32 ade6-M216 h ± ) were done as described (Ivanov et al., 1998c) . The plasmid for GST±SPA expression was made by PCR amplifying SPA (all but the ®rst codon of ORF1 through the downstream ORF2) from an in-frame template and cloning the product into the EcoRI and XhoI restriction sites of pGEX-5X-3 plasmid. The antizyme frameshift site in the BMV-coatantizyme fusion construct (C3NE) (Matsufuji et al., 1995) was mutated with a two-step PCR. To generate the two knockout strains, Dspa::URA4 and Dspa::LEU2, both ORFs of SPA were replaced exactly with the ORF of either URA4 or LEU2. To accomplish this, two pairs of primers ampli®ed URA4 and LEU2 such that 50±60 nucleotides, which normallȳ ank the two ORFs of SPA,¯ank the ORFs of the two genes. The ampli®ed DNA products were gel puri®ed and 2 mg of each were used to electroporate into ura4-D18 leu1-32 ade6-M216 h ± cells. URA + and LEU + transformants were selected by growth on URA ± and LEU ± media, respectively. PCR screen and partial sequencing, with primers¯anking the regions used for the homologous recombination, con®rmed the SPA disruptions. All DNA clones were sequenced with automated sequencing machines (ABI 100).\nSchizosaccharomyces pombe ODC active crude extracts were prepared as follows: S.pombe (strain 1519, leu1-32, h ± ) provided by R.Rowley was grown to OD 600 0.7 in 50 ml of minimal media + LEU. Ten milligrams of lysing enzymes (Sigma) were added, followed by continued incubation for 30 min at 30°C. Cells were harvested and washed once with cold homogenization buffer [25 mM Tris±HCl pH 7, 0.25 M sucrose, 1 mM dithiothreitol (DTT), 20 mM pyridoxal-5-phosphate, 2 mM EDTA] then resuspended in 0.75 ml of homogenization buffer. Cells were broken open and the lysate was clari®ed by centrifugation at 10 000 r.p.m. for 15 min at 4°C. Extracts were dialyzed overnight in dialysis buffer (25 mM Tris± HCl pH 7.4, 1 mM DTT, 20 mM pyridoxal-5-phosphate, 0.1 mM EDTA). A volume of 25 ml of extract was used for each ODC assay. ODC activity was assayed by measuring the release of 14 CO 2 from L-[1-14 C]ornithine (Amersham) as described (Nishiyama et al., 1988) . Each reaction took 1 h. Pre-incubation of S.pombe extract with 0.1 mM di¯uoromethyl ornithine (DFMO) for 15 min led to >99% inhibition of 14 CO 2 release.\nThe cells were collected by centrifugation, washed twice with 1 ml of phosphate-buffered saline (PBS) and then the pellet was frozen at ±80°C until use. The pellet was resuspended in 0.1 ml of PBS. An aliquot of the suspension was mixed with an equal volume of 8% perchloric acid, vortexed for 1 min, kept on ice for 5 min and centrifuged at 15 000 r.p.m., 4°C for 5 min. Ten microliters of the supernatant were subjected to polyamine analysis using¯uorometry on high-performance liquid chromatography as described previously (Murakami et al., 1989) . Protein concentrations were determined with the BCA protein assay kit (Pierce).\nThe experiments with the BMV-coat-antizyme fusion constructs were performed as described previously (Matsufuji et al., 1995) . All other plasmid DNA templates were prepared using QIAGEN Miniprep Kit and then digested with HindIII. Transcripts for SPA in vitro translation were made from PCR templates that had a T7 promoter incorporated into the PCR primers. Linearized DNA (1 mg) was used as a template for in vitro transcription with Ambion MEGAshortscript TM T7 Kit. The DNasetreated RNAs were recovered and resuspended in 40 ml of RNase-free water. One microliter of each speci®ed transcript suspension was used in each in vitro translation reaction [0.5 ml of 1 mM amino acid mix ±Met, 7 ml of reticulocyte lysate (Promega), 0.5 ml of [ 35 S]Met (Amersham)] to a total volume of 10 ml. The reactions were stopped by adding 1 ml of RNase (10 mg/ml). The frameshift ef®ciencies were quanti®ed as described (Ivanov et al., 1998a) ." }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: Introduction hnRNP A1 is an RNA-binding protein that contains two RNA-binding domains (RBDs) and a glycine-rich domain responsible for protein±protein interaction. It is involved in pre-mRNA splicing and transport of cellular RNAs (reviewed by Dreyfuss et al., 1993) . It is predominantly located in the nucleus, but also shuttles between the nucleus and the cytoplasm (Pin Äol-Roma and Dreyfuss, 1992) . The signal that mediates shuttling has been identi®ed as a 38 amino acid sequence, termed M9, located near the C-terminus of hnRNP A1 between amino acids 268 and 305 (Michael et al., 1995; Siomi and Dreyfuss, 1995; Weighardt et al., 1995) . Yeast two-hybrid screening with M9 as bait resulted in the discovery of a novel transportin-mediated pathway for nuclear import of hnRNP A1 (Pollard et al., 1996; Fridell et al., 1997; Siomi et al., 1997) . The function of the cytoplasmic hnRNP A1 has not been well de®ned. Studies have shown that cytoplasmic and nuclear hnRNP A1 exhibit different RNA-binding pro®les. Cytoplasmic hnRNP A1 is capable of high-af®nity binding to AU-rich elements that modulate mRNA turnover and translation (Hamilton et al., 1993 (Hamilton et al., , 1997 Henics et al., 1994) . It has also been shown to promote ribosome binding to mRNAs by a cap-mediated mechanism, and prevent spurious initiation at aberrant translation start sites (Svitkin et al., 1996) .\nMHV belongs to the Coronaviridae family of positivesense, single-stranded RNA viruses. MHV replication and transcription occur exclusively in the cytoplasm of infected cells via the viral RNA-dependent RNA polymerase (RdRp) (reviewed by Lai and Cavanagh, 1997) . Initially, the 5¢-most gene 1 of the viral genome is translated into the viral RdRp, which then replicates the viral genomic RNAs into negative-strand RNAs. Subsequently, the negative-strand RNAs are used as templates to transcribe mRNAs, which include a genomic-sized RNA and a nested set of subgenomic mRNA transcripts, all with an identical 5¢ non-translated leader sequence of 72±77 nucleotides and 3¢ co-terminal polyadenylated ends. The subgenomic mRNA transcription of MHV utilizes a unique discontinuous mechanism in which the leader sequence, often derived from a different molecule, is fused to RNAs at the intergenic (IG) sites (i.e. transcription initiation site) to generate subgenomic mRNAs (Jeong and Makino, 1994; Liao and Lai, 1994; Zhang et al., 1994) . The exact mechanism of how these mRNAs are made is still controversial. However, it has been shown that the process of discontinuous RNA transcription is regulated by several viral RNA elements, including the cis-and trans-acting leader RNA Zhang et al., 1994) , IG sequence (Makino et al., 1991) and 3¢-end untranslated sequence (Lin et al., 1996) . There is considerable biochemical evidence suggesting possible direct or indirect interactions between the various RNA regulatory elements. hnRNP A1 binds MHV negative (±)-strand leader and IG sequences (Furuya and Lai, 1993; Li et al., 1997) . Site-directed mutagenesis of the IG sequences demonstrated that the extent of binding of hnRNP A1 to the IG sequences correlated with the ef®ciency of transcription from the IG site (Zhang and Lai, 1995; Li et al., 1997) . Immunostaining of hnRNP A1 showed that hnRNP A1 relocated to the cytoplasm of MHV-infected cells, where viral RNA synthesis occurs (Li et al., 1997) . hnRNP A1 also mediates the formation of a ribonucleoprotein complex containing the MHV (±)-strand leader and IG sequences . These results suggest that hnRNP A1 may serve as a protein mediator for distant RNA regions to interact with each other. Heterogeneous nuclear ribonucleoprotein A1 regulates RNA synthesis of a cytoplasmic virus The EMBO Journal Vol. 19 No. 17 pp. 4701±4711, 2000 ã European Molecular Biology Organization Many cellular proteins, including calreticulin (Singh et al., 1994) , polypyrimidine tract-binding protein (PTB) (Hellen et al., 1994; Wu-Baer et al., 1996) , La protein (Pardigon and Strauss, 1996), Sam68 (McBride et al., 1996) , poly(rC)-binding protein (Parsley et al., 1997) and nucleolin (Waggoner and Sarnow, 1998) , have been implicated to be involved in viral RNA transcription or replication. In addition to MHV, hnRNP A1 has also been reported to interact with human cytomegalovirus immediate-early gene 2 protein, which plays an important role in the regulation of virus replication (Wang et al., 1997) . Furthermore, a yeast protein related to human core RNA splicing factors, Lsm1p, has been shown to be required for the ef®cient replication of brome mosaic virus RNA (Diez et al., 2000) . Recently, Reddy and colleagues demonstrated an inhibition of HIV replication by dominant-negative mutants of Sam68 (Reddy et al., 1999) . However, none of these cellular proteins has been shown experimentally to participate directly in RNA-dependent RNA synthesis.\nIn order to demonstrate the involvement of hnRNP A1 in MHV RNA replication and transcription, we established several DBT cell lines stably expressing either the wildtype (wt) hnRNP A1 or a C-terminus-truncated mutant lacking the M9 sequence and part of the glycine-rich domain. We showed that the mutant hnRNP A1, which was localized predominantly in the cytoplasm, exhibited dominant-negative effects on viral genomic RNA replication and subgenomic mRNA transcription. In contrast, overexpression of the wt hnRNP A1 accelerated the synthesis of all viral RNAs. Our results provide strong evidence that hnRNP A1 is directly or indirectly involved in MHV RNA synthesis in the cytoplasm and that the C-terminal part of the protein is important for its function. This ®nding thus reveals a novel function for hnRNP A1 in the cytoplasm.\nCharacterization of stable cell lines expressing the wt and a C-terminus-truncated hnRNP A1 To explore a potential role for hnRNP A1 in MHV RNA synthesis, we established murine DBT cell lines stably expressing the Flag-tagged wt hnRNP A1 (DBT-A1) or a mutant hnRNP A1, which has a 75 amino acid deletion from the C-terminus (DBT-A1DC) ( Figure 1A ). This mutant lacks part of the glycine-rich domain and the M9 sequence responsible for shuttling hnRNP A1 between the nucleus and the cytoplasm. Immunoblot of the whole-cell lysates with an anti-Flag antibody detected a 34 kDa protein in DBT-A1 cells and a 27 kDa protein in three independent clones of DBT-A1DC cells ( Figure 1B ), whereas no protein was cross-reactive to the anti-Flag antibody in the control cell line stably transfected with the pcDNA3.1 vector (DBT-VEC). The amounts of the Flagtagged wt and truncated hnRNP A1 were comparable in these cell lines. A chicken polyclonal antibody against hnRNP A1 detected two endogenous hnRNP A1 isoforms or hnRNP A1-related proteins in the whole-cell lysates of all of the cell lines. The bottom band (34 kDa) overlaps the Flag-tagged wt hnRNP A1 in DBT-A1 cells. There was only a slight increase in the overall amount of hnRNP A1 in DBT-A1 cells as compared with DBT-VEC cells, indicating that the exogenous hnRNP A1 constituted a small fraction of the total hnRNP A1 in the cells. In DBT-A1DC cells, an additional band of smaller size (27 kDa) corresponding to the mutant hnRNP A1 was detected. The overall expression levels of the exogenous hnRNP A1 and hnRNP A1DC were~3-fold lower than that of the endogenous hnRNP A1 in whole-cell lysates ( Figure 1B ). Similar to the endogenous hnRNP A1 protein (Pin Äol-Roma and Dreyfuss, 1992) , the Flag-tagged wt hnRNP A1 was localized almost exclusively in the nucleus ( Figure 1C ). The mutant hnRNP A1, however, was localized predominantly in the cytoplasm ( Figure 1C) , consistent with the previous ®nding that the M9 nuclear localization signal is necessary to localize hnRNP A1 to the nucleus Weighardt et al., 1995) . Thus, hnRNP A1DC was much more abundant than the endogenous hnRNP A1 in the cytoplasm. The expression levels of the wt or mutant hnRNP A1 varied among individual cells based on immuno¯uorescent staining ( Figure 1C ). The growth rate ( Figure 1D ) and cell morphology (data not shown) were similar among the different cell lines.\nThe effects of overexpression of the wt and mutant hnRNP A1 on syncytium formation and virus production We ®rst assessed the effects of hnRNP A1 overexpression on the morphological changes induced by MHV-A59 infection using several different clones of DBT cell lines. Virus infection was performed at a multiplicity of infection (m.o.i.) of 0.5 to detect the subtle morphological differences among the different cell lines. Syncytia appeared at~7 h post-infection (p.i.) in DBT-VEC cells and~1 h earlier in DBT-A1 cells. At both 8 and 14 h p.i., syncytia were signi®cantly larger and more spread out in DBT-A1 cells than those in DBT-VEC cells ( Figure 2A ). Similar differences were observed with two additional clones of DBT-A1 cells (data not shown). In contrast, no syncytium was observed in three different clones of DBT-A1DC cells, even at 14 h p.i. At 24 h p.i., almost all DBT-A1 cells detached from the plate, but~10±20% of DBT-VEC cells still remained on the plate (data not shown). Remarkably, there was no sign of syncytium formation in DBT-A1DC cells until 24 h after virus infection, when the overall morphology of the cells was similar to that of DBT-VEC cells at 7 h p.i. (data not shown). All of the DBT-A1DC cells were eventually killed at~48 h p.i., suggesting that the inhibition of viral replication was not a result of the disruption of the MHV receptor. Correspondingly, virus production from these cell lines was signi®cantly different. Between 6 and 14 h p.i., virus production from DBT-A1DC cells was 100-to 1000-fold less than that from DBT-VEC and DBT-A1 cells ( Figure 2B ). DBT-A1 cells produced twice as many viruses as those from DBT-VEC cells during that time period.\nRelocalization of hnRNP A1 during MHV infection MHV RNA synthesis occurs exclusively in the cytoplasm of infected cells. In order for hnRNP A1 to participate directly in viral transcription, it has to be recruited to the site of RNA synthesis. Although hnRNP A1 shuttles between the nucleus and the cytoplasm in normal cells (Pin Äol-Roma and Dreyfuss, 1992) , the level of cytoplasmic hnRNP A1 is very low. We have demonstrated previously that hnRNP A1 relocates from the nucleus to the cytoplasm of MHV-infected cells (Li et al., 1997) . To determine whether the overexpressed hnRNP A1 may participate in MHV RNA synthesis, we performed immunostaining experiments using an anti-Flag antibody to localize Flag-tagged hnRNP A1. In DBT-A1 cells, a signi®cant increase in the cytoplasmic level of hnRNP A1 and a corresponding decrease of nuclear hnRNP A1 were observed in virus-infected cell syncytia at 7 h p.i.\n( Figure 3B ); these cells express the MHV nucleocapsid (N) protein in the cytoplasm ( Figure 3A ). By comparison, in the uninfected cells, which did not have N protein staining, hnRNP A1 was predominantly localized to the nucleus (arrow in Figure 3B ). In DBT-A1DC cells, very few cells were stained positive for the MHV N protein at 7 h p.i. ( Figure 3C ). Signi®cantly, the viral N protein was detected only in the cells that were stained weakly or not at all for Flag-hnRNP A1 ( Figure 3D ), suggesting that the expression of a high level of hnRNP A1DC interfered with viral replication. The effects of wt and mutant hnRNP A1 on MHV protein production We further investigated the effects of the wt and mutant hnRNP A1 on the production of MHV structural and nonstructural proteins. Cytoplasmic protein was extracted from infected cell lines at different time points after infection for immunoblot analysis to detect an open reading frame (ORF) 1a product, p22 (Lu et al., 1998) and the N protein. p22 expression in DBT-VEC cells was clearly detected at 6 h p.i. and peaked at~16 h p.i. ( Figure 4A ). In DBT-A1 cells, p22 appeared at 5 h p.i. and peaked at~8 h p.i. In DBT-A1DC cells, no p22 protein was detected until 16 h p.i. Similar patterns of differences were observed for the N protein in these three cell lines. Actin levels in different cell lines remained relatively constant throughout the infection, except that, in DBT-A1 cells, actin was not detected at 16 and 24 h p.i. due to the loss of the dead cells ( Figure 4A ). These results clearly demonstrated that overexpression of the wt hnRNP A1 accelerated viral protein production, whereas expression of the mutant hnRNP A1 delayed it. We also performed immuno¯uorescent staining of the N protein at 7 h p.i. to further con®rm the western blot results. As represented by images shown in Figure 4B , there were more DBT-A1 cells stained positive for the N protein than DBT-VEC cells. Very few cells were found to express the N protein in DBT-A1DC cells. The p22 and N proteins appeared as doublets in some of the lanes of Figure 4A , but the results varied from experiment to experiment. The N protein is known to be phosphorylated (Stohlman and Lai, 1979) . Whether p22 is post-translationally modi®ed is not known. Figure 5A ). DBT-A1 cells showed a signi®cantly higher level of [ 3 H]uridine incorporation, which peaked at~8 h p.i. DBT-A1DC cells did not show any detectable level of incorporation of the radioactivity. These results suggest that hnRNP A1 regulates MHV RNA synthesis. We further assessed the production of genomic and subgenomic MHV RNAs in these cell lines by northern blot analysis. The genomic and the six subgenomic RNA species were detected at 8 h p.i. in both DBT-VEC and DBT-A1 cells; there were signi®cantly higher steady-state levels of all of the RNA species in DBT-A1 cells ( Figure 5B ). In contrast, no viral RNA was detected in DBT-A1DC cells at that time point. At 16 h p.i., MHV RNA levels in DBT-VEC and DBT-A1 cells decreased generally because of the loss of the dead cells, while the smaller subgenomic RNAs became detectable in DBT-A1DC cells. By 24 h p.i., most viral RNA species became detectable in DBT-A1DC cells ( Figure 5B , lane 10), while most of the DBT-A1 cells were dead (lane 9). These results con®rmed that the synthesis of all of the viral RNA species is accelerated by overexpression of the wt hnRNP A1 and delayed by a dominant-negative mutant of hnRNP A1.\nIn this analysis, we also detected an additional RNA species (arrow in Figure 5B ), which was determined to be a defective-interfering (DI) RNA by northern blot analysis using a probe representing the 5¢-untranslated region (without the leader), which is present only in genomic and DI RNAs (data not shown). Interestingly, this DI RNA was inhibited to a greater extent than other RNA species in DBT-A1DC cells. This result suggests that the replication of DI RNAs is more sensitive to the dominant-negative inhibition by cytoplasmic hnRNP A1.\nTo demonstrate further that MHV RNA transcription machinery is defective in cells expressing the mutant hnRNP A1, we studied transcription of an MHV DI RNA, 25CAT, which contains a transcription promoter (derived from the IG sequence for mRNA 7, IG7) and a chloramphenicol acetyltransferase (CAT) reporter gene . CAT activity can be expressed from At 1 h p.i., serum-free medium was replaced by virus growth medium containing 1% NCS and 5 mg/ml actinomycin D. [ 3 H]uridine (100 mCi/ml) was added to the infected cells at 2, 3, 4, 5, 6, 7, 8, 9, 16 and 24 h p.i. After 1 h labeling, cytoplasmic extracts were prepared and precipitated with 5% TCA. The TCA-precipitable counts were measured in a scintillation counter. (B) Northern blot analysis of MHV genomic and subgenomic RNA synthesis in DBT cells. Cytoplasmic RNA was extracted from MHV-A59-infected cells at 8, 16 and 24 h p.i. for northern blot analysis. The naturally occurring DI RNA of MHV-A59 is indicated by an arrow. this DI RNA only if a subgenomic mRNA containing CAT sequences is produced . The 25CAT RNA was transfected into MHV-A59-infected cells 1 h after infection. At 8 h p.i., CAT activity in DBT-A1 cells was signi®cantly higher than that in DBT-VEC cells ( Figure 6A ). On the other hand, CAT activity was very low in DBT-A1DC cells. At 24 h p.i., CAT activity in DBT-A1 cells became slightly lower than that in DBT-VEC cells because of the loss of the dead DBT-A1 cells. The CAT activity in DBT-A1DC was still signi®cantly lower than that in DBT-VEC or DBT-A1 cells. These results established that mRNA transcription from the DI RNA was also inhibited by hnRNP A1DC.\nThe results shown above ( Figure 5B ) also suggest that DI RNA replication is more sensitive to the inhibitory effects of the hnRNP A1 mutant. To con®rm this result, we further studied replication of another DI RNA during serial virus passages. DBT cells were infected with MHV-A59 and transfected with DIssE RNA derived from JHM virus (Makino and Lai, 1989) ; the virus released (P0) was passaged twice in DBT cells to generate P1 and P2 viruses. DBT cells were infected with these viruses, and cytoplasmic RNA was extracted for northern blot analysis using glyoxalated RNA for a better resolution of smaller RNAs. For DBT-A1DC cells, RNA was extracted at 36 h p.i. since viral RNA synthesis was delayed in this cell line. Cells infected with P0 viruses did not yield detectable amounts of DIssE, but contained the naturally occurring A59 DI RNA, whose replication was inhibited more strongly than the synthesis of MHV genomic and subgenomic RNAs in DBT-A1DC cells ( Figures 5B, lanes 8±10 and 6B, lanes 1± 3). However, this A59 DI RNA was not detectable in cells infected with P1 and P2 viruses ( Figure 6B , lanes 4±9). In contrast, DIssE appeared in cells infected with P1 viruses and further increased in cells infected with P2 viruses, indicating that the replication of the smaller DIssE may have an inhibitory effect on the replication of the larger A59 DI RNA (Jeong and Makino, 1992) . Similar to the A59 DI RNA, the replication of DIssE RNA was much more strongly inhibited than that of MHV genomic and subgenomic RNAs in DBT-A1DC cells ( Figure 6B , lanes 6 and 9). Our results thus suggest that MHV DI RNA replication is more dependent on the function of cytoplasmic hnRNP A1.\nThe mechanism of dominant-negative inhibition by the C-terminal deletion mutant of hnRNP A1 To understand the underlying mechanism of the inhibition of MHV RNA transcription by the C-terminal-deletion mutant of hnRNP A1, we ®rst examined the RNA-and protein-binding properties of this mutant protein.\nElectrophoretic mobility shift assay demonstrated that hnRNP A1DC retained the ability to bind the MHV (±)strand leader RNA and to form multimers with itself, similar to the wt hnRNP A1 (data not shown); this is consistent with the fact that both of its RBDs are intact ( Figure 1A) . Furthermore, UV-crosslinking experiments showed that increasing amounts of puri®ed glutathione S-transferase (GST)±hnRNP A1DC ef®ciently competed with the endogenous hnRNP A1 for the binding of the MHV (±)-strand leader RNA ( Figure 7A ), indicating that the binding of hnRNP A1DC to RNA was not affected. These results suggest that the RNA-binding properties of hnRNP A1DC were intact.\nWe next examined the protein-binding properties of hnRNP A1DC. Since hnRNP A1 has been shown to interact with the N protein, which also participates in MHV RNA synthesis (Compton et al., 1987; Wang and Zhang, 1999) , we ®rst determined whether the dominantnegative mutant of hnRNP A1 retained the ability to interact with the N protein in vitro. GST pull-down assay using various truncation mutants of hnRNP A1 showed that the N protein bound the N-terminal domain (aa 1±163) of hnRNP A1 ( Figure 7B) ; thus, the binding of hnRNP A1DC [equivalent to hnRNP A1(1±245)] to the N protein was not affected. We next examined the in vivo interaction of the wt and mutant hnRNP A1 with an MHV ORF 1a product, p22, which has been shown to co-localize with the de novo synthesized viral RNA (S.T.Shi and The viruses were passaged twice in wt DBT cells to obtain P1 and P2 viruses. Cytoplasmic RNA was extracted from the DBT cells infected with P0, P1 and P2 viruses and treated with glyoxal before electrophoresis and northern blot analysis using a 32 P-labeled (±)-strand mRNA 7 as a probe. The A59 DI RNA and DIssE RNA are indicated by arrows.\nM.M.C.Lai, unpublished results) and associate with the viral replicase complex (Gibson Bost et al., 2000) . Cytoplasmic extracts from MHV-A59-infected cells were immunoprecipitated with anti-Flag antibody-conjugated beads, followed by western blotting with a rabbit polyclonal antibody against p22. At 8 h p.i., p22 was co-precipitated with the Flag-tagged hnRNP A1 from DBT-A1 cells, whereas no precipitation of p22 was observed in DBT-VEC cells ( Figure 7C ). For DBT-A1DC cells, co-immunoprecipitation was performed at 24 h p.i., when abundant MHV proteins were synthesized. p22 was shown to co-precipitate with hnRNP A1DC, indicating that hnRNP A1DC still formed a complex with the viral polymerase gene product. These results suggest that the ability of hnRNP A1DC to interact with the N and polymerase proteins was not altered.\nWe next investigated whether the mutant hnRNP A1 is de®cient in the interaction with any other cellular proteins in this RNA±protein complex. We labeled proteins in MHV-infected cells or mock-infected cells at different time points after infection and immunoprecipitated with the anti-Flag antibody. Signi®cantly, a cellular protein of 250 kDa was shown to be associated only with the wt hnRNP A1, but not the mutant hnRNP A1 ( Figure 7D ), suggesting that hnRNP A1 binds to this protein through its C-terminal domain. We propose that this cellular protein is another important component of the MHV RNA transcription/replication complex.\nThere is an accumulating body of evidence signifying the importance of cellular factors in RNA synthesis of RNA viruses (reviewed by Lai, 1998) . Previous studies have shown that hnRNP A1 binds to the cis-acting sequences of MHV template RNA and that this interaction correlates with the transcription ef®ciency of viral RNA in vivo (Zhang and Lai, 1995; Li et al., 1997) . In addition, hnRNP A1 is also implicated in viral RNA replication by the recent ®nding that hnRNP A1 interacts with the 3¢-ends of both positive-and negative-strand MHV RNA (P.Huang and M.M.C.Lai, unpublished results). However, hnRNP A1 modulates cytoplasmic viral RNA synthesis the functional importance of hnRNP A1 in viral RNA synthesis has so far not been directly demonstrated. In the present study, we established that MHV RNA transcription and replication were enhanced by overexpression of the wt hnRNP A1 protein, but inhibited by expression of a dominant-negative hnRNP A1 mutant in DBT cell lines.\nOur results suggest that hnRNP A1 is a host protein involved in the formation of a cytoplasmic transcription/ replication complex for viral RNA synthesis. This represents a novel function for hnRNP A1 in the cytoplasm.\nOur results indicate that the inhibitory effects on MHV replication exhibited by the dominant-negative mutant of hnRNP A1 were relatively more prominent than the enhancement effects by overexpression of the wt hnRNP A1. This is consistent with the subcellular localization patterns of the wt and mutant hnRNP A1 proteins. The overexpressed exogenous wt hnRNP A1 in DBT-A1 cells was predominantly localized in the nucleus, similar to the endogenous hnRNP A1 ( Figure 1C ). The C-terminal-deletion mutant, however, was localized mainly in the cytoplasm. Thus, the level of hnRNP A1DC was much higher than the endogenous wt hnRNP A1 in the cytoplasm of DBT-A1DC cells, where MHV replication occurs. This result explains why hnRNP A1DC could have a strong dominant-negative inhibitory effect, despite the fact that it was expressed at a lower level than the endogenous hnRNP A1 ( Figure 1B) .\nThe effects of the expression of the wt and mutant hnRNP A1 on virus production ( Figure 2B ), viral protein synthesis ( Figure 4A ) and viral RNA synthesis ( Figure 5A ) correlated with each other. Furthermore, hnRNP A1DC caused not only a global inhibition of genomic RNA replication and subgenomic mRNA transcription, but also a preferential inhibition of at least two DI RNA species. These results suggest that the inhibition of MHV replication by the hnRNP A1 mutant was most likely a direct effect on viral RNA synthesis rather than an indirect effect on other aspects of cellular or viral functions. Since hnRNP A1 binds directly to the cis-acting MHV RNA sequences critical for MHV RNA transcription (Li et al., 1997) and replication (P. Huang and M.M.C.Lai, unpublished results) , it is most likely that hnRNP A1 may participate in the formation of the transcription/replication complex. Indeed, our data show that hnRNP A1 interacts directly or indirectly with the N protein and a gene 1 product, p22, both of which are probably associated with the viral transcription/replication complex (Compton et al., 1987; Wang and Zhang, 1999; Gibson Bost et al., 2000) . hnRNP A1 may participate directly in viral RNA synthesis in a similar role to that of transcription factors in DNAdependent RNA synthesis, e.g. by maintaining favorable RNA conformation for RNA synthesis. Alternatively, hnRNP A1 may modulate MHV RNA transcription or replication by participating in the processing, transport and controlling the stability of viral RNAs. It has been reported that RNA processing of retroviruses, human T-cell leukemia virus type 2 (Black et al., 1995) and HIV-1 (Black et al., 1996) , is altered by the binding of hnRNP A1 to the viral RNA regulatory elements. It is also possible that hnRNP A1 may participate in MHV RNA synthesis indirectly by affecting the production of other host cell proteins, which may, in turn, regulate MHV RNA synthesis. Since hnRNP A1 is a dose-dependent altern-ative splicing factor (Caceres et al., 1994) , even small changes in the intracellular level of hnRNP A1 can alter the splicing of other cellular proteins. Regardless of the mechanism, our study established the importance of cellular factors in viral RNA-dependent RNA synthesis.\nThe transcription from 25CAT RNA was strongly inhibited by the dominant-negative mutant of hnRNP A1, as shown by CAT assays ( Figure 6A ). In addition, the replication of the naturally occurring A59 DI RNA and the arti®cial DIssE RNA was completely abolished ( Figure 6B) . Surprisingly, the replication of MHV DI RNAs suffered a stronger inhibition by the dominantnegative mutant of hnRNP A1 than the synthesis of MHV genomic and subgenomic RNAs, suggesting that DI RNA replication may be more dependent on hnRNP A1. Although DI RNAs contain all of the cis-acting replication signals that are essential for their replication in normal cells (Kim and Makino, 1995) , the small size of DI RNA may cause it to require more hnRNP A1 to maintain a critical RNA structure. It has been shown that different DI RNAs require different cis-acting signals for RNA replication (Kim and Makino, 1995) .\nOur results demonstrate that the C-terminal domain of hnRNP A1, including the M9 sequence and the glycinerich region, is important for MHV RNA transcription and replication, but the mechanism of the dominant-negative effects of hnRNP A1DC is still not clear. hnRNP A1DC retains the RNA-binding and self-association ability and is capable of binding the viral proteins N and p22, which are associated with the transcription/replication complex. It is possible that hnRNP A1DC is not productive due to its inability to interact with other viral or cellular proteins that are involved in MHV RNA synthesis. We have found a protein of~250 kDa that binds only the wt, but not the mutant hnRNP A1 ( Figure 7D ). It remains to be shown whether this cellular protein is involved in MHV RNA synthesis.\nIn our preliminary study, we found that MHV could replicate in an erythroleukemia cell line, CB3, which was reported to lack detectable hnRNP A1 expression as a result of a retrovirus integration in one allele and loss of the other allele (Ben-David et al., 1992) . Since hnRNP A1 protein is involved in a variety of important cellular functions, including RNA splicing, transport, turnover and translation, it is conceivable that other redundant gene products may substitute for the function of hnRNP A1 in CB3 cells. Indeed, UV-crosslinking assays using CB3 cell extracts detected two proteins comparable to hnRNP A1 in size that could interact with the MHV negative-strand leader RNA (data not shown). These proteins may represent hnRNP A1-related proteins, since many of such hnRNPs exist in the cells (Buvoli et al., 1988; Burd et al., 1989) . Therefore, multiple cellular proteins may have the capacity to be involved in MHV RNA synthesis.\nBased on previous ®ndings (Kim and Makino, 1995; Zhang and Lai, 1995; Li et al., 1997) and the results from this study, we propose a model for the regulation of transcription/replication of MHV RNA by hnRNP A1. We hypothesize that hnRNP A1 is one of the components of the MHV RNA transcription or replication complex, and the crosstalk between hnRNP A1 and another viral or cellular RNA-binding protein (designated X in Figure 8 ) is essential for MHV replication and transcription. The X protein binds to the C-terminus of hnRNP A1 and cooperates with hnRNP A1 to recruit more proteins to form the transcription or replication complex. The C-terminaldeletion mutant of hnRNP A1 loses the ability to interact with the X protein and to bring it into the initiation complex, resulting in an inhibition of MHV RNA transcription and replication. The residual replication and transcription activities of MHV RNA in the absence of functional hnRNP A1 may be due to a limited af®nity of the X protein to a cis-acting signal that is only present in MHV genomic RNA (site B). On the other hand, DI RNAs may lack this cis-acting signal. When the crosstalk between the X protein and hnRNP A1 is abolished by the dominant-negative mutant of hnRNP A1, the X protein can no longer participate in the formation of the initiation complex, resulting in a complete loss of DI RNA replication.\nIn summary, our data provide direct experimental evidence that hnRNP A1 is involved directly or indirectly in MHV RNA synthesis, probably by participating in the formation of an RNA transcription/replication complex. This ®nding reveals a novel cytoplasmic function for hnRNP A1.\nCells and viruses DBT cells, a mouse astrocytoma cell line (Hirano et al., 1974) , were cultured in Eagle's minimal essential medium (MEM) supplemented with 7% newborn calf serum (NCS) and 10% tryptone phosphate broth. MHV strain A59 (Robb and Bond, 1979) was propagated in DBT cells and maintained in virus growth medium containing 1% NCS.\nPlasmid construction and establishment of DBT stable cell lines The cDNA of the murine hnRNP A1 gene was ampli®ed by RT±PCR using RNA extracted from DBT cells and a set of primers representing the 5¢-and 3¢-ends of hnRNP A1-coding region, and cloned into pcDNA3.1 (Invitrogen, Carlsbad, CA). The 8 amino acid Flag tag was attached to the N-terminus of hnRNP A1 by including the Flag tag in the forward PCR primer. The truncated hnRNP A1DC was similarly constructed using a PCR-ampli®ed fragment that represents hnRNP A1 (aa 1±245).\nFor the establishment of permanent DBT cell lines, pcDNA3.1 alone or the plasmid containing the Flag-tagged hnRNP A1 or hnRNP A1DC was transfected into 60% con¯uent DBT cells using DOTAP according to the manufacturer's instructions (Boehringer Mannheim, Indianapolis, IN) . After 4 h, the transfected cells were selected in DBT cell medium containing 0.5 mg/ml Geneticin (G418) (Omega Scienti®c, Tarzana, CA) for 10 days. Single colonies were then collected and cultured individually for 10 additional days before screening for the expression of Flag-tagged proteins.\nThe polyclonal rabbit antibody against p22 was a gift from Dr Susan C.Baker at Loyola University, IL. The chicken polyclonal antibody against hnRNP A1 was produced by Aves Labs, Inc. (Tigard, OR) by immunizing chickens with the puri®ed mouse hnRNP A1 protein expressed in bacteria. The polyclonal anti-Flag antibody was purchased from Af®nity Bioreagents (Golden, CO). The goat polyclonal antibody against actin was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The mouse monoclonal antibody against the N protein has been described previously (Fleming et al., 1983) .\nExamination of growth rate of permanent DBT cells Equal numbers (1 3 10 5 ) of DBT-VEC, DBT-A1 and DBT-A1DC cells were plated in 10-cm culture plates and maintained in culture medium for 4 days. Cells were trypsinized, stained with Trypan Blue (Gibco-BRL, Grand Island, NY) and counted at 24-h intervals with a hemacytometer (Hausser Scienti®c, Horsham, PA).\nPlaque assay DBT cells in 10-cm plates were infected with MHV-A59 at an m.o.i. of 2. After 1 h for virus adsorption, the cells were washed three times with serum-free MEM, which was then replaced with virus growth medium containing 1% serum. At 1, 6, 8, 10, 14 and 24 h p.i., 1 ml of medium was taken from each plate for plaque assay.\n[ 3 H]uridine labeling of MHV RNA Cells plated in 6-well plates were infected with MHV-A59 at an m.o.i. of 2. At 1 h p.i., 5 mg/ml actinomycin D was added to the virus growth medium to inhibit cellular RNA synthesis. To label newly synthesized MHV RNA, 100 mCi/ml of [ 3 H]uridine (NEN, Boston, MA) were added to the medium at hourly intervals. After 1 h of labeling, the cells were washed twice in ice-cold PBS and scraped off the plates in 1 ml of PBS. The cells were then collected by centrifugation and incubated in 200 ml of NTE buffer (150 mM NaCl, 50 mM Tris pH 7.5, 1 mM EDTA) containing 0.5% NP-40, 0.5 mM dithiothreitol (DTT) and 400 U/ml of RNasin on ice for 15 min. After centrifugation, 5 ml of the cytoplasmic extract were spotted on a piece of 3 mm paper and incubated with 5% trichloroacetic acid (TCA). The radioactivity remaining on the 3 mm paper was measured in a scintillation counter.\nNorthern blot analysis DBT cells were infected with MHV-A59 at an m.o.i. of 2. At 8, 16 and 24 h p.i., cytoplasmic extract was prepared as described above and subjected to phenol/chloroform extraction and ethanol precipitation to purify cytoplasmic RNA. Approximately 10 mg of RNA were separated by electrophoresis on a 1.2% formaldehyde-containing agarose gel and transferred to a nitrocellulose membrane. For a better resolution of the DIssE RNA ( Figure 6B ), RNA was glyoxalated before being electrophoresed on a 1% agarose gel. An in vitro transcribed, 32 P-labeled negative-strand mRNA 7 of MHV-JHM was used as a probe to detect MHV genomic and subgenomic RNAs. For detecting DI RNA species, RNA blots were probed with an RNA representing a sequence complementary to the sequence of the 5¢-untranslated region of MHV-JHM RNA, but excluding the leader sequence.\nWestern blot analysis DBT cells in 6-well plates were infected with MHV-A59 and cytoplasmic extracts were prepared as described previously (Li et al., 1997) at various hnRNP A1 modulates cytoplasmic viral RNA synthesis time points p.i. The extracts were electrophoresed on a 12% polyacrylamide gel and transferred to a nitrocellulose membrane for western blotting.\nImmuno¯uorescence staining Cells were washed in phosphate-buffered saline (PBS) and ®xed in 4% formaldehyde for 20 min at room temperature, followed by 5 min in ±20°C acetone. Primary antibodies were diluted in 5% bovine serum albumin and incubated with cells for 1 h at room temperature. After three washes in PBS,¯uorescein-conjugated secondary antibodies were added to cells at 1:200 dilution for 1 h at room temperature. FITC-or TRITCconjugated secondary antibodies were used to generate green or red uorescence. Cells were then washed in PBS and mounted in Vectashield (Vector Laboratories, Burlingame, CA).\nUV-crosslinking assay UV-crosslinking assay was performed as described previously (Huang and Lai, 1999) . In brief, DBT cell extracts (30 mg protein), 200 mg/ml tRNA and 10 4 c.p.m. of an in vitro transcribed, 32 P-labeled negativestrand MHV 5¢-end RNA (182 bp) were incubated for 10 min at 30°C. Increasing amounts of puri®ed GST (0, 0.5, 1.5 and 5 ng) or recombinant GST±hnRNP A1 fusion protein (0, 1, 3 and 10 ng) were included in the reaction to compete with the endogenous hnRNP A1 for binding. The reaction mixture was placed on ice and UV-irradiated in a UV Stratalinker 2400 (Stratagene) for 10 min, followed by digestion with 400 mg/ml RNase A for 15 min at 37°C. The protein±RNA complexes were then separated on a 10% SDS±polyacrylamide gel and visualized by autoradiography.\nGST pull-down assay GST pull-down was performed as described previously (Tu et al., 1999) . In brief, GST±hnRNP A1 fusion proteins on glutathione beads (Pierce, Rockford, IL) were incubated with the in vitro translated, 35 S-labeled N protein in 0.3 ml of GST-binding buffer containing 0.1% NP-40 for 2 h at 4°C. The beads were washed ®ve times with the GST-binding buffer containing 0.3% NP-40. Proteins bound to beads were eluted by boiling in Laemmli buffer for 5 min and separated on a 10% polyacrylamide gel.\n[ 35 S]methionine labeling and immunoprecipitation DBT cells were infected with MHV-A59 at an m.o.i. of 2. The cells were incubated with methionine-free medium for 30 min before labeling and were labeled in 100 mCi/ml [ 35 S]methionine starting at 1.5, 7 or 24 h p.i. After labeling for 2 h at each time point, the cells were harvested for protein extraction as described previously (Li et al., 1997) . The protein extracts were immunoprecipitated with anti-Flag antibody-conjugated beads (Sigma, St Louis, MO) in Tm 10 buffer (50 mM Tris±HCl pH 7.9, 0.1 M KCl, 12.5 mM MgCl 2 , 1 mM EDTA, 10% glycerol, 1 mM DTT, 0.1% NP-40, 1 mM phenylmethylsulfonyl¯uoride) at 4°C for 2 h. The immunoprecipitates were washed and separated on a 4±15% gradient SDS±polyacrylamide gel and visualized by autoradiography.\nPlasmid 25CAT was linearized by XbaI and in vitro transcribed with T7 RNA polymerase to produce the DI RNA . The DI RNA was transfected into MHV-A59-infected DBT cells using DOTAP as described previously (Huang and Lai, 1999) . In brief,~80% con¯uent DBT cells were infected by MHV-A59 at an m.o.i. of 10. At 1 h p.i., the cells were transfected with 5 mg of in vitro transcribed DI RNA and incubated at 37°C for the desired lengths of time. To amplify the DI RNA, viruses (P0) were passaged twice in wt DBT cells to generate P1 and P2 viruses.\nCells were harvested at 8 or 24 h p.i. and lysed by freezing and thawing for three times. After centrifugation at 12 000 r.p.m. for 10 min, the supernatant was used in a CAT assay as described previously (Lin et al., 1996) ." }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: p62 is a novel cellular protein which was initially identified in humans as a phosphotyrosine independent ligand of the src homology 2 (SH2) domain of p56 lck (1, 2) . p56 lck is a member of the c-src family of cytoplasmic tyrosine kinases that is found predominantly in cells of lymphoid origin (3, 4) . In addition to the interaction with p56 lck , p62 also associates with the Ser/Thr kinase (1, 2) , atypical protein kinase C (5, 6) , and ubiquitin (7) . In addition to the SH2 domain, p62 possesses several structural motifs, including a ubiquitin associated (UBA) domain that is capable of binding ubiquitin nonconvalently (8, 9) . Ubiquitin (Ub) is a small polypeptide of 76 amino acids that can be convalently attached to other proteins at specific lysine residues through chains composed of one (mono) or several ubiquitin moieties (poly). In addition to its classical role in protein degradation, ubiquitin is emerging as a signal for protein transport and processing (10) (11) (12) . Conjugation of ubiquitin to substrate proteins requires three enzymes: a ubiquitin activating enzyme E1, a ubiquitin-conjugating enzyme E2, and a ubiquitin ligase E3. Initially, E1 activates ubiquitin by forming a high energy thioester intermediate with the C-terminal glycine using ATP. The activated ubiquitin is sequentially transferred to E2, then to E3 which catalyzes isopeptide bond formation between the activated C-terminal glycine of ubiquitin and ε-amino group of a lysine residue of the substrate. Following the linkage of the first ubiquitin chain, additional molecules of ubiquitin are attached to lysine side chains of the previously conjugated moiety to form branched polyubiquitin chains. The fate of ubiquitinated substrates depends on the number of ubiquitin moieties conjugated, as well as, the lysine linkage of Ub-Ub conjugation. The conjugation of ubiquitin to eukaryotic intracellular proteins is one way in which those proteins are targeted to the proteasome for subsequent rapid degradation. This mechanism is particularly important for short-lived regulatory proteins such as cyclins, cyclin-dependent protein kinase-inhibitors, p53, the nuclear factor kappa B precursor, and IκB (13) . The ubiquitinproteasome system consists of two steps: 1) the target protein is conjugated with polyubiquitin molecules, which mark the substrate for degradation; 2) the target protein is transferred to the 26S proteasome, unfolded and degraded.\nThe UBA domain is a conserved sequence motif among proteins that can bind polyubiquitin. It is comprised of ~45 amino acids (13) . The amino acids 386-434 of p62, which bind polyubiquitin, has been shown to possess homology to other recently described UBA domains (9) . Interestingly, proteins with UBA domains are more likely to bind polyubiquitin chains over monoubiquitin, such as the yeast UBA protein Rad23, a highly conserved protein involved in nucleotide excision repair (13) . Recently, it has been shown that yeast cells lacking two UBA proteins (Dsk2 and Rad23) are deficient in protein degradation and that the UBA motif is essential for their function in proteolysis (14) .\nIn addition to the important role in recycling of amino acids from damaged or misfolded proteins, ubiquitin-protein conjugation also has functions unrelated to proteasomal targeting. For example, polyubiquitination is required for the internalization of several yeast and mammalian cell surface proteins into the endocytic pathway (15, 16) . Interestingly, p62 appears to sequester ubiquitinated substrates into a cytoplasmic structure referred to as a sequestosome, into which excess ubiquitinated proteins are segregated (17) . In addition, p62 is an immediate early response gene product for a variety of signals (18) . Thus, p62 appears to play a novel regulatory role for polyubiquitinated proteins and may have an essential function in cell proliferation and differentiation. We have developed a method that will enable identification of protein(s) that interact with p62's UBA domain.\nHuman adult brain library 10×96 well plates with 100 cDNAs per well and Gold TNT SP6 \nTo search for novel proteins that bind to the UBA domain of p62, we performed in vitro expression cloning (IVEC) using the ProteoLink IVEC system. The human adult brain library 96 well plates with 100 cDNAs per well was transcribed and translated employing the Gold TNT SP6 Express 96 plate and [ 35 S] methionine. The TNT Quick-coupled transcription-translation system contained a rabbit reticulocyte lysate pre-mixed with most of the reaction components necessary to carry out transcription/translation in the lysate, including all of the amino acids except methionine. [ 35 S] Methionine was used to label newly synthesized proteins. The reactions were set up according to the manufacturer's instructions. Rabbit reticulocyte lysate has been shown to be capable of carrying out ubiquitination of proteins that were translated in such an in vitro translation system (19, 20) . The reactions mixtures also contained ubiquitin so that the newly synthesized proteins could be ubiquitinated. The reactions were incubated at 30°C for 2 hours. The resulting proteins were assayed to determine their binding ability with p62's UBA domain. Potential positive \"hits\" were further subdivided and reassayed to link individual clones to the protein of interest (Fig. 1 ). \nEach translated pool was resuspended in binding buffer (25 mM Tris pH 7.5, 125 mM NaCl, 0.1% NP-40) and used as a source of protein in p62 UBA pull down assays. Proteins that specifically interact with the UBA domain of p62 were isolated by interaction with agarose-immobilised p62-UBA peptide (amino acid 387-436 of p62) (5 µg) for 2 hours at 4ºC, then washed three times in washing buffer (25 mM Tris pH 7.6, 100 mM NaCl, 1% NP-40). Bound proteins were released by addition of SDS-sample buffer and separated by SDS-PAGE. The SDS-PAGE gels were fixed in 50% methanol, 10% acetic acid for 30 min, stained in 0.2% Commassie Brilliant Blue R-250, 45% methanol, 10% acetic acid for 15 min, destained in 10% acetic acid, 50% methanol overnight, and enhanced in autoradiography enhancer En 3 HANCE for 1 hr and exposed to X-Ray film.\nBy combining 4 pools as one mixed pool, 96 protein pools were divided into 24 mixed protein pools for use in p62 UBA pull down assays. Positive mixed protein pools were selected and individual pools were retested for its ability to bind p62's UBA domain. The individual cDNA pool from which the positive protein pool was generated was transformed into JM109 competent cells and plated on LB ampicillin plate. Individual colonies were chosen to grow overnight in 1 ml of LB media plus ampicillin. Plasmid DNA was purified from the cell culture and used for TNT Quick coupled in vitro transcription/ translation. The individual protein synthesized from each plasmid DNA chosen was screened for its ability to bind p62's UBA domain.\nTo confirm the interaction with p62's UBA domain, the final resulting individual proteins were used in the coupled TNT/p62 UBA pull down assays. The cDNA inserts were sequenced in the Genomics Core Facility at Auburn University and the sequences were compared with known sequences in NCBI database by BLAST analysis.\nHuman embryonic kidney 293 (HEK 293) cells were cultured in high glucose Dulbecco's modified Eagle's medium (DMEM) containing 10% heat-inactivated fetal calf serum and transfected with myc-tagged HSP70 plasmid using the Mammalian Cell Transfection Kit. Cells were harvested and lysed in 1 ml of SDS lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10 mM NaF, 0.5% TX-100, 1 mM Na 3 VO 4 , 2 µg/ml aprotinin, 2 µg/ml leupeptin, 1 mM PMSF, 1% SDS) for 30 min on ice, followed by centrifugation at 14000 rpm for 15 min at 4°C to remove the insoluble fraction. The protein concentration of the supernatant was determined using the Bio-Rad DC protein assay reagent with bovine serum albumin (BSA) as standard. Equal amount of protein (750 µg) was immunoprecipitated with anti-myc and collected with agarose-coupled secondary antibody. To the agarose beads containing the immunoprecipitated HSP70, 50 µl of reaction buffer (50 mM Tris-HCl pH 7.5, 2.5 mM MgCl 2 , 2 mM DTT, 2 mM ATP) was added containing 100 ng E1, 200 ng E2 (UbcH7), and 100 µg of E3 (Flag-tagged TRAF6) along with 5 µg GST-WT-Ub, GST-K29R Ub, GST-K48R Ub, GST-K63R Ub, or K63 Ub. Control samples without HSP70, E1, E2, E3, or GST-WT-Ub were also included. Reactions were carried out by continuous shaking at 37°C for 2 hours and then washed three times with reaction buffer. The proteins were released by boiling for 2 min in SDS-PAGE sample buffer, separated on 7.5% SDS-PAGE and Western blotted for anti-ubiquitin.\nTo search for novel proteins that bind to the UBA domain of p62, we performed in vitro expression cloning (IVEC) using the ProteoLink IVEC system from Promega (Cat. No. L6500). The human adult brain library 96 well plates with 100 cDNAs per well were transcribed and translated employing the Gold TNT SP6 Express 96 plate in the presence of [ 35 S] methionine and ubiquitin (25 µg/µl, Sigma). By combining 4 protein pools as one mixed pool, 96 protein pools were divided into 24 mixed pools ( Fig. 2A, 2B ). Each lane contained more than 100 proteins (theoretically 400) with different molecular weight. Therefore, each lane appeared as a smear, indicating that the in vitro transcription/translation system from Promega worked successfully. In order to examine whether proteins synthesized in the IVEC system are also ubiquitinated, Western blot analysis was performed by blotting the newly synthesized proteins (in the presence of cold methionine instead of 35 S methionine) with ubiquitin monoclonal antibody. In the mixed protein pools, each of the 24 lanes appeared as a smear, indicating that proteins synthesized by the IVEC system are also ubiquitinated (Fig. 3A) . Furthermore, the rabbit reticulocyte lysate in the IVEC system can utilize different lysine linkages of ubiquitin (i.e., Ub K29, Ub K48, and Ub K63) for ubiquitination (Fig. 3B) . In order to investigate whether the agarose-immobilised p62 UBA peptide has binding specificity, a mixed protein pool synthesized by IVEC system was tested in a pull down assay in the presence of agarose beads alone or in the presence of p62 UBA agarose beads (Fig. 3C ). Our results revealed that proteins that bound to p62's UBA domain could not be pulled down by agarose beads alone, indicating that the agarose-immobilised p62 UBA peptide had binding specificity. \nIn order to identify proteins that bind to p62's UBA domain, p62 UBA pull down assays were performed. Out of the 24 mixed protein pools, several pools contained [ 35 S] methionine-labeled bands in the primary p62 UBA pull down assays (Fig. 4) . We chose 6 pools (pool # 2, 4, 8, 14, 20, 21) because of their stronger signal to specifically identify which individual protein pool in the mixed pools has the ability to bind to p62's UBA domain. Therefore, a secondary screen was conducted on the 6 positive individual mixed pools (representing 24 individual protein pools) which bound with p62's UBA domain (Fig. 5) . Mixed protein pool # 2 generated a positive protein with molecular weight of 51 KDa (Fig. 4) , and only individual protein pool \"c\" out of the four protein pools (a, b, c, d) that comprised protein pool #2 had a protein with the same molecular weight (Fig. 5) . Depending on the size of the protein pulled down in the secondary screen compared to the primary screen (Fig. 4) , individual protein pools \"c\", \"h\", \"i\", \"o\", \"t\", and \"v\" were identified (Fig. 5) . To specifically identify which protein in the individual protein pool has the ability to bind p62's UBA domain, the cDNAs from the positive individual protein pools were then transformed into JM109 competent cells and plated out on LB ampicillin plates. Individual colonies were chosen to grow overnight in 1 ml of LB media plus ampicillin. Plasmid DNAs were purified from the cell culture and used for TNT Quick coupled in vitro transcription/translation. The individual protein synthesized from each plasmid DNA was retested for its ability to bind p62's UBA domain. By synthesizing individual protein from individual plasmid using the Gold TNT Quick coupled in vitro transcription/translation system and subjecting them to p62 UBA pull-down assays, 11 positive clones were isolated from the 6 positive individual pools. It is not surprising that 5 more clones showed binding ability with p62's UBA domain since there are 100 cDNAs in each positive individual pool and some of them could have lower binding ability and therefore showed weak signal in the mixed protein pool. It is also possible that they are not as efficiently synthesized in the mixed TNT reaction as in the individual TNT reaction in which only one cDNA was used as template. The 11 positive plasmids were sequenced and compared with known cDNA sequences in NCBI database using BLAST analysis with results shown in Table 1 .\nInterestingly, the proteins identified in the screen fall into three distinct categories. One set are proteins that are associated with Alzheimer's disease, including myelin basic protein, 14-3-3 protein, syntaxin binding protein munc18, transketolase, heat shock protein HSP70, reelin, and calcium/calmodulin kinase II (Table 1 ). Significant decrease in the amount of myelin basic protein has been reported in the white matter of Alzheimer's disease patients, accompanied by increased quantities of βamyloid peptides (21) . The presence of β-amyloid peptides containing senile plaques and neurofibrillary tangles are the two major pathological features in the brain of patients with Alzheimer's disease (22) . Interestingly, 14-3-3 proteins have also been demonstrated to be components of neurofibrillary tangles of Alzheimer's disease brains (23) . Syntaxin binding protein munc18 can powerfully regulate amyloid precursor protein metabolism and β-amyloid secretion through direct and indirect interactions with X11 proteins (24) . The activity of transketolase has been reported to be reduced in dementia of Alzheimer's type brain (25) . Heat shock protein HSP70 expression is significantly increased in the temporal cortex of patients with Alzheimer's disease (26) . Besides HSP70, other heat shock proteins are also linked with Alzheimer's disease. For example, increased synthesis of HSP27 has been suggested to play a role in preventing neuronal injury in AD (27) , and alpha-crystallin heat shock protein has a close relationship with neurofibrillary tangles of AD brains (28) . Reelin is a large secreted protein that controls cortical layering by signaling through the very low density lipoprotein receptor and apolipoprotein E receptor 2, thereby inducing tyrosine phosphorylation of the adaptor protein Disabled-1 (Dab1) and suppressing tau phosphorylation (29) . Neurofibrillary tangles comprised of highly phosphorylated tau proteins are a key component of Alzheimer's disease (30) . Enhanced activity of calcium/calmodulin kinase II has been suggested to contribute to phosphorylation of tau protein and lead to neurofibrillary tangle deposition and neuronal death in Alzheimer's disease (31) . Although the relationship between p62 and neurofibrillary tangles or neuritic plaques is unclear, both neurofibrillary tangles and dystrophic neuritis of neuritic plaques are associated with ubiquitin (32) , suggesting that dysfunction in ubiquitin-mediated proteolysis and the resulting accumulation of ubiquitinconjugated proteins may contribute to the origination of dystrophic neuritis and neurofibrillary tangles. Furthermore, p62 has been recently reported to accumulate early in neurofibrillary tangles in Alzheimer's disease (33) , suggesting that p62 may play an important role in Alzheimer's disease by interacting with those proteins through its UBA domain.\nA second set of proteins identified in the screen that bind to p62's UBA domain are associated with brain development, including homeobox protein Meis2 and unc51 like kinase II (Table 1) . Although Meis proteins are not extensively studied in humans, these proteins have been shown to be required for hindbrain development in the zebrafish (34) . Unc51 like kinase II has been demonstrated to play a role in axonal elongation (35, 36) , which is needed for the formation of complicated neuronal networks. The third set of proteins that exhibit ability to bind p62's UBA domain are proteins that are linked with other neurodegenerative diseases, including FK506 binding proteins and nuclear receptor corepressor I (Table 1) . FK506 (tacrolimus) is a potent immunosuppressive drug used in the treatment of patients after organ transplantation and in selected autoimmune disorders (37) . FK506 is activated upon binding to members of the immunophilin family of proteins, which were designated as FK506 binding proteins (38) . Immunophilins are chaperone proteins and FK506 binding proteins have been suggested as therapeutics for neurological disorders (39, 40) . Nuclear receptor corepressor I has been suggested to play a role in Huntington's disease because it is able to interact with huntingtin (41) . The proteins identified here suggest that p62's UBA domain has the ability to interact with multiple proteins that play important roles in neurodegenerative diseases. Further screening from the whole genome-wide perspective will be necessary to define the important role that p62's UBA domain plays. \nIt has been reported that polyubiquitin chains assembled through lysine 48 of ubiquitin act as a signal for substrate proteolysis by the 26S proteasome (42) (43) (44) . In order to understand whether the proteins identified in our screen bind to the p62's UBA domain through lysine 48 (K48), polyubiquitin K48 chains were added to the p62 UBA pull down assay (Fig. 6) . Inclusion of polyubiquitin K48 chains in the assay should compete for the binding of substrate to the p62's UBA domain and reduce the interaction of those proteins with the p62's UBA domain if those proteins are assembled through K48 chains. An alternative interpretation for polyubiquitin K48 chain competition is that the ubiquitin chains are competing for the same binding site as the binding partners which are either ubiquitinated or non-ubiquitinated. We randomly chose five proteins out of the 11 binding partners for the competition pull down (Fig. 6) . Out of the five proteins, four proteins (# 2, 3, 4, and 5) showed reduced binding ability with p62's UBA domain when polyubiquitin K48 chains were included (Fig. 6A, 6B ). However, K48 chains failed to compete with HSP70, suggesting that p62's UBA domain binds to HSP70 through a ubiquitin lysine linkage other than K48. Interestingly, it has been reported that heat shock protein 70 cognate (HSP70) is ubiquitinated by CHIP (carboxyl terminus of Hsc70-interacting protein) via ubiquitin chain synthesis that uses either K29 or K63 (45) . In order to examine which lysine linkage utilized by HSP70 binds to p62's UBA domain, in vitro ubiquitination assay was performed by incubating lysates from HEK cells expressing HSP70 with E1, E2, and E3 in reaction buffer (50 mM Tris-HCl pH 7.5, 2.5 mM MgCl 2 , 2 mM DTT, 2 mM ATP). As control, the ubiquitination of HSP70 utilizing the rabbit reticulocyte lysate was also investigated by Western blot analysis. Our results revealed that HSP70 was ubiquitinated in the IVEC system (Fig. 7A, 7B) , and the rabbit reticulocyte lysate contained enzymes such as TRAF6 (E3) and UbcH7 (E2) for in vitro ubiquitination (Fig. 7C ). TRAF6 was chosen as an E3 in this in vitro ubiquitination assay due to its RING domain, a common feature of E3 ligases, and the observation that p62 is a scaffold for TRAF6 interaction (46) . Therefore, in vitro ubiquitination assays using the E1-E2-E3 system were performed in the presence of either ubiquitin wild type or ubiquitin mutants (K29R, K48R, and K63R). If one lysine mutant blocks the ubiquitination of HSP70, it would suggest that the ubiquitination of HSP70 utilizes that specific lysine linkage.\nOur results revealed that HSP70 utilizes K63 linkage to assemble polyubiquitin chains to bind to p62's UBA domain since only the K63R ubiquitin mutant blocked the ubiquitination of HSP70 (Fig. 8A) . A similar result was also observed when reactions were conducted with wild type ubiquitin or mutant ubiquitin with all lysines mutated to arginines except K63 and the ubiquitination of HSP70 occurred only in the reaction that has either intact K63 ubiquitin or wild type ubiquitin (Fig. 8B ). This finding is consistent with previous reports (45) , demonstrating that HSP70 is K63-polyubiquitinated. Furthermore, the in vivo interaction of HSP70 and p62 was confirmed by transfecting myc-tagged HSP70 into HEK 293 cells in the presence of the proteasome inhibitor MG132 and subjecting cell lysates to p62 immunoprecipitation and Western blot with anti-myc antibody (Fig. 8C) . The interaction between HSP70 and p62 in vivo took place only when MG132 was included, suggesting that the interaction in vivo is dependent upon the ubiquitination of HSP70. The specific type of polyubiquitin chain recognized by p62's UBA domain is not yet known and studies are underway lab to determine p62's interaction with specific polyubiquitin chains, however, our preliminary studies suggest that p62's UBA domain may recognize K63 linked polyubiquitin chains. Protein was synthesized employing TNT Quick Coupled in vitro transcription/translation system in the presence of ubiquitin, resolved on 10% SDS-PAGE gels, transferred to nitrocellulose membrane and western blotted with ubiquitin monoclonal antibody. B: HSP70 Protein was synthesized employing TNT Quick Coupled in vitro transcription/translation system in the presence of ubiquitin and 35 S-methionine, resolved on 10% SDS-PAGE and exposed to X-ray film. C: Western blot of rabbit reticulocyte lysate with TRAF6 (E3) and UbcH7 (E2). In summary, for the first time, we demonstrate a systematic approach to identify UBA domain binding proteins from a proteome wide perspective. This approach could be readily adapted to high throughput screening. Using the rabbit reticulocyte lysate in vitro expression cloning system, we have successfully identified eleven proteins in the human adult brain that interact with the UBA domain of p62, and the majority of the eleven proteins are associated with neurodegenerative disorders, such as Alzheimer's disease. This is a very interesting finding since 9600 cDNAs have been screened and only 11 of them showed binding specificity with p62's UBA domain. Studies are underway to unfold the functional roles of p62 in the ubiquitin system. Our approach provides an easy route to the characterization of UBA domain binding proteins at the level of the whole proteome, its application will unfold the important roles that p62's UBA domain plays. This method could be easily adapted to identify proteins that interact with other UBA domains as well." }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: Intracellular bacterial and viral pathogens have evolved numerous mechanisms to appropriate and exploit different systems of the host during their life cycles in order to facilitate their spread during entry and exit from the host (Cudmore et al., 1997; Finlay and Cossart, 1997; Dramsi and Cossart, 1998) . In the case of viruses, perhaps the best studied example is the exploitation of the actin cytoskeleton by vaccinia virus during its exit from infected cells (Cudmore et al., 1997) . Vaccinia virus is a large DNA virus with a genome of~191 kb encoding 260 open reading frames (ORFs) that is a close relative of variola virus, the causative agent of smallpox (Johnson et al., 1993; Massung et al., 1993) . Vaccinia virus morphogenesis is a complex process which occurs in the cytoplasm of infected cells and results in the formation of the intracellular mature virus (IMV) and the intracellular enveloped virus (IEV). IMV consist of a viral core of DNA and protein enveloped in a membrane cisterna derived from the intermediate compartment (Sodeik et al., 1993) . The IMV core contains ®ve major proteins, A3L, A4L, A10L, F17R and L4R (Vanslyke and Hruby, 1994; Jensen et al., 1996a) , while 12 proteins, A12L, A13L, A14L, A14.5L, A17L, A27L, D8L, G4L, G7L, H3L, I5L and L1R, are associated with the membranes around the virus particle (Jensen et al., 1996a; Betakova et al., 2000) . Depending on the virus strain and cell type, a proportion of IMV can become enwrapped by a membrane cisterna derived from the trans-Golgi apparatus to give rise to IEV particles (Schmelz et al., 1994) . To date, six IEV-speci®c proteins, A33R (Roper et al., 1996) , A34R (Duncan and Smith, 1992) , A36R (Parkinson and Smith, 1994) , A56R (Payne and Norrby, 1976; Shida, 1986) , B5R (Engelstad et al., 1992; Isaacs et al., 1992) and F13L (Hirt et al., 1986) , have been identi®ed. Studies using recombinant viruses have shown that A33R, A34R, B5R and F13L play an important role in IEV assembly (Blasco and Moss, 1991; Engelstad and Smith, 1993; Wolffe et al., 1993 Wolffe et al., , 1997 Roper et al., 1998; Sanderson et al., 1998a; Ro Èttger et al., 1999) . Vaccinia virus is thought to leave the cell by fusion of the outer IEV membrane with the plasma membrane, to give rise to the extracellular enveloped virus (EEV) (Morgan, 1976; Payne, 1980; Blasco and Moss, 1991) or the cell-associated enveloped viruses (CEV) which remain associated with the outer surface of the plasma membrane (Blasco and Moss, 1992) .\nDuring the complex vaccinia infection process, the actin cytoskeleton is dramatically reorganized and numerous actin comet-like tails are induced by IEV particles (Cudmore et al., 1995; Ro Èttger et al., 1999) . Using actin polymerization as the driving force, IEV particles are propelled on actin tails until they contact the plasma membrane and extend outwards, thereby facilitating infection of neighbouring cells (Cudmore et al., 1995) . In addition, vaccinia infection results in stimulation of cell motility, loss of contact inhibition and changes in cell adhesion (Sanderson and Smith, 1998; Sanderson et al., 1998b) . Vaccinia virus-induced cell motility can be subdivided further into cell migration and extension of neurite-like projections, the latter of which is dependent on microtubules (Sanderson et al., 1998b) . The dependence of neurite-like projection formation on microtubules suggests that the microtubule cytoskeleton may also play a role during the life cycle of vaccinia virus. Indeed, recently, the vaccinia A27L protein and microtubules have been shown to be required for ef®cient IMV dispersion (Sanderson et al., 2000) . Furthermore, in the absence of vaccinia actin-based motility, cell to cell spread still occurs although it is less ef®cient (Wolffe et al., 1997 Sanderson et al., 1998a) , suggesting that additional transport mechanisms must exist.\nGiven these observations, we wondered whether the microtubule cytoskeleton has a function during the life Vaccinia virus infection disrupts microtubule organization and centrosome function The EMBO Journal Vol. 19 No. 15 pp. 3932±3944, 2000 cycle of vaccinia virus. We now report that the microtubule cytoskeleton and the dynein±dynactin complex play an important role during the early stages of vaccinia infection. However, later during the infection cycle, loss of centrosome function and accumulation of viral-encoded microtubule-associated proteins (MAPs) result in a dramatic rearrangement of the microtubule cytoskeleton.\nVaccinia localization in the vicinity of the MTOC depends on microtubules and the dynein±dynactin complex Indirect immuno¯uorescence labelling shows that by 6 h post-infection the majority of vaccinia virus particles are concentrated in the area coinciding with the centre of the microtubule aster ( Figure 1A and C). To examine whether this localization is indeed microtubule dependent, we infected cells pre-treated with nocodazole to depolymerize microtubules. In the absence of microtubules, virus particles were distributed throughout the cytoplasm ( Figure 1B and D) . The accumulation of virus particles in the area around the centre of the microtubule aster suggested that a microtubule minus end-directed motor may be involved in establishing the position of the virus in this location. To examine this possibility, we infected cells overexpressing p50/dynamitin which acts as a dominantnegative for dynein±dynactin function (Echeverri et al., 1996) . We found in cells overexpressing p50/dynamitin that virus particles did not accumulate at the centre of the microtubule aster but rather throughout the cytoplasm, as occurs in the absence of microtubules (compare Figure 2B with Figure 1D ).\nAs vaccinia morphogenesis involves wrapping by host membranes, it was possible that the effects of nocodazole and p50/dynamitin on virus localization were in fact due to disruption of the intermediate compartment and Golgi apparatus by these reagents (Burkhardt et al., 1997) . However, two independent experiments showed that this is not the case. First, in cells infected in the absence of microtubules, the Golgi apparatus as well as vaccinia virus particles are dispersed throughout the cytoplasm but do not co-localize ( Figure 3F and O). Secondly, vaccinia particles remain in the vicinity of the microtubule-organizing centre (MTOC) when the Golgi but not the microtubules was disrupted by treatment with brefeldin A ( Figure 3G and P). Similar results were obtained using other markers: A17L for vaccinia, galactosyltransferase for the Golgi or ERGIC53 for the intermediate compartment (data not shown). Taken together, our data indicate that the microtubule cytoskeleton is required for the localization of newly assembled virus particles in the vicinity of the MTOC during vaccinia infection.\nFormation of functional IEV, but not IMV, is microtubule dependent Given the requirement for microtubules in vaccinia localization, we subsequently examined whether this localization has a role in morphogenesis of the two different intracellular forms of vaccinia virus, IMV and IEV. From electron microscopic examination of cells infected in the presence of nocodazole, it became clear that IMV particles which are morphologically indistinguishable from controls are formed ( Figure 4 ). Although IMV particles are assembled in the absence of microtubules, we wondered whether their number is reduced and whether those that are formed are infectious, since the integrity of the intermediate compartment depends on microtubules (Burkhardt et al., 1997) . To address this question, three independent virus stocks were prepared in the presence or absence of nocodazole. To simplify the interpretation of the data, we used the recombinant vaccinia virus mutant DF13L, which is unable to form IEV (Blasco and Moss, 1991) . The ®nal concentration of virus particles produced, Vaccinia uses and abuses the microtubule cytoskeleton as determined by the method of Joklik (1962) , was 30.2 6 5.2 3 10 10 particles/ml in the presence of microtubules and 9.0 6 6.7 3 10 10 particles/ml in the absence of microtubules. Although there is a 3-fold decrease in the number of virus particles formed in the absence of microtubules, the particles that are formed are infectious (data not shown).\nWhile infectious IMV are formed in the absence of microtubules, we found no evidence for IEV formation, based on electron microscope examination of cells infected in the presence of nocodazole ( Figure 4 ). We did, however, observe IMV particles partially wrapped in trans-Golgi membranes most probably in the process of abortive IEV formation ( Figure 4D ). Given these data, we examined by indirect immuno¯uorescence whether low amounts of IEV particles are formed in the absence of microtubules. However, we could ®nd no evidence for colocalization of the IEV protein markers A36R, A34R or A33R with vaccinia particles formed in the presence of nocodazole ( Figure 5F ). We also found no evidence for IEV formation, based on their ability to nucleate actin tails ( Figure 5O ). As IEV particle assembly involves wrapping by the Golgi apparatus (Schmelz et al., 1994) , we examined the effects of only disrupting this membrane compartment using brefeldin A. We could ®nd no evidence for IEV formation, based on co-localization of IEV protein markers with virus particles and actin tails in cells infected in the presence of brefeldin A ( Figure 5G±I and P±R). Indeed, in brefeldin A-treated cells, the IEV membrane proteins required for assembly were observed in the endoplasmic reticulum and not the trans-Golgi ( Figure 5H ). In summary, our data indicate that the microtubule cytoskeleton is required for ef®cient IMV assembly and is essential for IEV formation.\nIn the course of our experiments, it became obvious that the Golgi apparatus becomes progressively dispersed during infection co-concominantly with disruption of the microtubule network ( Figure 6 ). Further analysis showed that during infection the normal morphology of the microtubule cytoskeleton is replaced by morphologically aberrant microtubule forms, which vary among each other but have in common the absence of a discrete MTOC ( Figure 7 ). These aberrant forms can be broadly classi®ed into three types: (i) cells with a disorganized microtubule network where microtubules seem randomly oriented ( Figure 7E ); (ii) cells in which microtubules form rings around the nucleus and throughout the cytoplasm ( Figure 7H ); or (iii) cells with long projections consisting of microtubule bundles ( Figure 7K ). We quanti®ed the appearance of the different morphological forms in ®ve independent infection experiments, in which 200 cells were counted for each time point for each experiment ( Figure 7C , F, I and L). Small compact cells, representing 20.7 6 2.6, 21.8 6 12.4 and 29.7 6 15.2% for 5, 8 and 24 h post-infection, respectively, in which the microtubule cytoskeleton morphology was not evident were not included in the analysis. Already by 5 h post-infection, when virus particle assembly has occurred, the normal aster microtubule con®guration has been disrupted and replaced in the majority of cells by microtubules without obvious organization from the MTOC ( Figure 7F ). Furthermore,~10% of cells have microtubule rings and 5% of cells have long projections by this time point ( Figure 7I and L). As the infection proceeds, microtubules become progressively more disrupted and bundled ( Figure 7I and L).\nFrom our observations, there seems to be no obvious connection between the disruption and changes in the actin and the microtubule cytoskeletons ( Figure 7) . Moreover, the same reorganization of the microtubule network occurs in cells infected with the vaccinia deletion mutants DF13L and DA36R which do not make actin tails (data not shown). The effects of vaccinia virus infection on the reorganization of the microtubule cytoskeleton were also observed in all cell lines we examined (BHK-21, C 2 C 12 , PtK2, RK 13 and Swiss 3T3) to varying degrees (data not shown). Our data show that vaccinia infection results in severe disruption of the normal morphology of the microtubule cytoskeleton.\nThe formation of microtubule bundles and the loss of organization from the MTOC in vaccinia-infected cells is strongly reminiscent of the phenotype observed in cells overexpressing a MAP (Weisshaar et al., 1992; Togel et al., 1998) . As overexpression of MAPs stabilizes microtubules, we examined whether the microtubule cytoskeleton in infected cells was more resistant to depolymerization by nocodazole or cold treatment ( Figure 8 ). This was indeed the case, suggesting that the virus genome may encode viral proteins with MAP-like properties. To identify viral proteins which exhibit microtubule-binding properties, we performed microtubule co-sedimentation assays using extracts prepared from uninfected and vaccinia-infected cells (Figure 9 ). Initial experiments, however, revealed that intact virus particles in the extracts were prone to pellet even in the absence of microtubules, making identi®cation of viral MAPs impossible. To avoid this problem, we prepared extracts from cells infected in the presence of rifampicin, a drug that inhibits vaccinia virus particle assembly but does not affect viral protein expression (Moss et al., 1969; Tan and McAuslan, 1970) . The morphological effects of vaccinia infection on the microtubule cytoskeleton were the same in the presence or absence of rifampicin (data not shown). Comparison of the proteins present in pellets from microtubule co-sedimentation assays reveals that a number of additional prominent and minor bands are present in extracts prepared from vaccinia-infected but not from uninfected cells (Figure 9 ). Co-sedimentation assays Vaccinia uses and abuses the microtubule cytoskeleton performed in the presence of nocodazole or with coldtreated extracts reveal that the majority of these additional bands disappear in the absence of microtubules. To identify the viral proteins co-sedimenting with microtubules, we performed in-gel protease digestion followed by analysis of the resulting peptides by MALDI mass spectrometry. Using this approach, we identi®ed a number of potential vaccinia-encoded MAPs: A10L (a structural protein), I1L and L4R (which are DNA-binding proteins), all of which are associated with viral cores (Vanslyke and Hruby, 1994; Jensen et al., 1996a; Klemperer et al., 1997) , and A6L which is conserved in all poxvirus genomes but is of unknown function (Figure 9 ).\nA10L and L4R associate with microtubules in vivo and mediate binding of viral cores to microtubules in vitro Using available antibodies, we examined the localization of A10L, L4R and I1L in infected cells to see whether they associate with microtubules in vivo, in addition to their essential role in the virus core (Vanslyke and Hruby, 1994; Jensen et al., 1996a) . As a negative control, we also examined the localization of the A3L core protein which was identi®ed as the prominent 70 kDa protein pelleting in the absence of microtubules (Figure 9 ). Indirect immunouorescence analysis showed that A10L and L4R are associated with microtubules, in both the presence and absence of rifampicin ( Figure 10 ). As expected, A10L and L4R were also associated with viral particles (data not shown). In contrast, I1L and A3L were never observed in association with microtubules, regardless of the ®xation conditions, but were localized to viral factories and viral particles, respectively (data not shown). Interestingly, A10L and L4R were not associated with all microtubules but were co-localized with a subset of acetylated microtubules ( Figure 10) .\nThe association of A10L and L4R with virus particles and microtubules raises the question of whether there is a role for this microtubule-binding activity during infection. We wondered whether these two proteins mediate the interaction of incoming viral cores with microtubules at the beginning of infection, as cores and not virus particles are released in the cytoplasm at the start of the infection cycle (Ichihashi, 1996; Vanderpasschen et al., 1998; Pedersen et al., 2000) . To examine this possibility, we investigated whether puri®ed viral cores would bind microtubules in vitro. We found that viral cores were able to bind microtubules, while protease-treated cores showed no association ( Figure 11A and B) . Pre-incubation of puri®ed viral cores with antibodies against A10L and L4R speci®cally inhibited the interaction of viral cores with microtubules ( Figure 11C and D); in contrast, IgG or antibodies against A3L had no inhibitory effect ( Figure 11E and F). Taken together, our data suggest that A10L and L4R have MAP-like properties and may play a role in mediating interactions of incoming viral cores with microtubules.\nThe dramatic rearrangement of the microtubule cytoskeleton which occurs during vaccinia infection is unlikely to be attributed exclusively to the action of A10L and L4R since they only associate with a subset of microtubules ( Figure 10) . Furthermore, the loss of microtubule organization precedes detectable association of A10L and L4R with microtubules, which occurs from~8 h post-infection. We therefore wondered whether vaccinia infection disrupts centrosome function, given the loss of microtubule aster con®guration during infection (Figure 7 ). Since microtubules are nucleated by the centrosome in animal cells, we examined whether vaccinia infection affects g-tubulin, which is critically required for this process (Stearns and Kirschner, 1994) . We observed that g-tubulin labelling of the centrosome is greatly reduced from as early as 2 h post-infection ( Figure 12 ). The same result was obtained when we infected PtK1 cells stably expressing green¯uorescent protein (GFP)-labelled g-tubulin (Khodjakov and Rieder, 1999) . In addition, the centrosomal and centriolar components pericentrin, C-Nap 1, Nek 2 and centrin are reduced by immuno¯uorescence in the centrosomes/centrioles of vaccinia-infected cells ( Figure 12) . Furthermore, the reduction of centrosomal markers requires viral protein synthesis as their levels are not affected when cells are infected in the presence of cycloheximide (data not shown).\nThe dramatic reduction of g-tubulin from the centrosome implies that vaccinia infection perturbs centrosome Vaccinia uses and abuses the microtubule cytoskeleton function. To test this hypothesis, we examined whether the centrosome in vaccinia-infected cells could re-nucleate microtubules, following their depolymerization by nocodazole. We found that by 2 h post-infection, when we already see a reduction in g-tubulin, microtubule nucleation from the centrosome was very inef®cient, as compared with uninfected controls, indicating that vaccinia has disrupted`normal' centrosome function (Figure 13 ). At later times post-infection, microtubule re-nucleation ef®ciency from the centrosome was even lower (data not shown). However, following nocodazole washout, microtubules eventually are repolymerized throughout the cytoplasm of infected cells but do not display any organization from the MTOC, as do controls (compare Figure 13I and K).\nThe size of virus particles is such that they are unlikely to move within and between cells by diffusion alone, suggesting that their movements will require interactions with the host cytoskeleton. Previous data have shown that vaccinia virus both disrupts and hijacks the actin cytoskeleton to facilitate movement of the intracellular enveloped form of vaccinia virus (Cudmore et al., 1995; Ro Èttger et al., 1999) and of the infected cell itself (Sanderson and Smith, 1998; Sanderson et al., 1998b) . The data described here now show that vaccinia also uses and subsequently disrupts the microtubule cytoskeleton during its infection cycle. It is clear from our experiments and the previous observations of Ulaeto et al. (1995) that microtubules are required to maintain the integrity of the Golgi apparatus which is in turn required for IMV wrapping to form IEV (Schmelz et al., 1994) . In contrast, IMV are assembled in the absence of microtubules, albeit at reduced levels. While microtubules are not required for IMV assembly, they are required together with the dynein±dynactin complex for virion accumulation in the vicinity of the microtubule aster. One can envisage that minus enddirected microtubule-dependent movements of IMV particles from their site of assembly in the viral factory towards the MTOC, by the dynein±dynactin complex, would enhance the possibility of wrapping with the Golgi apparatus and subsequent IEV formation. Recently it has been shown that the IMV protein A27L and microtubules are required for ef®cient IMV dispersion from the viral factories (Sanderson et al., 2000) . In the absence of A27L, mature IMV particles accumulate at the periphery of the virus factory but do not subsequently wrap to form IEV, presumably because they are unable to move on microtubules (Sanderson et al., 2000) .\nThe microtubule-and dynein±dynactin-dependent accumulation of vaccinia in the vicinity of the MTOC is analogous to the microtubule-dependent movements required for herpes simplex virus 1 (HSV-1) and adenovirus to reach their site of replication in the nucleus (Sodeik et al., 1997; Suomalainen et al., 1999; Leopold et al., 2000) . In the case of HSV-1, the UL34 protein, 9 . Vaccinia encodes proteins that co-sediment with microtubules. Analysis of pellets from in vitro microtubule co-sedimentation assays performed with protein extracts from vaccinia-infected (inf.) and uninfected (uninf.) cells. Twice the amount of pellet has been loaded in control assays performed in the absence of microtubules (nocodazole or 4°C). Proteins co-sedimenting with microtubules that were only present in extracts from infected cells are indicated by an asterisk. The identity of proteins determined by in-gel proteolysis MALDI mass spectrometry is indicated (arrowheads).\nwhich is associated with the incoming nucleocapsids, interacts with the intermediate chain of cytoplasmic dynein (IC-1a) (Ye et al., 2000) . It has also been reported that incoming nucleocapsids of pseudorabies virus, an alphaherpes virus closely related to HSV-1, are associated with and dependent on microtubules for their movement to the nucleus (Kaelin et al., 2000) . This interaction may be mediated by the UL25 protein, a minor but essential component of the capsid, which co-localizes with microtubules and accumulates at the MTOC (Kaelin et al., 2000) . The accumulation of UL25 at the MTOC is consistent with a possible interaction with the dynein± dynactin motor complex which is known to be localized at the MTOC (Echeverri et al., 1996) . It would not be surprising, based on observations with HSV-1 and pseudorabies, if microtubules and dynein±dynactin were also involved in establishing the infection cycle of cytomegalovirus (CMV), Epstein±Barr virus and varicella-zoster virus, all of which are herpes viruses. The other clear example of microtubule-dependent virus movements during the establishment of infection is that of incoming human foamy virus (HFV) which is dependent on microtubules and presumably a minus end-directed microtubule motor to get to its nuclear replication site (Saib et al., 1997) . In the absence of protein expression, HFV Gag proteins, which are associated with the viral genome, accumulate at the centrosome in a microtubuledependent fashion prior to nuclear import (Saib et al., 1997) . The centrosomal accumulation of Gag proteins of HFV, however, appears to be unique for this class of retroviruses as no similar localization has been reported for human immunode®ciency virus (HIV) or other retroviruses. On the other hand, the Gag protein of murine leukaemia virus and HIV has been shown to interact with KIF4, a microtubule plus end-directed kinesin motor, both in vitro and in vivo (Kim et al., 1998; Tang et al., 1999) , suggesting that additional roles may exist for microtubules and motors during the outward movement of virus particles. Indeed, vaccinia virus particles are able to reach the cell periphery in the absence of actin-based motility (see images in Wolffe et al., 1997; Sanderson et al., 1998a Sanderson et al., , 2000 Ro Èttger et al., 1999) , suggesting that viral particles can also move out on microtubules (Sanderson et al., 2000) . Microtubule-dependent motordriven movements of virus particles represent an ef®cient mechanism to achieve a peri-nuclear localization, required to facilitate entry into the nucleus during establishment of infection. They also provide an excellent way for newly assembled virus particles to reach the cell periphery, facilitating the continued spread of infection.\nOur data show that although vaccinia virus uses the microtubule cytoskeleton to achieve a peri-nuclear localization, microtubule and Golgi organization becomes disrupted later during the infection process. Interestingly, HSV-1 and CMV have also been reported to disrupt the microtubule cytoskeleton and Golgi organization in their infection cycles (Avitabile et al., 1995; Fish et al., 1996) . While disruption of the microtubule network might at ®rst sight not appear to be bene®cial to the virus, it may not actually hinder viral spread but could enhance it. First, extensive virus assembly and spread to the cell periphery have already occurred by the time the microtubule cytoskeleton and Golgi organization are disrupted. Secondly, disruption of microtubule organization may overcome potential microtubule motor anchoring effects at the MTOC, thus allowing viral spread to the periphery to occur more easily. Lastly, the formation of long projections of up to 200 mm supported by extensive microtubule bundles provides a means to achieve long range spread of virus particles (Sanderson et al., 1998b) .\nIt is clear that disruption and reorganization of the microtubule cytoskeleton by vaccinia virus is mediated by the combined effects of viral proteins with MAP-like properties and loss of microtubule-organizing function from the MTOC. The same may also be true for HSV-1, although disruption of centrosome function remains to be established, as late in infection microtubules are organized in bundles around the nucleus and do not show MTOCorchestrated organization (Avitabile et al., 1995) . The identi®cation of viral proteins with MAP-like properties is not unique to vaccinia virus. The VP22 tegument protein from HSV-1 co-localizes with microtubules in infected cells and induces microtubule bundles when expressed in uninfected cells (Elliott and O'Hare, 1998) . Other examples of viral MAPs based on their in vivo localization Fig. 11 . Vaccinia cores bind directly to microtubules in vitro. Puri®ed viral cores labelled by DAPI (green) bind to rhodamine-labelled microtubules (red) in the absence of ®xation (A). Binding to microtubules is not observed if cores are pre-treated with protease (B) or pre-incubated with antibodies against the A10L (C) or L4R (D) proteins. In contrast, pre-incubation of puri®ed viral cores with control IgG (E) or antibody against the A3L protein (F) does not inhibit their interaction with microtubules. Scale bar = 5 mm.\nVaccinia uses and abuses the microtubule cytoskeleton or in vitro association with microtubules are the N protein from murine coronavirus (Kalicharran and Dales, 1996) , the movement protein from tobamovirus (Heinlein et al., 1995) , the aphid transmission factor from cauli¯ower mosaic virus (Blanc et al., 1996) , the UL25 protein from pseudorabies virus (Kaelin et al., 2000) , the VP4 spike protein from rotavirus (Nejmeddine et al., 2000) and the M protein of vesicular stomatitis virus (VSV) (Melki et al., 1994) . The identi®cation of A10L and L4R, two viral core proteins, as MAP-like proteins was, however, unexpected given their previously characterized role in viral morphogenesis (Vanslyke and Hruby, 1994) . The interaction of A10L and L4R with microtubules in vivo, together with the in vitro microtubule-binding data, suggest a potential mechanism for the association of viral cores with microtubules. One could envisage that viral cores which are released into the cytoplasm at the beginning of infection (Ichihashi, 1996; Vanderpasschen et al., 1998; Pedersen et al., 2000) bind directly to microtubules in a manner analogous to adenovirus or HSV-1 nucleocapsids. Further work is required to determine whether incoming cores do in fact move towards the MTOC by the dynein± dynactin complex and/or use the complex for anchoring on microtubules.\nThe loss of centrosome function must enhance disruption of the microtubule cytoskeleton during infection. Indeed, the loss of microtubule organization from the MTOC precedes detectable association of A10L and L4R with microtubules, which occurs from~8 h post-infection. Vaccinia-induced loss of centrosomal proteins is inhibited by cycloheximide, indicating that viral protein expression is required for disruption of the centrosome microtubule nucleation activity. To our knowledge, vaccinia virus infection represents the ®rst example of virus-induced disruption of centrosome function, although we would predict that HSV-1 may have a similar effect. The mechanism by which vaccinia virus disrupts the centrosome requires further study; nevertheless, it is clear that understanding the molecular basis of this disruption will provide important insights into the regulation and stability of centrosome function which currently is the subject of intense research (Ohta et al., 1993; Lane and Nigg, 1997; Karsenti, 1999) .\nHeLa cells (ATCC CCL2) were infected with the wild-type vaccinia virus strain Western Reserve (WR) or with the vaccinia deletion mutants DF13L (vRB12) (Blasco and Moss, 1991) or DA36R (Parkinson and Smith, 1994) at a multiplicity of infection of 1 p.f.u. (plaque-forming unit) per cell, as described previously (Ro Èttger et al., 1999) . Nocodazole dissolved in dimethyl sulfoxide (DMSO) and brefeldin A dissolved in ethanol were added to the culture medium to ®nal concentration of 10 mM and 5 mg/ml, respectively unless otherwise stated. In non-treated controls, an equal volume of DMSO or ethanol was added. Cells transfected with a myc-tagged p50/dynamitin expression construct (Echeverri et al., 1996) were infected 24 h later with WR and subsequently ®xed 6 h postinfection. All experiments described have been repeated 3±10 times.\nThe following antibodies were kindly provided: anti-a-tubulin by Dr E.Karsenti, anti-centrin (20H5) by Professor J.L.Salisbury (Sanders and Salisbury, 1994; Paoletti et al., 1996) , anti-Nek2 and anti-C-Nap1 by Professor E.Nigg (Fry et al., 1998a,b) , anti-myc and anti-gp27 by Dr T.Nilsson (Fu Èllekrug et al., 1999) and antibodies against the corresponding vaccinia proteins: A3L, A10L and L4R by Professor D.Hruby (Vanslyke and Hruby, 1994) , I1L by Professor P.Traktman (Klemperer et al., 1997) , A27L (C3) by Dr M.Esteban (Rodriguez et al., 1985) , A33R, A34R and A36R (Ro Èttger et al., 1999) . In addition, the following antibodies were obtained from commercial sources: anti-a-tubulin (N356) (Amersham International, UK), anti-acetylated a-tubulin (6-11B-1) (Sigma, USA), anti-g-tubulin (GTU-88; Sigma), anti-pericentrin and anti-TGN46 (BAbCO, USA), and rabbit IgG (Sigma). Actin was visualized with¯uorescently labelled phalloidin derivatives (Molecular Probes, USA).\nCells were ®xed in ±20°C methanol or in 5% paraformaldehyde in BRB80 (80 mM PIPES pH 6.8, 1 mM MgCl 2 , 1 mM EGTA) followed by 0.1% Triton X-100 permeabilization. Fixed cells were processed for immuno¯uorescence, viewed and images recorded as described previously (Ro Èttger et al., 1999) .\nHeLa cells were pre-incubated with 25 mM nocodazole in the medium for 1 h to depolymerize microtubules, prior to infection with vaccinia DF13L at 1 p.f.u./cell. Nocodazole was kept in the medium throughout the infection, while an equal volume of DMSO was added to the controls. At 24 h post-infection, the cells were scraped from the¯asks into the medium and sedimented by centrifugation (300 g, 7 min, 4°C). The cell membranes were disrupted and the nuclei were removed by centrifugation. The resulting post-nuclear supernatant was centrifuged through a 36% sucrose cushion (76 000 g, 30 min, 4°C). The virus pellet was resuspended in 10 mM Tris pH 9; the virus was collected by centrifugation (76 000 g, 30 min, 4°C), resuspended in 10 mM Tris pH 9 and stored at ±80°C. The concentration of the virus (elemental bodies) was determined by OD 260 measurement (Joklik, 1962) . Fig. 13 . Vaccinia infection reduces centrosome microtubule nucleation ef®ciency. In uninfected cells, microtubules (A, E and I) nucleate from centrosomes (B, F and J) after nocodazole washout for the times indicated. In contrast, 2 h after infection with vaccinia, microtubules (C, G and K) are nucleated inef®ciently from centrosomes (D, H and L). All images were collected with identical camera settings, to allow comparison of uorescence intensity between centrosomes. Inserts (B, D, F, H, J and L) are adjusted as in Figure 12 to facilitate visualization of the weak g-tubulin centrosomal labelling. Arrowheads indicate the position of the centrosome. Scale bar = 10 mm.\nIn vitro microtubule binding assays Puri®ed EEV particles were prepared as described previously (Ro Èttger et al., 1999) and subsequently were used to prepare virus cores following the method of Cudmore et al. (1996) . Rhodamine-labelled microtubules were prepared according to Hyman et al. (1991) . Vaccinia virus cores were incubated with rhodamine-labelled microtubules in BRB80 buffer containing 10 mM taxol for 5 min at room temperature. 4¢,6-diamidino-2phenylindole (DAPI) was added subsequently to a ®nal concentration of 0.1 mg/ml to label the virus cores. Finally, the mixture was diluted 1:1± 1:10 with antifade solution (0.1 mg/ml catalase, 0.1 mg/ml glucose oxidase, 10 mM glucose) and viewed without ®xation. Proteinase K or trypsin treatment of core particles prior to incubation with microtubules was performed as described previously (Roos et al., 1996) . Anti-A3L, A10L, L4R or control IgG antibodies were incubated with puri®ed cores for 1 h at room temperature prior to incubation with microtubules.\nCell extracts and microtubule co-sedimentation assay Extracts from HeLa cells infected for 24 h or uninfected controls maintained in the presence of 0.1 mg/ml rifampicin were prepared as described previously (Ro Èttger et al., 1999) . The extract was clari®ed by centrifugation at 150 000 g for 20 min at 4°C and cytochalasin D added to a ®nal concentration of 1 mg/ml to depolymerize actin ®laments. Endogenous tubulin in the extract was polymerized in a two-step procedure. First, the extract supernatant was supplemented with protease inhibitors, 2 mM MgGTP and 5 mM taxol and incubated for 5 min at room temperature; subsequently, an additional 15 mM taxol was added to the mix and the reaction incubated at 33°C for 30 min. For controls, no taxol was added at any stage and microtubule polymerization was inhibited either by the addition of nocodazole to a ®nal concentration of 40 mM or by maintaining the extract at 4°C throughout the experiment. Following microtubule assembly, each 400 mg extract reaction was diluted 5-fold in BRB80 buffer (containing protease inhibitors and 20 mM taxol) and centrifuged through a 10% sucrose cushion containing protease inhibitors and 20 mM taxol at 165 000 g for 20 min at 25°C. The microtubule pellet was solubilized in SDS±PAGE sample buffer and analysed by SDS± PAGE.\nIn-gel proteolytic cleavage was performed automatically in the`Progest' as described (Houthaeve et al., 1997) [Genomic Solutions Cambridge (http://www.genomicsolutions.com)] and the peptides obtained were analysed on a Bruker REFLEX MALDI mass spectrometer (Bruker Analytik, Germany) (Jensen et al., 1996b) . Proteins were identi®ed by peptide mass ®ngerprinting (Jensen et al., 1997) using the program PeptideSearch (http://www.narrador.embl-heidelberg.de/Services/ PeptideSearch/PeptideSearchIntro.html.\nAt 1 h post-infection, nocodazole was added to the culture medium to a ®nal concentration of 25 mM to depolymerize microtubules. At 2 h postinfection, the cells were washed 3±4 times in warm medium to remove nocodazole. Washed cells were incubated in medium without nocodazole for the indicated time at 37°C to re-initiate microtubule polymerization; they were then washed brie¯y in warm phosphate-buffered saline (PBS) and immediately ®xed. In parallel, samples were also removed at the same time point, brie¯y rinsed in ice-cold PBS, ®xed and processed for immuno¯uorescence to con®rm complete microtubule depolymerization before initiation of microtubule assembly. Uninfected control HeLa cells were treated and processed in an identical fashion. The same numbers of images were integrated using identical camera settings to allow direct comparison between infected and uninfected samples from the same experiment." }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: The 1918-1919 influenza pandemic killed more people than any other outbreak of disease in human history. The lowest estimate of the death toll is 21 million, while recent scholarship estimates from 50 to 100 million dead. World population was then only 28% what is today, and most deaths occurred in a sixteen week period, from mid-September to mid-December of 1918.\nIt has never been clear, however, where this pandemic began. Since influenza is an endemic disease, not simply an epidemic one, it is impossible to answer this question with absolute certainty. Nonetheless, in seven years of work on a history of the pandemic, this author conducted an extensive survey of contemporary medical and lay literature searching for epidemiological evidence -the only evidence available. That review suggests that the most likely site of origin was Haskell County, Kansas, an isolated and sparsely populated county in the southwest corner of the state, in January 1918 [1] . If this hypothesis is correct, it has public policy implications.\nBut before presenting the evidence for Haskell County it is useful to review other hypotheses of the site of origin. Some medical historians and epidemiologists have theorized that the 1918 pandemic began in Asia, citing a lethal outbreak of pulmonary disease in China as the forerunner of the pandemic. Others have speculated the virus was spread by Chinese or Vietnamese laborers either crossing the United States or working in France.\nMore recently, British scientist J.S. Oxford has hypothesized that the 1918 pandemic originated in a British Army post in France, where a disease British physicians called \"purulent bronchitis\" erupted in 1916. Autopsy reports of soldiers killed by this outbreak -today we would classify the cause of death as ARDS -bear a striking resemblance to those killed by influenza in 1918 [2] .\nBut these alternative hypotheses have problems. After the 1918-1919 pandemic, many investigators searched for the source of the disease. The American Medical Association sponsored what is generally considered the best of several comprehensive international studies of the pandemic conducted by Dr. Edwin Jordan, editor of The Journal of Infectious Disease. He spent years reviewing evidence from all over the world; the AMA published his work in 1927.\nSince several influenza pandemics in preceding centuries were already well-known and had come from the orient, Jordan first considered Asia as the source. But he found no evidence. Influenza did surface in early 1918 in China, but the outbreaks were minor, did not spread, and contemporary Chinese scientists, trained by Rockefeller Institute for Medical Research (now Rockefeller University) investigators, stated they believed these outbreaks were endemic disease unrelated to the pandemic [3] . Jordan also looked at the lethal pulmonary disease cited by some historians as influenza, but this was diagnosed by contemporary scientists as pneumonic plague. By 1918 the plague bacillus could be easily and conclusively identified in the laboratory [3] . So after tracing all known outbreaks of respiratory disease in China, Jordan concluded that none of them \"could be reasonably regarded as the true forerunner\" of the pandemic [3] .\nJordan also considered Oxford's theory that the \"purulent bronchitis\" in British Army camps in 1916 and 1917 was the source. He rejected it for several reasons. The disease had flared up, true, but had not spread rapidly or widely outside the affected bases; instead, it seemed to disappear [3] . As we now know a mutation in an existing influenza virus can account for a virulent flare-up. In the summer of 2002, for example, an influenza epidemic erupted in parts of Madagascar with an extremely high mortality and morbidity; in some towns it sickened an outright majority -in one instance sixty-seven percent -of the population. But the virus causing this epidemic was an H3N2 virus that normally caused mild disease. In fact, the epidemic affected only thirteen of 111 health districts in Madagascar before fading away [4]. Something similar may have happened in the British base.\nJordan considered other possible origins of the pandemic in early 1918 in France and India. He concluded that it was highly unlikely that the pandemic began in any of them [3] .\nThat left the United States. Jordan looked at a series of spring outbreaks there. The evidence seemed far stronger. One could see influenza jumping from Army camp to camp, then into cities, and traveling with troops to Europe. His conclusion: the United States was the site of origin.\nA later equally comprehensive, multi-volume British study of the pandemic agreed with Jordan. It too found no evidence for the influenza's origin in the Orient, it too rejected the 1916 outbreak among British troops, and it too concluded, \"The disease was probably carried from the United States to Europe [5] .\" Australian Nobel laureate MacFarlane Burnet spent most of his scientific career working on influenza and studied the pandemic closely. He too concluded that the evidence was \"strongly suggestive\" that the disease started in the United States and spread with \"the arrival of American troops in France [6] .\"\nBefore dismissing the conclusions of these contemporary investigators who lived through and studied the pandemic, one must remember how good many of them were. They were very good indeed.\nThe Rockefeller Institute, whose investigators were intimately involved in the problem, alone included extraordinary people. By 1912 its head Simon Flexner -his brother wrote the \"Flexner report\" that revolutionized American medical education -used immune serum to bring the mortality rate for meningococcal meningitis down from over 80% to 18%; by contrast, in the 1990s at Massachusetts General Hospital a study found a 25% mortality rate for bacterial meningitis. Peyton Rous won the Nobel Prize in 1966 for work he did at the institute in 1911; he was that far ahead of the scientific consensus. By 1918 Oswald Avery and others at Rockefeller Institute had already produced both an effective curative serum and a vaccine for the most common pneumococcal pneumonias. At least partly because of the pandemic, Avery would spend the rest of his career studying pneumonia. That work led directly to his discovery of the \"transforming principle\"his discovery that DNA carries the genetic code.\nThe observations of investigators of this quality cannot be dismissed lightly. Jordan was of this quality.\nMore evidence against Oxford's hypothesis comes from Dr. Jeffrey Taubenberger, well-known for his work extracting samples of the 1918 virus from preserved tissue and sequencing its genome. He initially believed, based on statistical analysis of the rate of mutation of the virus that it existed for two or three years prior to the pandemic. But further work convinced him that the virus emerged only a few months prior to the pandemic (personal communication with the author from J Taubenberger, June 5 th 2003).\nSo if the contemporary observers were correct, if American troops carried the virus to Europe, where in the United States did it begin?\nBoth contemporary epidemiological studies and lay histories of the pandemic have identified the first known outbreak of epidemic influenza as occurring at Camp Funston, now Ft. Riley, in Kansas. But there was one place where a previously unknown -and remarkable -epidemic of influenza occurred.\nHaskell County, Kansas, lay three hundred miles to the west of Funston. There the smell of manure meant civilization. People raised grains, poultry, cattle, and hogs. Sod-houses were so common that even one of the county's few post offices was located in a dug-out sod home. In 1918 the population was just 1,720, spread over 578 square miles. But primitive and raw as life could be there, science had penetrated the county in the form of Dr. Loring Miner. Enamored of ancient Greece -he periodically reread the classics in Greek -he epitomized William Welch's comment that \"the results [of medical education] were better than the system.\" His son was also a doctor, trained in fully scientific ways, serving in the Navy in Boston.\nIn late January and early February 1918 Miner was suddenly faced with an epidemic of influenza, but an influenza unlike any he had ever seen before. Soon dozens of his patients -the strongest, the healthiest, the most robust people in the county -were being struck down as suddenly as if they had been shot. Then one patient pro-gressed to pneumonia. Then another. And they began to die. The epidemic got worse. Then, as abruptly as it came, it disappeared. Men and women returned to work. Children returned to school. And the war regained its hold on people's thoughts.\nThe disease did not, however, slip from Miner's thoughts. Influenza was neither a reportable disease, nor a disease that any state or federal public health agency tracked. Yet Miner considered this incarnation of the disease so dangerous that he warned national public health officials about it. Public Health Reports (now Morbidity and Mortality Weekly Report), a weekly journal produced by the U.S. Public Health Service to alert health officials to outbreaks of communicable diseases throughout the world, published his warning. In the first six months of 1918, this would be the only reference in that journal to influenza anywhere in the world.\nHistorians and epidemiologists have previously ignored Haskell most likely because his report was not published until April and it referred to deaths on March 30, after influenza outbreaks elsewhere. In actuality, by then the county was free of influenza. Haskell County, Kansas, is the first recorded instance anywhere in the world of an outbreak of influenza so unusual that a physician warned public health officials. It remains the first recorded instance suggesting that a new virus was adapting, violently, to man.\nIf the virus did not originate in Haskell, there is no good explanation for how it arrived there. There were no other known outbreaks anywhere in the United States from which someone could have carried the disease to Haskell, and no suggestions of influenza outbreaks in either newspapers or reflected in vital statistics anywhere else in the region. And unlike the 1916 outbreak in France, one can trace with perfect definiteness the route of the virus from Haskell to the outside world.\nAll Army personnel from the county reported to Funston for training. Friends and family visited them at Funston. Soldiers came home on leave, then returned to Funston.\nThe Monitor reported in late February, \"Most everybody over the country is having lagrippe or pneumonia (Santa Fe Monitor, February 21 st 1918).\" It also noted, \"Dean Nilson surprised his friends by arriving at home from Camp Funston on a five days furlough. Dean looks like soldier life agrees with him.\" He soon returned to the camp. Ernest Elliot left to visit his brother at Funston as his child fell ill. On February 28, John Bottom left for Funston. \"We predict John will make an ideal soldier,\" said the paper (Santa Fe Monitor February 28 th , 1918).\nThese men, and probably others unnamed by the paper, were exposed to influenza and would have arrived in Funston between February 26 and March 2. On March 4 the first soldier at the camp reported ill with influenza at sick call. The camp held an average of 56,222 troops. Within three weeks more than eleven hundred others were sick enough to require hospitalization, and thousands morethe precise number was not recorded -needed treatment at infirmaries scattered around the base.\nWhether or not the Haskell virus did spread across the world, the timing of the Funston explosion strongly suggests that the influenza outbreak there did come from Haskell. Meanwhile Funston fed a constant stream of men to other American locations and to Europe, men whose business was killing. They would be more proficient at it than they knew.\nSoldiers moved uninterrupted between Funston and the outside world, especially to other Army bases and France. On March 18, Camps Forrest and Greenleaf in Georgia saw their first cases of influenza and by the end of April twenty-four of the thirty-six main Army camps suffered an influenza epidemic [3] . Thirty of the fifty largest cities in the country also had an April spike in excess mortality from influenza and pneumonia [7] . Although this spring wave was generally mild -the killing second wave struck in the fall -there were still some disturbing findings. A subsequent Army study said, \"At this time the fulminating pneumonia, with wet hemorrhagic lungs, fatal in from 24 to 48 hours, was first observed [8] .\" (Pathology reports suggest what we now call ARDS.) The first recorded autopsy in Chicago of an influenza victim was conducted in early April. The pathologist noted, \"The lungs were full of hemorrhages.\" He found this unusual enough to ask the then-editor of The Journal of Infectious Diseases \"to look over it as a new disease\" [3] .\nBy then, influenza was erupting in France, first at Brest, the single largest port of disembarkation for American troops. By then, as MacFarlane Burnet later said, \"It is convenient to follow the story of influenza at this period mainly in regard to the army experiences in America and Europe [6] .\"\nThe fact that the 1918 pandemic likely began in the United States matters because it tells investigators where to look for a new virus. They must look everywhere.\nIn recent years the World Health Organization and local public health authorities have intervened several times when new influenza viruses have infected man. These interventions have prevented the viruses from adapting to man and igniting a new pandemic. But only 83 countries in the world -less than half -participate in WHO's surveillance system (WHO's flunet website http:// rhone.b3e.jussieu.fr/flunet/www/docs.html). While some monitoring occurs even in those countries not formally affiliated with WHO's surveillance system, it is hardly adequate. If the virus did cross into man in a sparsely populated region of Kansas, and not in a densely populated region of Asia, then such an animal-to-man cross-over can happen anywhere. And unless WHO gets more resources and political leaders move aggressively on the diplomatic front, then a new pandemic really is all too inevitable." }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: There are hundreds of viruses that infect different human organs and cause diseases. Some fatal emerging viral infections have become serious public health issues worldwide. Early diagnosis and subsequent treatment are therefore essential for fighting viral infections. Current diagnostic techniques frequently employ polymerase chain reaction (PCR)-based methods to quickly detect the pathogenic viruses and establish the etiology of the disease or illness. However, the fast PCR method suffers from many drawbacks such as a high false-positive rate and the ability to detect only one or a few gene targets at a time. Microarray technology solves the problems of the PCR limitations and can be effectively applied to all fields of molecular medicine. Recently, a report in Retrovirology described a multi-virus DNA array that contains more than 250 open reading frames from eight human viruses including human immunodeficiency virus type 1. This array can be used to detect multiple viral co-infections in cells and in vivo. Another benefit of this kind of multi-virus array is in studying promoter activity and viral gene expression and correlating such readouts with the progression of disease and reactivation of latent infections. Thus, the virus DNA-chip development reported in Retrovirology is an important advance in diagnostic application which could be a potent clinical tool for characterizing viral co-infections in AIDS as well as other patients.\nMicroarray technology has been proven to be a powerful tool with great potential for biological and medical uses. In this technique, recombinant DNA fragments or synthesized oligonucleotides affixed on the surface of glass slides or nylon membranes are used for detecting complementary nucleic acid sequences (frequently representing a few hundred to >10,000 genes/expressed sequence tags) as well as for genotyping microorganisms and for profiling the gene-expression patterns in cells from higher organisms [1].\nA new report by Ghedin, et al. [2] in Retrovirology describes the successful use of a multi-virus array (termed multivi-rus-chip) to detect multiple viral co-infections in cultured cells as well as to study viral gene expression and promoter activities (Figure 1 ). Ghedin's multivirus-chip contains genes from eight human viruses including human immunodeficiency virus type 1 (HIV-1). Conceptually, this chip can be used to detect viral co-infections in AIDS patients who are frequently rendered susceptible to additional opportunistic infections. In developing their multivirus-chip, Ghedin, et al. tested more than 250 ORFs from HIV-1, human T cell leukemia virus types 1 (HTLV-1) and 2 (HTLV-2), hepatitis C virus (HCV), Epstein-Barr virus (EBV), human herpesvirus 6A (HHV6A) and 6B (HHV6B), and Kaposi's sarcoma-associated herpesvirus (KSHV) which were PCR-amplified and spotted on glass slides. They then hybridized their slides with Cy3-or Cy5labeled genomic DNA or cDNAs derived from various virus-infected cells. Their multivirus-chip was found to be highly specific and sensitive for detecting different viral genomic sequences in cell lines. Moreover, the chip could also detect the effect of various drugs on viral gene expression. In such instance, cell lines latently infected with HIV-1 and KSHV were used to generate profiles of viral gene expression in the presence of cyclin-dependent Schematic drawing of the multivirus-chip that possesses multiple functions kinase inhibitor (CKI), Roscovitine, which was applied to cells to suppress the reactivation of latently infected viruses.\nGhedin, et al. [2] also studied the role of cellular chromatin structure on viral gene expression using their multivirus-chip.\nThey employed the chromatin immunoprecipitation technique (ChIP) [3] to isolate cellular DNA fragments that were bound to phosphorylated histone H3 (P-H3). These DNA fragments were hybridized to the viral ORFs contained on the multivirus-chip to investigate the role of phospho-H3 on viral gene expression. They showed that whether transcriptionally active or silent the chromatin state played a role in regulating the expression of KSHV genes under the different cellular context. Current routine clinical diagnostics employ PCR, Southern blotting, Northern blotting, DNA sequencing and microarray hybridization to detect and characterize genes of interest in biomedicine. PCR is generally regarded as the most sensitive diagnostic method. However, Iyer, et al. [4] have shown that the sensitivity of cDNA-chip hybridization is comparable to that of TaqMan-driven quantitative PCR assay, and that the microarray hybridization technique is less likely to be complicated by high false positive rates due to carry-over contaminations. Furthermore, using microarrays, the viral gene transcripts in infected cells can be easily detected by hybridization without any prior amplification steps, and the microarray technique requires much less experimental material when compared to Southern or Northern blotting and can provide high sensitivity in the setting of large throughput.\nIn view of the above, the multivirus-chip described in Retrovirology [2] holds several advantages over other more commonly used techniques (e.g. PCR, DNA sequencing) for the diagnosis of viral infections. First, this chip provides a more accurate diagnosis of viral infection by simultaneously evaluating the transcription of all viral genes, and can use such cumulative data to correlate infection with clinical disease manifestations. Second, the high throughput and flexible synthesis nature of DNA microarray construction can allow scientists to tailor-make and rapidly alter arrays to match evolving emergence of new pathogens. The SARS genome chip made by the US NIAID, NIH is a good example [5] of how diagnostic arrays can be developed quickly and be used in a timely manner.\nFinally, the most novel application described by Ghedin, et al. is their use of microarrays to correlate the cellular \"histone code\" [6] with the promoter activity of KSHV. Usually the transcription of a gene located on chromosomal DNA is influenced not only by the cis-acting ele-ments (or DNA-binding motifs), but also by the structure of chromatin. The latter can be vary depending on the post-translational modifications of histone proteins. Methylation, acetylation, and/or phosphorylation of certain amino acid residues at the amino terminal \"tails\" of histone H3 and/or H4 can indeed influence chromatin structure. Thus accumulating evidence has shown that chromatin-associated proteins and their modifications play vital roles in many physiological processes such as growth, differentiation, and development in mammals, plants and fungi [6, 7] . Many studies have used DNA array technology to investigate viral gene expression or to genotype viral isolates; however, none has used this technique to study the influence of cellular chromatin structure on viral gene expression [1]. Ghedin, et al. [2] demonstrated that only DNA fragments derived from ChIP of latent BCBL-1 cell genomic DNA captured using phospho-H3 antibody bound specifically to the KSHV ORF on the multivirus-chip. This result suggests that latent KSHV genome in BCBL-1 cells is packed into a nucleosomal structure and that histone H3 proteins near the viral promoter can be phosphorylated at serines to make the DNA at the promoter region less tightly packed with histones and more easily accessible to transcription factors.\nIn conclusion, the multivirus-chip improvements developed by Ghedin, et al. [2] provide versatile clinical and basic uses. In the near future, such chips are likely to be used to detect viral co-infections in many different clinical settings. " }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: In some critically ill patients herpes simplex virus (HSV)-1 is isolated from the upper or lower respiratory tract [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] . Immunodepressed patients may be susceptible to transmission and acquisition of viral diseases; alternatively, viral reactivation may occur and may contribute relatively little to morbidity and mortality. Indeed, reactivation of human herpesvirus-6 is common in critically ill patients and does not worsen outcome [16, 17] . In immunocompetent patients, however, isolation of HSV-1 may be associated with viral pneumonia, even if reactivation rather than primary infection is responsible [6, 8, 18] . HSV-1 has been associated with acute respiratory distress syndrome (ARDS) and ventilator-associated pneumonia in the critically ill [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] , as either a primary or a superimposed infection. However, there are few reports of the virus eliciting an infectious host response, as demonstrated by a rise in serum antibodies, by bronchoscopic airway disease, by 'typical' findings on computed tomography of the lungs, or by the presence of giant cells or nuclear inclusion bodies on cytology or biopsy of the lower respiratory tract [3, 5, 9, 10, 18] . Indeed, Tuxen and coworkers [4] observed that prophylactic antiviral therapy in ARDS prevented respiratory HSV-1 emergence but it had no impact on duration of mechanical ventilation or on patient outcome. The pathogenicity of the virus therefore remains unknown, and the rare association in the critically ill of HSV-1 isolation with mortality may represent reactivation of the virus in immunodepressed patients with multiple organ failure and poor outcome [1, 2, 11, 14, 15] , rather than a symptomatic primary infection or superinfection contributing to death.\nAssessing pulmonary capillary protein permeability noninvasively at the bedside to yield the pulmonary leak index (PLI) could help in determining the extent of tissue injury, as was previously described [18] [19] [20] . This radionuclide technique involves gallium-67-labelled transferrin ( 67 Gatransferrin) and technetium-99m-labelled red blood cells ( 99m Tc-RBCs). In bacterial pneumonia, for instance, the PLI is elevated and the increase above normal directly relates to the severity of pneumonia, expressed as the lung injury score (LIS) [19] . In patients with acute lung injury (ALI) or ARDS during the course of bacterial pneumonia, the PLI is uniformly and greatly elevated above normal (up to 14.1 × 10 -3 /min) when LIS is greater than 2.5; the PLI is also elevated in 80% of patients with mild injury and a LIS between 1.5 and 2.5 [19] . Hence, the technique is a direct measure of permeability and an indirect measure of capillary injury in the lungs. The PLI is also elevated in interstitial lung disease [21] .\nIn order to help differentiate between symptomatic and asymptomatic viral shedding and spread, which could inform the decision regarding whether to institute antiviral therapy and help in determining the pathogenicity of the virus, we measured the PLI in four consecutive critically ill patients with persistent pulmonary infiltrates of unknown origin on ventilatory support, in whom a HSV-1 had been isolated.\nWe studied a small series of consecutive patients in whom respiratory secretions, sent for viral culture because of persistent pulmonary infiltrates of unknown origin, were found to be positive for HSV-1 (Table 1) . Tracheal aspirates or bronchoalveolar lavage fluid were transported directly to the microbiology laboratory or placed in viral transport medium (Copan Diagnostics Inc., Corona, CA, USA). For isolation of HSV-1, specimens were inoculated using standard procedures in triplicate flat bottom tubes on human embryonal lung fibroblasts and incubated at 37°C. Cultures were studied three times weekly for 10 days to identify the presence of a cytopathic effect. If a cytopathic effect, indicating the presence of HSV-1, was apparent or otherwise at days 2 and 7, the cells were fixed in methanol:acetone (1:1) and typed by immunofluorescence with labelled specific HSV-1 and HSV-2 antibodies (Syva Mikrotac HSV-1/HSV-2 typing kit, Palo Alto, CA, USA). In the four patients studied, the results were available within 3 days after samples had been inoculated in culture medium.\nOn the day of specimen collection for viral culture, demographic, chest radiographic and respiratory data were recorded, as were clinical features. In three out of four patients on mechanical ventilation after intubation, the total respiratory compliance was calculated from ventilator settings as follows (ml/cmH 2 O): tidal volume/(plateau -end-expiratory pressure). From the radiographic score (ranging from 0 to 4 depending on the number of quadrants with radiographic opacities), the ratio of arterial oxygen tension to fractional inspired oxygen, the level of positive end-expiratory pressure and the compliance, the LIS was calculated [22] . (LIS ranges between 0 and 4, with values up to 2.5 denoting ALI and those above 2.5 ARDS.) None of the patients had visible oropharyngeal vesicles.\nTo characterize further the persistent pulmonary infiltrates, the PLI was measured using a modification to a method described previously [19, 20] . Because this is a routine procedure, informed consent was waived. Autologous RBCs were labeled with 99m Tc (11 MBq, physical half-life 6 hours; Mallincrodt Diagnostica, Petten, The Netherlands), using a modified in vitro method. Ten minutes after injection of the labelled RBCs, transferrin was labelled in vivo, following intravenous injection of 67 Ga-citrate (6 MBq, physical half-life 78 hours; Mallincrodt Diagnostica). Patients were in the supine position, and two scintillation detection probes were positioned over the right and left lung apices. The probe system (manufactured by Eurorad C.T.T., Strasbourg, France) consists of two small cesium iodide scintillators (15 × 15 × 15 mm 3 ), each in a 2-mm tungsten and 1-mm aluminium housing cover (35 mm in diameter and 40 mm in height). The front end of each probe has an aluminium flange attached (3 mm in thickness and 70 mm in diameter) to facilitate easy fixation to the patient's chest with tape. Each probe weighs approximately 255 g. The probe signals are led into a dual amplifier, from which the output is fed into a multichannel analyzer system connected to a personal computer. Because the probes have separate channels, there is no electronic crossover.\nStarting at the time of the intravenous injection of 67 Ga, radioactivity was measured each minute for 1 hour. For each measurement interval, the entire spectrum of photon energies was stored on disk. During processing, the 99m Tc and 67 Ga -and plotted against time. The PLI was calculated, using linear regression analysis, from the slope of increase of the radioactivity ratio divided by the intercept, in order to correct for physical factors in radioactivity detection.\nBy taking pulmonary blood volume and thus presumably surface area into account, the radioactivity ratio represents the ratio of extravascular to intravascular 67 Ga radioactivity. The PLI represents the transport rate of 67 Ga-transferrin from the intravascular to the extravascular spaces in the lungs, and it is therefore a measure of pulmonary capillary permeability to transferrin [19, 20] . The mean PLI from the two lungs was taken. The upper limit of normal PLI is 14.1 × 10 -3 /min. Where appropriate, numbers are summarized as median (range).\nPatient data are presented in Table 1 . The patients had stayed for some time in the hospital or intensive care unit before HSV-1 was isolated, and they had been admitted primarily because of respiratory insufficiency during the course of pneumonia. Patient 4 was admitted into the coronary care unit a few days before intensive care unit admission for cardiogenic pulmonary oedema. All patients had been dependent on mechanical ventilatory support for some time before sampling. They had received adequate antibiotic therapy for pneumonia and had ALI at the time of sampling, which was of otherwise unknown origin. Table 1 shows that patients had radiographic abnormalities but without an increased PLI. Central venous pressure was not elevated, which suggests that the persistent pulmonary infiltrates were not caused by overhydration. In patients 1 and 3 a high-resolution computed tomography scan of the lungs with contrast was obtained; the findings were nonspecific, however, with alveolar consolidations and pleural fluid, even in the presence of interstitial abnormalities with a ground glass appearance in patient 3. In patient 1 a bronchoscopy was performed and there were no mucosal lesions. There was a normal distribution of lymphocyte subtypes in the lavage fluid. A transbronchial biopsy revealed interstitial inflammation with many macrophage deposits, and immunohistochemical staining for HSV-1 was negative. No multinucleated cells or cell inclusions were observed, either in bronchoalveolar lavage fluid from patient 1 or in tracheal aspirates from the other patients. In patients 1-3 concomitant isolation of bacteria by culture was regarded as bacterial colonization. Antibody testing was not done in patients 2-4 but was found to be positive for anti-HSV-1 IgG in patient 1, which is indicative of prior HSV-1 infection.\nThe antiviral agent aciclovir (10 mg/kg three times daily) was started when cultures became positive in two patients, at the discretion of the treating physician. Aciclovir was withheld in the other two patients because it was presumed that the pulmonary infiltrates were not caused by HSV-1, on the basis of a normal PLI among other findings. In patient 1, who had a normal PLI, a course of steroids was initiated on the day after the PLI was measured, and was continued despite positivity for HSV-1, reported 5 days later. All patients survived until discharge from the intensive care unit.\nThe 67 Ga-transferrin PLI is a sensitive and specific measure of pulmonary capillary permeability, which is utilized for noninvasive assessment of severity of a broad range of pulmonary conditions [19] [20] [21] . The PLI roughly parallels clinical severity (i.e. the LIS) [19, 20] . Although it involves the use of relatively routine equipment, the diagnostic method has not gained broad application, partly because of its laborious nature [20] . It has the advantage that bedside measurements are possible in mechanically ventilated critically ill patients, who cannot easily be transported. Pulmonary inflammation, of whatever cause, increases the PLI up to four times normal values in the most severe forms of lung injury, including ARDS. In less severe injury, such as impending ARDS and interstitial lung disease, the PLI is also elevated, albeit to a lesser extent, as reported by us and other groups [20, 21] .\nThe patients had in common a prior infectious episode, followed by a relatively prolonged period of respiratory insufficiency. They had persistent and nonspecific pulmonary infiltrates of unknown origin, after treatment of their primary disease, which prompted viral culture. The normal PLI observed suggests the involvement of a relatively harmless reactivation of HSV-1, rather than the presence of a primary and damaging infection. Indeed, critically ill patients with sepsis may have late immunodepression, with lymphocytic apoptosis, lymphocytopenia and T-cell anergy, promoting viral reactivation [23, 24] . Apparently, the virus must have been latent in the nerve endings of the mucous membranes of the upper respiratory tract in these patients [2, 15] . Herpesviruses (HSV-1) have frequently been isolated in vivo from respiratory secretions of patients with ARDS [3, 4] and detected in surveillance cultures from the respiratory tract of patients following burns, trauma, transplantation, major surgery and others. However, these viruses are detected in only 3% of lung biopsies from patients with prolonged and unresolving ARDS [3, 7, [9] [10] [11] [12] [13] 15] . The literature is thus widely divergent on the precise role of the virus in pulmonary disease in the critically ill and its contribution to patient morbidity and mortality [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] .\nWe believe that the tracheal aspirates were representative of lower respiratory tract secretions, in the absence of herpes orolabialis and oral epithelial cells in smears for Gram stain of the secretions. Concurrent colonization with other pathogens has previously been described [5, 13] . Because there was no overlap in the duration of stay of the patients, transmission of the virus from one patient to another can be excluded. This further suggests that respiratory HSV-1 infections in the critically ill may result from relatively harmless endogenous reactivation. Although the normal PLI argues against pulmonary parenchymal pathogenicity, tracheobronchitis caused by the virus [18, 25] cannot be ruled out, even in the absence of orolabial lesions, because bronchoscopy was not performed in three of the four patients, even though it was unremarkable in patient 1. The persistent pulmonary infiltrates in our patients may thus relate to slow radiographic resolution of prior bacterial or aspiration pneumonia, rather then superimposed infection. Moreover, computed tomographic images of the lung may be largely nonspecific [26] , and so the precise diagnostic criteria for HSV-1 pneumonia remain unclear. When properly standardized, for instance with respect to cell numbers in bronchoalveolar fluid or tracheal aspirates, quantitative cultures, viral RNA and DNA by polymerase chain reaction, could be helpful together with the PLI in further studies to quantitate viral load and the ratio of replication to shedding, and therefore the pathogenicity of the virus in the lower respiratory tract.\nIn conclusion, the anecdotal data presented here suggest that isolation of HSV-1 from respiratory secretions in the critically ill patient with a persistent pulmonary infiltrate may warrant evaluation of tissue injury potentially caused by the virus to judge its pathogenicity. This could be done using a radionuclide PLI measurement, and would help to inform decisions regarding antiviral therapy, which may have adverse effects. In some patients a normal PLI may argue against viral pathogenicity, and withholding of aciclovir in such patients may be safe." }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: Concerns about intentional releases of smallpox have prompted extensive preparations to improve our ability to detect and respond to an outbreak of smallpox [1, 3, 4, 2] . Many factors contribute to the public health challenge of understanding and preparing for smallpox, including the age and quality of epidemiological data on native smallpox and the smallpox vaccine, the difficulty of extrapolating that data to our current populations, the possible terrorist use of altered smallpox, our ignorance of terrorist methods of release, and the relatively high risk of adverse events caused by the smallpox vaccine.\nThe Centers for Disease Control and Prevention (CDC) established ring vaccination (selective epidemiological control [5] ), a strategy in which contacts of cases are identified and vaccinated, as the preferred control measure in the event of a smallpox outbreak (interim plan). The successful use of ring vaccination during the smallpox eradication campaign and its logical emphasis of case-contacts for immediate vaccination support its use (though the attribution of the success of the eradication program to ring vaccination has been challenged [6] ). Health Officers should initiate ring vaccination upon identification of the first cases of smallpox. However, there are legitimate concerns regarding the ability of public health practitioners to mount a quick, comprehensive and successful ring vaccination program, particularly in the face of a moderatesized or large smallpox outbreak. To guide preparation efforts and inform incident decision-making, we attempt to identify outbreak characteristics and response capacities that significantly impact the ability of ring vaccination to control a smallpox outbreak and to determine whether ring vaccination is useful in the presence of a mass vaccination campaign. Our analysis uses a newly developed mathematical model: a continuous-time, event-driven network simulation model of smallpox ring vaccination.\nMathematical models can advance our understanding of how a smallpox outbreak might progress. Several mathematical and computer models address the question of smallpox transmission [7] [8] [9] [10] [11] [12] [13] . The first model to appear [8] concluded that ring vaccination would be effective, but did not treat response logistics in detail; the model was linear and did not treat the depletion of susceptibles as the epidemic progressed (appropriate, however, for assessing control early in an epidemic, when the number infected is small compared to the number of susceptibles, e.g. [14] ). The innovative model by Kaplan et al. [9] emphasized the importance of resource limitation and the logistics of smallpox response, but assumed that full infectiousness began before the onset of symptoms (and the subsequent identification and removal), and did not separately monitor close epidemiological contacts of patients (which are at greatest risk, but also easiest to find and vac-cinate); the conclusions were highly critical of ring vaccination. The model by Halloran et al. [11] , a stochastic, discrete-time network model omitted the explicit inclusion of response logistics while otherwise used parameter values similar to those in Kaplan et al. [9] ; the inclusion of residual immunity from individuals vaccinated prior to the discontinuation of routine vaccination, however, led to a more favorable view of ring vaccination. The model by Bozzette et al. [12] assumed that ring vaccination would reduce the number of transmissions and focused on health care workers (but did not explicitly include the network structure of the population nor the response logistics of ring vaccination). The model by Eichner [15] did not explicitly include the network structure of the population nor the logistics of ring vaccination, but did use parameters based on data from an outbreak in Nigeria, and did distinguish close and casual contacts, case isolation, and surveillance of contacts; it concluded that case isolation and contact tracing could prevent the spread of smallpox. Finally, the individual-based model by Epstein et al. [16] presented scenarios illustrating certain alternatives to pure mass vaccination and ring vaccination of contacts of cases in preventing smallpox transmission in small populations of 800 individuals; this model includes no homogeneity assumptions, but did not analyze tracing of contacts of contacts.\nBecause none of the available models includes both network structure (with explicit contact tracing) and response logistics limited by the number of available disease control investigators [9] , we included these features in a continuous-time event-driven network simulation model of smallpox ring vaccination. Specifically, the model we developed includes the following features: exposed individuals and vaccinate them in time, resulting in a \"race to trace\" [9] .\nMild, ambulatory cases of smallpox may spread disease because such cases may be harder to recognize.\nVaccination of individuals prior to the discontinuation of routine vaccination may provide some, possibly considerable, protection against infection [11, 23, 24] , although it may also result in more mild cases which may be harder to detect.\nPublic awareness may lead to more rapid detection of cases.\nWe use this model to determine what factors promote or hinder the success of ring vaccination during a smallpox outbreak, and whether ring vaccination is useful in the presence of a mass vaccination campaign. In particular, the goal of this paper is to examine the control of smallpox by contact tracing and ring vaccination using a network model which includes response logistics [9] .\nNatural history of smallpox We briefly review relevant features of the natural history and epidemiology of smallpox [17, [25] [26] [27] 8, 28] . Following infection by the variola virus, individuals exhibited an incubation period of approximately 7-19 days with 10-14 being most typical. Sudden onset of fever and malaise, often with accompanying headache and backache, began the initial (or pre-eruptive) phase of smallpox. After 2-3 (or perhaps 4) days, individuals with the most common form, ordinary type smallpox, developed the characteristic focal rash, preceded in many cases by oropharyngeal lesions. In fatal cases of ordinary smallpox, death often occurred between the tenth and sixteenth day of symptoms; among survivors, most scabs had separated by day 22-27 of illness [26] .\nThe course of smallpox varied widely between individuals, and several different clinical classifications were developed [29] [30] [31] 17, 26] . Consideration of the clinical features and severity of smallpox is important from the standpoint of mathematical transmission modeling because (1) the clinical features affect the ease of diagnosis (and thus of case identification), (2) more severe forms of smallpox may result in more transmission, (3) vaccinated individuals may develop less severe disease. We utilize a modified or simplified version of the classification system developed by Rao [32, 31, 26] ; for the mathematical model, we will classify smallpox into five categories: early hemorrhagic, flat and late hemorrhagic, ordinary, modified, and mild. However, the clinical features and severity of smallpox in different populations may have been affected by underlying host factors, differences in viral strains, or differences in the infectious dose owing to different prevailing modes of transmission, and thus robust and precise quantitative estimates of the effects of (pre-or post-exposure) vaccination on the resulting smallpox severity, or of the infectivity differences between individuals exhibiting different forms of smallpox, are not available. The significance of such differences will be revealed through sensitivity analysis. Further details are given in Appendix 1 [see Additional file: 1].\nVaccination with vaccinia virus provided substantial protection against infection. Dixon assessed the risk of infection for an individual successfully vaccinated 3 years prior to exposure to be 0.1% the infection risk of an unvaccinated individual [17] . However, smallpox vaccination did not always take when applied, and moreover, in many instances, individuals who experienced a repeated vaccination failure developed severe smallpox upon exposure. The probability of a successful take depended on the vaccination method used; we assume that the take rate is between 95% and 100% [22, 28] . In addition to protection against infection, vaccination could in many cases modify the course of infection and reduce the severity. Vaccine protection waned over time, but individuals vaccinated 20 years prior to exposure were believed to still have half the infection probability that an unvaccinated person had [17] , and to have some protection against the most severe manifestations of smallpox. Dixon [17] believed that vaccine protection had at least three components, which decayed at different rates; for the purpose of this paper, we will assume that the severity of smallpox in previously any (recently or otherwise) vaccinated individuals follows the same distribution as for the vaccinated subjects seen in the case series observed by Rao in Madras [26] , except that anywhere from 0 to 5% of vaccinated subjects develop smallpox too mild to diagnose without special surveillance or awareness. Observe that the vaccinated cases studied by Rao were vaccinated (at some point in their lives) before exposure, rather than after exposure to smallpox.\nSmallpox was largely a disease of close contacts [17, 26, 33] , spread primarily through face to face contact with an infected person (or occasionally through contaminated clothing). Individuals in the incubation period of smallpox were not infectious, and long term carriers did not exist. Patients were believed to be infectious following the development of oropharyngeal lesions, which could precede the rash by 24 hours [26] . However, patients were believed to be most infectious during the first week of the rash [26] ; Dixon (1962) believed that patients could be infectious from the onset of acute viremia, but most evidence suggested that little transmission occurred prior to the development of the rash [26, 33] . The more severe the case, the more infectious they appeared to be [34] ; mild cases were believed to have very little infectiousness. While scabs contained infectious material and patients were considered to be infectious until the last scab fell off, in practice patients were not highly infectious during the scabbing phase. Importantly, patients who had been vaccinated were found to cause fewer secondary cases [34] . Very severe cases, such as hemorrhagic or flat smallpox, occasionally resulted in considerable transmission, owing to diagnostic difficulties; mild cases, in which the patient remained ambulant during the course of the disease, could cause considerable spread as well [35, 36] . Within a household or family dwelling, the secondary attack rate of unvaccinated susceptibles depended on the time and place, occasionally below 50% [29] , but often approaching 100% [37] . Drier conditions were often believed to favor transmission [17, 27] , so that lower rates of transmission derived from tropical regions may not be applicable to the temperate zone [38] . The number of secondary cases resulting from a given importation into Europe varied widely [39] , with most importations yielding few cases, but with the occasional large outbreak being seen.\nMathematically, we represent the course of smallpox according to Figure 1 . We distinguish eight epidemiologically relevant states: (1) just following exposure, during which time vaccination could afford complete protection against disease, (2) a period of several days during which vaccination will not prevent disease, but may still reduce the severity of disease, (3) still prior to the development of symptoms, but too late for vaccination, (4) the beginning of the pre-eruptive period, during which the patient exhibits fever, malaise, and possibly other symptoms, but is not yet infectious, (5) a short period prior to the appearance of the rash, during which the appearance of oropharyngeal lesions will permit variola transmission, (6) the first week of the rash, during which time the patient is most infectious, (7) and (8), succeeding stages of the rash, during which time the patient is less infectious. For each of these states, we assume that conditional on surviving, the waiting time until the next stage is chosen from a uniform distribution as indicated in Appendix 2 [see Additional file: 2], except that the incubation period (the time from infection until Stage 4) is derived from estimates of the incubation distribution of smallpox based on importation cases in Europe [26] (see Appendix 2 [see additional file 2] for details). We chose to sample from a uniform distribution as a simple way to ensure a minimum waiting time in each state; many alternatives to this choice are possible.\nWe simulate the transmission of smallpox on a \"smallworlds\" network (highly clustered, but with short characteristic path lengths) [40] . Specifically, we assume that each person is located in a single household, and that the transmission rates were greatest in the household. We also assume that a fraction of the population are grouped into workplace or social groups, in which transmission may also occur, but with a lower rate per unit time than for household contacts. Finally, we assume that with a still smaller probability, any individual may transmit infection to any other individual in the population (casual contacts).\nIn general, in a network-structured model, the number of secondary cases caused by an index case in a completely susceptible population is not a useful index of epidemic potential [41, 42] (for a simple example, see [43] ), since (for instance) an individual could infect everyone in his or her household, and not cause a widespread epidemic unless between-household transmission were sufficiently frequent. Rather than constructing the appropriate generalized basic reproduction number for our model (leading to highly cumbersome expressions), we chose an alternative (ad hoc) index of epidemic potential. For any given scenario of interest, we simulated the introduction of 10 index cases at random into a population of size 10000, and operationally defined \"containment\" to occur whenever the final size of the epidemic was less than 500 cases within 250 days (we showed, in the discussion of Figure Smallpox stages used in the simulation model Figure 1 Smallpox stages used in the simulation model. Flat and ordinary smallpox rashes are indicated with more dots than modified and \"mild\" smallpox, suggesting potentially greater infectiousness. Hemorrhagic smallpox is indicated by horizontal line shading. Further details are provided in Table 6 . 5A below, that in nearly all cases, the 250-day window differs very little from a 1000-day window). Because we simulate a disease with a finite duration on a finite and nonrenewing population, epidemic extinction always occurs in finite time.\nWe assume that even in the absence of specific case investigations, the presence of smallpox symptoms will prompt patients to be diagnosed; we assume, however, a higher diagnosis rate for all forms of ordinary smallpox than for the severe flat and hemorrhagic forms, or for the mildest form. We assume that once an individual is diagnosed, their household and workplace contacts are investigated and detected with some probability; we assume that a high fraction (such as 95%) of household contacts are assumed to be traceable (see below). We assume that the fraction of workplace/social contacts that are traceable is less than the fraction of household contacts that are traceable; we assume that no casual contacts are traceable.\nHigh contact-finding rates may be plausible; we examined San Francisco Department of Public Health records of contact investigations for meningococcal disease (like smallpox, a potentially fatal disease for which rapid intervention may prevent mortality and morbidity). Records were available from December 2001 to April 2002; 13 such investigations during this period resulted in identification of 62 household contacts, all of which were contacted; out of 38 workplace/social contacts identified, 32 were contacted (84%).\nIn our model, we assume that identified asymptomatic contacts are vaccinated, quarantined, and monitored for symptom development, while symptomatic patients are isolated and treated as necessary [9] ; thus, the modeled interventions include more than ring vaccination alone. Finally, we include the possibility that all contacts (of both symptomatic and asymptomatic) traced and the same procedure applied, so that all contacts of contacts would be investigated. We assume that uninfected or asymptomatic individuals who are visited or traced individuals will be diagnosed more rapidly than if they had not been traced; in fact, such individuals would be isolated and would not be able to continue a chain of transmission. We follow previous models [9] in assuming a limited vaccination capability of K r per day for ring vaccination. We assumed one of two strategies for contact tracing: (1) tracing only of direct contacts of diagnosed cases, and (2) tracing of contacts of contacts of diagnosed cases as well as direct contacts.\nThe contact structure of the network is illustrated in Figure 2 . Observe that individuals b and c are household contacts of individual a, so that if individual a were a smallpox case, an attempt would be made to find and vaccinate individuals b and c as household contacts of a case. If individuals a and b were both cases, then two attempts could be made to find individual c. We have modeled the effect of multiple contact-finding attempts conservatively in the sense that if the first attempt to find an individual as a household contact (of a case or of a contact) is determined to fail, no further attempts will be made. This maintains the failure rate of contact tracing (looked at from the standpoint of finding individuals) even in large households. Similar considerations apply to workplace/social groups. Figure 2 Network structure shown for households (joined by thick lines) of size 3 and workplace/social groups of size 4 (joined by thin lines); a small portion of the network is shown. Individual a has two household contacts (b and c), and three workplace/social contacts (d, e, and f). If individual a were a smallpox case, the household contacts would be at highest risk for acquiring smallpox, followed by workplace/social contacts; all individuals in the population are at a low risk of casual transmission from individual a. Case investigation of individual a would identify the direct contacts b-f with probabilities that depend on whether the contact is household or workplace/social; if such individuals are identified, they will be vaccinated. If contacts of contacts are being traced, the investigation will subsequently identify individuals g-p. \nWe analyzed the model in three ways. First, we selected a Latin Hypercube sample [44] [45] [46] of parameters chosen uniformly from the parameter ranges given in Appendix 2 [see additional file 2] , and simulated the transmission and control of smallpox to determine which parameters were most important for contact tracing and ring vaccination to be effective. Second, we used the same Latin Hypercube Sample of input parameters, but assumed that all disease control efforts were inactive. We used these parameters to simulate smallpox transmission, but then iteratively selected transmission parameters so that (1) between 1% and 10% of new infections resulted from casual (random) transmission, and (2) each index case resulted in between two and five secondary cases (thought to be plausible for historic smallpox; [8] suggest three secondary cases). For each of the resulting smallpox parameter sets using 100 stochastic simulations per set, we determined the daily ring vaccination/case tracking capacity needed to contain all simulated smallpox epidemics (i.e., keep the total number of cases below 500 within 250 days). Third and finally, we chose parameter values to yield an moderately large smallpox epidemic (with each index case causing approximately six secondary cases), and present illustrative scenarios for ring vaccination. These scenarios are intended to complement the simulations which were calibrated to historic smallpox, since the characteristics of smallpox that may be used in a deliberate release are not known. It is important to realize that in our model, the case finding time determines the fraction of contacts that will become infected, and that our model parameters have been chosen to yield quite rapid transmission to close contacts; in reality, much transmission of natural smallpox occurred through \"sickbed\" routes which would not occur in a modern setting [47] , so that in this sense our model errs considerably on the side of caution and pessimism.\nTo determine which of the input parameters were most important in determining the total number of smallpox cases, we selected a Latin Hypercube sample of size 1000 from the input parameter ranges indicated in Appendix 2 [see additional file 2] and simulated the mean number of cases within 250 days in a population of 10000. We then computed the partial rank correlation coefficient [46] (PRCC; see Appendix 2 [see additional file 2]) between each input parameter and the number of smallpox cases; when the PRCC is close to zero, the value of the parameter has little relation to the simulation output; when the PRCC is close to +1.0 or -1.0, the value of the parameter is highly important in determining the simulation output. Neglecting the number of index cases (which is directly related to the number of new cases), those parameters whose PRCC exceeds 0.1 are shown in Table 2 . Most of these parameters identified as important are related to the density of available contacts (mean household size, prior vaccination fraction, and protection due to prior vaccination) or the transmission rate and infectivity (including the length of the pre-eruptive infectious period (stage 5 in Figure 2 )). Note, however, that the speed of ring vaccination (household tracing delay) and faster diagnosis due to awareness of the outbreak are important parameters. Additionally, the infectivity of mild cases appears as an important parameter as well.\nTo explore factors which contribute to the success of ring vaccination, we chose smallpox scenarios which resulted in severe and fast-moving epidemics in the absence of disease control; these simulated epidemics are considerably more severe than is believed likely under present circumstances.\nWe used these parameters to simulate smallpox epidemics beginning with 10 cases, for a variety of levels of ring vaccination capacity per day (contact tracing capacity per day), as shown in Figure 3A . In this Figure, we assume that the population size is 10000, and that the epidemic began with 10 infected individuals. The mean household size is assumed to be 4, the mean size of the workplace/social contact group is 8, and contacts of contacts are traced. We assume that each day, the number of contacts that can be traced and vaccinated as a result of case investigation is 0, 10, 20, 30 and 40 per day; the probability of finding a workplace/social contact is assumed to be 80%. The Table 1 .\nBecause we assumed nonzero diagnosis probabilities during the prodromal period for all individuals in Figure 3A , we repeated the simulation assuming no diagnosis in the prodromal period unless individuals were under specific surveillance. The results were nearly identical: assuming 30 contact tracings (ring vaccinations) per day, we found 26% of the scenarios in Figure 3A exhibited decontainment, and 28% assuming no diagnosis during the prodromal period; assuming 40 contact tracings per day, we found 1 out of 100 scenarios showed loss of containment in Figure 3A and when we repeated the scenario of Figure 3A assuming no diagnosis during the prodromal period.\nIn Figure 3B , we illustrate control of an epidemic for which all parameters are identical to Figure 3A , except that the workplace/social group size is 12 (instead of 8, as in Figure 3A ), and the probability of finding workplace/ social contacts is 60% (instead of 80%, as in Figure 3A ). In this case, the larger size of the workplace/social groups and the lower contact finding probability makes it necessary to have a higher ring vaccination capacity to attain a high probability of containing the epidemic, and on average it takes longer for eradication to finally occur.\nFinally, in Figure 3C , we show control of an epidemic in a population of 100,000, beginning with 1000 initial infectives, keeping all other parameters the same as in Figure 3A . Each curve corresponds to the indicated number of possible ring vaccinations per day. This figure shows that assuming sufficient capacity, ring vaccination is in principle capable of containing even epidemics beginning with very many infected individuals. However, mass vaccination in such cases is justified because of the far larger number of individuals at risk and the inability to perform such extensive contact tracing.\nIn Figure 3D , we compare the effect of tracing contacts of contacts (as described in Appendix 2 [see additional file 2]) at different levels of ring vaccination capacity. Thin Figure 5A , 5B φ\nProb. of finding household contact 0.95 Delay, tracing household contacts 1-5 days Expanding severe smallpox epidemic Figure 3 3A -Expanding severe smallpox epidemic beginning with 10 initial cases, assuming 0, 10, 20, 30, and 40 possible ring vaccinations per day. The household size is 4 and the workplace/social group size is 8; we assume 95% of household contacts are traceable (with a mean delay of 1 day) and 80% of workplace/social contacts are traceable (with a mean delay of 2 days). We also assume that 25% of the population have 50% protection from infection resulting from vaccination prior to the discontinuation of routine vaccination. We assume that infection will be transmitted to close contacts with a mean time of 0.2 days, and that each person while infective causes on average 0.15 casual (untraceable) infections per day. We assume that individuals are 20% as infectious in the day just before the appearance of the rash as they will be during the first week of the rash, and that individuals are 20% as infectious as this (4% as infectious as during the first week of the rash) during the prodromal period. We assume that diagnosis rates will increase by a factor of 50% after smallpox becomes known to the community; we assume that each individual contacted during an investigation has a additional diagnosis or removal rate of 0.75 per day following the onset of symptoms (reflecting enhanced surveillance or contact isolation). Important parameters are summarized in Table 1 ; the full set of parameter choices is outlined in Tables 8-11 in Appendix 2 [see additional file 2] . Diagnosis times are discussed in Appendix 2 [see additional file 2]. 3B -An expanding severe smallpox epidemic under inadequate ring vaccination is shown for parameters identical to Figure 3A , except that workplace/social group sizes are 12 (instead of 8), and the probability of tracing workplace/social contacts is 0.6 (instead of 0.8).\n3C -A severe smallpox epidemic is controlled by ring vaccination despite the large number of initial cases. The parameters are identical to Figure 3A , except that 1000 index cases inaugurate the attack in these scenarios (and ring vaccination capacity is much greater, as indicated). While not recommended, ring vaccination may ultimately halt epidemics beginning with many index cases if sufficient vaccination capacity were available, contact finding feasible, and follow-up sufficient. 3D -Tracing contacts of contacts (red) is beneficial when sufficient contact tracing/ring vaccination capacity exists (dotted lines). In these scenarios, all parameters are the same as in Figure 3A ; the number of contact tracings possible per day is either 20 or 40 per day. Contacts of contacts are traced in two scenarios; in the other two, only direct contacts of cases are traced. For low levels of ring vaccination (20 per day), tracing contacts of contacts is harmful; for high levels (40 per day) of ring vaccination, it is beneficial to trace contacts of contacts. When the contact tracing/ring vaccination capacity is too small to adequately cover contacts of the cases themselves, diversion of resources to contacts of contacts is harmful; however, provided that sufficient capacity exists, tracing contacts of contacts helps outrun the chain of transmission. Each line corresponds to the average of 100 realizations. The average number infected on each day is plotted in the Figure. The figure illustrates that when ring vaccination capacity is low, tracing contacts of contacts (as modeled) yields a more severe average epidemic; when ring vaccination capacity is large, tracing contacts of contacts results in a less severe average epidemic; if the contact tracing/ring vaccination capacity is too low to cover adequately the contacts of contacts in addition to the contacts of cases, extension of tracing to the contacts of contacts (the second ring) is harmful; however, if there is sufficient capacity to cover the contacts of contacts, then the tracing of contacts of contacts is beneficial.\nFinally, in Figure 4 , we illustrate the considerable variability that may be seen from simulation to simulation. This figure shows twenty simulations when contacts of contacts are not traced. Stochastic variability between realizations is considerable, even when all parameters are held constant; this variability is expected to limit the ability to make inferences based on observation of a single realization of the process.\nBecause our baseline hazard for infection of individuals may be larger than would be expected for naturally occurring smallpox, we examined the effect of more realistic values of this hazard. In particular, we chose different levels of ring vaccination capacity (10, and 20) , and of the relative hazard for workplace/social contacts, and then chose values of the baseline hazard for infection varying from 0.5 per day (for a mean time to infection of 2 days) to 2 per day (for a mean time to infection of one half day), Table 3 : Estimated decontainment probability for different levels of ring vaccination capacity (Kr) and relative hazard for infection due to workplace/social contacts (h2), for different levels of the baseline hazard for infection from household contacts λ (based on replications of 100 simulations for each level). For each scenario, 10 index cases were introduced into a population of size 10000. All other parameters were the same as for Figure 3A . As before, we define decontainment to mean that the total number of cases from 10 index cases eventually exceeded 500 by day 250. and introduced 10 index cases into a population of 10000. We then repeated this 100 times, and reported the fraction of scenarios in which the number of infections ultimately exceeded 500 (as before, chosen as a cutoff to indicate the ultimate \"escape\" of containment of the epidemic). These results, shown in Table 3 , support the idea that ring vaccination can easily control introduced smallpox provided there is sufficient capacity and efficacy of tracing.\nBecause of considerable uncertainty in the model parameters, we chose a collection of parameter values, and for each, estimated the containment probability (operationally defined as fewer than 500 total cases as a result of 10 index cases, within 250 days). We estimated this containment probability by simulating the smallpox epidemic 100 times for the same parameter values, and computing the frequency out of these 100 realizations for which fewer than 500 index cases resulted within 250 days.\n(Using a 1000 day window produces slightly smaller containment estimates; for 3 out of 1000 parameter set choices, this difference was greater than 0.06; the maximum difference seen was 0.23; the mean absolute difference was 0.0029; in only one case out of 1000 did we see containment in all 100 cases for the 250-day window, but not in all 100 cases for the 1000-day window).\nOne thousand scenarios chosen from a Latin Hypercube sample were analyzed, and as indicated before, we chose the hazard for close contact transmission and the hazard for random transmission to guarantee that between 2 and 5 secondary cases per case occur, and that no more than 5% of cases are attributable to random transmission (we refer to this set as the \"calibrated\" scenarios further in this text). Having chosen this collection of 1000 parameter sets, we considered two levels of two different control parameters which were applied to each (so that each of the 1000 parameter sets were simulated under four different control conditions). The first of the two control parameters was the probability of workplace/social group contact finding; we chose values of 0.8 and 0.9 for this parameter (the household contact finding probability was 0.95 in all cases). The second of the control parameters was the rate of diagnosis (and effective removal) from the community of cases developing among previously identified and traced contacts who were initially asymptomatic (we refer to this as the monitored diagnosis rate); we assumed first a low level corresponding to a mean diagnosis time of one day from the onset of symptoms, and a high level corresponding to a mean time of 3 hours from the onset of symptoms (high levels of the monitored diagnosis rate correspond effectively to isolation of contacts). Finally, we assumed a doubling of the diagnosis rate after the beginning of widespread community awareness of smallpox. We then computed the containment fraction at different levels of ring vaccination capacity (contact tracing capacity per day). Thus, for each of 1000 scenarios (parameter set choices), we assigned the workplace/social group contact tracing success probability (υ 2 ), the monitored diagnosis rate φ (Appendix 2 [see additional file 2]), and the contact tracing/ring vaccination capacity per day (K r ). We then performed 100 realizations beginning with 10 index cases, and computed the containment fraction (fraction showing fewer than 500 cases in 250 days, beginning with 10 index cases). Thus, for each of the two choices each of υ 2 and φ, and for each value of K r we examined, we obtained 1000 values of the containment fraction. We use the resulting distributions in Figure 5A (averaging over these 1000 containment fractions), and Figure 5B (displaying the minimum value of the 1000 containment fractions).\nIn Figure 5A , we plot the mean containment fraction (averaging the containment fraction over all 1000 scenarios), as ring vaccination capacity varies, for the two levels of workplace/social group contact finding probabilities (0.8 and 0.9), and for the two levels of monitored diagnosis rate among initially asymptomatic contacts (1 day -1 and 8 day -1 ). For low levels of ring vaccination (traceable contacts per day), the epidemic is almost never contained, but for ring vaccination levels near 50-60 per day (5-6 per index case per day), the average containment fraction Figure 4 Stochastic variability is illustrated by plotting the number of infectives over time over multiple replications. In this example, most simulations exhibit rapid containment of smallpox. The mean number of cases (averaging over simulations) is influenced by a small number of simulations exhibiting an uncontained epidemic. The parameters are the same as in Figure 3A , except that contacts of contacts are not traced in these replications. \nThe mean containment probability Figure 5 5A -The mean containment probability increases as the number of ring vaccinations per day is increased. For this figure, the 1000 \"calibrated\" parameter sets were chosen, and for each parameter set, 100 realizations were simulated and the fraction of these for which the epidemic was contained to fewer than 500 cases was determined. The average of these 1000 containment fractions is plotted on the vertical axis. We assumed a household contact finding probability of 95% and that the diagnosis rates double after community awareness of the epidemic. We considered high levels of workplace/social (w/s) contact finding (0.9), as well as moderate levels (0.8). We also considered two levels of diagnosis of smallpox among investigated (alerted) contacts: high levels (corresponding to a 3 hour mean delay, indicated by \"high contact isolation\"), and moderate levels (corresponding to a one day delay, and indicated by \"less contact isolation\"). The figure shows four such conditions, a. high workplace/social contact finding probability and high contact isolation, b. moderate workplace/social contact finding probability and high contact isolation, c. high workplace/social contact finding probability and less contact isolation, and d. moderate workplace/social contact finding probability and less contact isolation. All other parameter values were chosen from the uncertainty analysis (the 1000 \"calibrated\" parameter sets). In this figure, \"contact isolation\" refers to the monitored diagnosis rate, i.e. the rate at which previously asymptomatic contacts who subsequently develop disease will be diagnosed (φ, Table 1 , Table 8 ). 5B -The minimum containment probability out of the same 1000 scenarios chosen in Figure 5A . Whereas in Figure 5A , we averaged the simulated containment frequency (out of 100 realizations for each scenario), in this figure we determined which of the 1000 scenarios led to the lowest containment frequency, and we plotted this single worst (out of 1000) containment frequency, at different levels of ring vaccination capacity, for the same four conditions as in Figure 5A : a. high workplace/ social contact finding probability (0.9) and high contact isolation (effective 3 hour delay following symptoms), b. moderate workplace/social contact finding probability (0.8) and high contact isolation, c. high workplace/social contact finding probability (0.9) and less contact isolation (effective one day delay), and d. moderate workplace/social contact finding probability (0.8) and less contact isolation. All parameters are the same as in Figure 5A (the household contact finding probability is 0.95 for all scenarios, and the diagnosis rates are doubled after the onset of community awareness). In this figure, \"contact isolation\" refers to the monitored diagnosis rate, i.e. the rate at which previously asymptomatic contacts who subsequently develop disease will be diagnosed (φ, c.\nd.\nbecame close to 1. However, this average conceals the fact that for some scenarios (parameter sets chosen from the calibrated uncertainty analysis), control remains difficult or impossible even at high levels of ring vaccination. Therefore, in Figure 5B , we plotted the single lowest containment fraction seen out of the 1000 computed; focusing on the single worst scenarios reveals a different picture, and shows that isolation of asymptomatic contacts and very high probabilities of finding workplace or social contacts would be needed to control smallpox under these most pessimistic parameter choices.\nRapid contact tracing in ring vaccination may play an important role in suppressing the epidemic, since the longer it takes to trace a contact, the less likely the vaccine is to be efficacious, and the more opportunities the infected individual may have to transmit disease before they are finally located, isolated, and vaccinated if appropriate. We illustrate this possibility in Figure 6 by examining the same scenario we showed earlier in Figure 3A (e.g. households of size 4, workplace/social groups of size 8, 95% of household contacts traceable, 80% of workplace/social groups traceable, an average time to infection for a household contact of an infective given by 0.2 days). We assume in one case that contacts may be traced quickly (1 day for a household contact, 2 days for a workplace/ social contact), and in the other that the contacts are on average found slowly (5 days for a household contact, 10 days for workplace/social contacts); we assumed 30 ring vaccinations (traceable contacts) possible per day. In this scenario, the epidemic is more severe and containment (as we have been defining it) less likely when contact tracing is slow: in the fast scenario, 238 infections occurred on average and the (estimated) containment probability was 99%; for the slow scenario, on average 3587 infections occurred and the (estimated) containment probability was only 1%.\nWhile Figure 6 illustrates the possibility that rapid contact tracing may be of decisive importance in some scenarios (parameter set choices), this is not always the case. For some parameter sets, the probability of tracing contacts (household or workplace/social) may be too low, or the transmission rate too high, for more rapid contact tracing to make any difference. Conversely, for other parameter sets, the smallpox transmission rate may be so low that smallpox is easily contained even with slow contact tracing. While rapid contact tracing is never harmful, overall, how typical are the results of Figure 6 (in which rapid contact tracing was important in ensuring the efficacy of ring vaccination)? To address this question, we simulated the growth of smallpox for the 1000 \"calibrated\" scenarios we used in Figure 5A and 5B. As before, we assumed ten initial cases, and (as in Figure 6 ) that 30 ring vaccinations were possible per day; then we simulated 100 epidemics assuming one day to find a household contact (and 2 days to find a workplace/social contact). We then simulated 100 epidemics assuming that it takes five days to find a household contact and 10 days to find a workplace/social contact (as in Figure 6 ). For each of these 1000 scenarios, we calculated the fraction of simulations for which the total number of cases is 500 or less within 250 days, i.e. the containment fraction. For nearly all scenarios (parameter set choices), the containment fraction was smaller (sometimes much smaller) when the contact finding time is faster (since faster contact finding, all else being equal, improves smallpox control, as illustrated in Figure 6 ). However, for 64.5% of the scenarios (parameter set choices) examined, the difference was less than 2.5% in absolute terms (smallpox was either contained or not contained depending on other factors, and rapid contact tracing did not make the difference). On the other hand, for 18.7% of the scenarios examined, the absolute difference in the containment probability was 20% or more; thus, a substantial difference in containment probability is occa-Faster contact tracing Figure 6 Faster contact tracing may improve the efficacy of ring vaccination. We assume the same baseline parameters as in Figure 3A (e.g. households of size 4, workplace/social groups of size 8, 95% of household contacts traceable, 80% of workplace/social contacts traceable), and 30 ring vaccinations available per day (with contacts of contacts not traced). The fast scenario corresponds to an average one day delay for household and two days for workplace/social contacts (as in Figure 3A ); the slow scenario corresponds to an average five day delay for household and ten day delay for workplace/ social contacts. This figure shows the average of one hundred realizations starting with ten index cases. Effect of more rapid diagnosis Public awareness of smallpox, leading to more rapid isolation and identification, may play an important role in eliminating the epidemic, as illustrated by the scenarios in Figure 7 . We assumed 20 ring vaccinations possible per day, a capacity too small to contain the epidemic in the absence of increased surveillance or diagnosis; the black line in the figure shows the steeply rising average number of cases for the first 100 days. If, however, surveillance or public awareness of the symptoms of smallpox increases the diagnosis rate by 50% (multiplies the baseline diagnosis rates by 1.5), containment becomes possible (blue line); with a doubling of the diagnosis rate (red line) the peak number of cases is lower still. In these scenarios, increased diagnostic rates markedly improve the ability of ring vaccination to control the epidemic, this suggest that any ring vaccination effort be accompanied by increased public awareness and surveillance.\nIn many cases, however, more rapid diagnosis was not required for ring vaccination to be effective. As before, we simulated smallpox epidemics for each of 1000 calibrated scenarios, performing 100 realizations each beginning with 10 index cases, and computed the fraction of scenarios for which the epidemic was always contained (as defined earlier), assuming no change in diagnosis rates. We assumed 80 ring vaccinators per day, contact finding probabilities of 0.95 for households and 0.8 for workplace/social contacts (as in Figure 3A ). Under these assumptions, for 83.4% of the scenarios, the epidemic was contained within 500 total cases in each of the 100 realizations, even with no change in diagnosis rates. Uncertainty analysis (using the 1000 calibrated scenarios, and based on the fraction of 100 replications showing decontainment) revealed the most important parameters which predict the failure of ring vaccination without more rapid diagnosis were the same as we found in the earlier uncertainty analysis; a higher fraction vaccinated before the epidemic, smaller households or workplace/social groups, less transmissibility, lowered infectivity prior to the rash, more rapid diagnosis, and a higher rate of diagnosis for alerted individuals all contribute to a greater containment probability even without an overall increase in the diagnosis rate.\nWe have been assuming that whenever an individual is contacted during an investigation, the individual will be diagnosed more quickly should they subsequently develop symptoms. When transmission is assumed to be very rapid (smallpox is assumed to be highly contagious), most individuals may already be infected when identified through contact tracing from an infective. Using the scenario we examined in Figure 3A , we see that continued surveillance of contacts is an essential component of effective ring vaccination designed to control rapidly spreading smallpox: if smallpox in a contact is not diagnosed any more quickly than for a non-contact, containment by ring vaccination requires over 98% contact finding probabilities for both household and workplace/ social contacts -even if unlimited numbers of ring vaccinators are available; containment cannot be guaranteed by adding additional ring vaccination capacity if the contact finding rates are too low and/or the follow-up for contacts is insufficient. Smallpox which is transmitted less rapidly to contacts would, however, be containable with a lower contact finding probability (results not shown).\nFinally, we used the \"calibrated\" scenarios (parameter set choices) to explore the levels of contact finding probability needed to contain the epidemic (as before, defined to mean 500 or fewer cases ultimately resulting from ten initial cases) ( Table 4 ). In these scenarios, we assumed that all traceable contacts were followed up very More rapid diagnosis Figure 7 More rapid diagnosis due to public awareness or increased surveillance may lead to far more effective epidemic control. We assume the same baseline parameters as in Figure 3A , and averaged 100 realizations of the epidemic beginning with 10 index cases and assumed a ring vaccination capacity of 20 per day (and contacts of contacts not traced).\nFor the black line, the diagnosis rate of cases does not change after the first case is identified (the multiplier is 1.0); for the blue line, the diagnosis rate increases by 50% (multiplier 1.5) after the first case is identified (as in Figure 3A ), resulting in substantially fewer cases; and for the red line, the diagnosis rate is doubled (multiplier 2.0) after the first case is identified, resulting in still fewer cases. quickly (1/a = 1 hour, so that cases arising in previously contacted persons almost never transmit the infection further). We chose different levels of household and workplace/social contact finding probabilities and different levels of ring vaccination capacity, and performed 100 replications of each of the 1000 different scenarios. In Table 4 we report the fraction of scenarios for which all 100 replications exhibited containment. Scenarios in which smallpox is highly contagious require high contact finding probability to ensure the containment of the epidemic.\nTransmission prior to the rash makes epidemic control more difficult. In Figure 8 , we show an expanding smallpox epidemic assuming differing levels of infectivity prior to the rash (adding increased infectivity prior to the rash, keeping constant the infectivity after the rash). We assume all parameters are the same as in Figure 3A (and that the ring vaccination capacity is 40 per day). Infectivity prior to the rash is modeled as the relative infectivity during the short (1 day) period of oropharyngeal lesions just prior to the rash (compared to the infectivity during the first week of the rash), and as the relative infectivity during the prodromal period (relative to the period just prior to the rash). We consider three scenarios: a relative infectivity during entire period is one (i.e., infectivity during the prodromal period and just prior to the rash is the same as during the first week of the rash), b the relative infectivity just prior to the rash is the same as during the first week of the rash, but during the prodromal period is 4% (as in Figure 3A ) of this value, and c the relative infectivity just prior to the rash is 20% of the infectivity during the first week of the rash, and during the prodromal period is 20% of this value. The figure shows that increased infectivity just prior to the rash leads to a larger epidemic (comparing b and c); in case b (high infectivity just prior to onset of rash), loss of containment occurs 36% of the time (but in none of the 100 realizations shown in case c (low infectivity prior to rash)). Scenario a (full infectivity during entire the prodromal period) showed loss of control in every realization. Increasing the ring vaccination capacity from 40 per day to 80 per day (results not shown) led to containment in all of the realizations with high infectivity just prior to the rash and low infectivity during the prodromal period (case b), but made no difference if the infectivity was as high during the prodromal period as during the rash (case a). While intuitively adding additional infectiousness must increase the number of secondary cases and make control more difficult, these results do illustrate that even a small amount of increased infectiousness prior to the rash (when diagnosis is more difficult) may substantially increase the difficulty of smallpox control.\nFinally, in Figure 9 , we present scenarios in which each of four other parameters are modified from the baseline values of Figure 3A , assuming 40 contact tracings (ring vaccinations) are possible per day (line a in the figure) . Specifically, we assume that severe smallpox (hemorrhagic and flat) on average takes four times longer to diagnose and isolate than ordinary smallpox (case b), Table 4 : Containment of severe smallpox at different levels of contact finding. The first three columns are assumed levels for the probability of finding a household contact, the probability of finding a workplace/social (W/S) contact, and for the number of contact tracings/ring vaccinations possible per day; the last two columns express (as percentages) the resulting probability of containment given the assumed contact finding probabilities and contact tracing capacities; two containment probabilities are given: the containment probability when only contacts of cases are traced (first column, \"Contacts\"), and the containment probability when contacts of contacts of cases are traced in addition to the contacts of cases (second column, \"Contacts of Contacts\"). All other parameters are the same as in Figure 3A . that no one in the population has prior vaccination protection (from before the discontinuation of routine vaccination, case c), that 10% more smallpox is too mild to diagnose (but still contagious, case d) compared to baseline, and finally that the vaccine is completely ineffective (case e). Each of these scenarios will be discussed further below.\nScenario b was motivated by the possibility that individuals with severe forms of smallpox may be more difficult to diagnose, and thus remain infectious in the community longer (despite the much greater degree of illness of such patients), or that such patients may be more infectious. In this particular case, quadrupling the mean diagnosis time led to one additional replication out of 100 in which containment was not achieved (2/100, compared to the baseline of 1/100). However, we assumed that community awareness of smallpox leads to the same relative rate of increased diagnosis among severe cases as for ordinary cases, and that the most severe forms are relatively rare. In addition to the scenario shown in the figure, we also replicated the same 1000 \"calibrated\" simulations, assuming that in each case 40 contact tracings per day are possible and that the diagnosis time for severe cases was four times that of ordinary cases. Finally, we repeated each \"calibrated\" scenario 100 times assuming long diagnosis times for severe cases, and not making this assumption, and found that the difference in the decontainment fraction was not large (results not shown).\nScenario c illustrates that vaccination prior to the discontinuation of routine vaccination does play a role in smallpox control by ring vaccination; there were more decontainment scenarios (5/100) when no prior Transmission prior to the rash Figure 8 Transmission prior to the rash makes epidemic control more difficult. The figure shows a expanding smallpox epidemic assuming differing levels of infectivity prior to the rash. We assume all parameters are the same as in Figure 3A (and that the ring vaccination capacity is 40 per day). Infectivity prior to the rash is modeled as the relative infectivity during the short (1 day) period of oropharyngeal lesions just prior to the rash (compared to the infectivity during the first week of the rash), and as the relative infectivity during the prodromal period (relative to the period just prior to the rash). For scenario a, relative infectivity during the prodromal period and just prior to the rash is the same as during the first week of the rash, for scenario b, the relative infectivity just prior to the rash is the same as during the first week of the rash, but during the prodromal period is 4% (as in Figure 3A ) of this value, and for scenario c, the relative infectivity just prior to the rash is 20% of the infectivity during the first week of the rash, and during the prodromal period is 20% of this value (these two parameters are the same as in Figure 3A ). Additional scenarios, assuming 40 ring vaccinations or con-tact tracings possible per day, and that contacts of contacts are traced; all parameters are identical to those in Figure 3A unless otherwise indicated Figure 9 Additional scenarios, assuming 40 ring vaccinations or contact tracings possible per day, and that contacts of contacts are traced; all parameters are identical to those in Figure 3A unless otherwise indicated. The figure shows the average of 100 replications of five scenarios (Case a repeats the result from Figure 3A for reference); the numbers in parentheses in the legend are the corresponding fraction of the 100 scenarios for which decontainment occurred. For case b, we assumed that flat and hemorrhagic smallpox cases took four times as long on average to diagnose as ordinary cases; for case c., we assumed that no one in the population had prior protection (as opposed to 25% for Figure 3A) ; for case d, we assumed that an additional 10% of individuals (13% instead of 3%) would develop mild smallpox (with 75% developing ordinary smallpox instead of 85% as in Figure 3A ); and for case e, we assumed that the vaccine is completely ineffective and provides no protection against infection. protection exists in the population. The results suggest that prior vaccination aids in the control of smallpox, but that it is not strictly necessary for control (in this scenario, 95% of the replications exhibited containment). In Figure 3A , we assumed 25% of individuals had protection due to vaccination prior to the discontinuation of routine vaccination; in scenario c of Figure 9 , we assumed this fraction was zero.\nScenario d demonstrates that if 10% more smallpox infections (in absolute terms, i.e. 13% compared to 3% in Figure 3A ) lead to mild cases among individuals with no prior protection, the epidemic is more difficult to contain (13/100 replications showed loss of containment).\nFinally, scenario e demonstrates that containment is still possible even when the vaccine is completely ineffective in everyone -because of case isolation and isolation of contacts (and of contacts of contacts). Here, with 40 contact tracings possible per day, 55% of the replications nevertheless exhibited containment even with a vaccine which offered no protection whatever. With 90 contact tracings possible per day, all replications exhibited containment even assuming no vaccine protection.\nAlthough less efficient than ring vaccination in the sense that more vaccinations must be delivered to eliminate infection, comprehensive mass vaccination following the introduction of smallpox is sufficient to eliminate the infection. In Figure 10 , we show the probability of achieving containment (defined to be fewer than 500 total cases resulting from 10 index cases) for different levels of ring vaccination (0, 5, 10, and 20 vaccinations per day) and mass vaccination (0, 0.5%, 1%, and 2%; compare with the 10%-20% per day many jurisdictions in the United States are planning to vaccinate). Specifically, for each level of ring vaccination and mass vaccination, we used the same 1000 parameter sets used in Figure 5 , and performed 100 simulated epidemics for each parameter set. On the vertical axis, we plot the fraction of the 1000 scenarios for which each of the 100 simulated epidemics was contained. We further computed the fraction of scenarios for which none of the 100 simulated epidemics was contained; this is indicated by the colored segment in the small pie chart at each symbol. When the mass vaccination rate was 2% per day, the mean number of deaths (averaging over all scenarios and all simulations within each scenario) was 47.7, 33.7, 26.4, and 20.1 for a ring vaccination level of 0, 5, 10, and 20 per day (respectively) out of a population of 10000. Moreover, when we increased the mass vaccination level to 3%, an average of 28.9 deaths occurred when no ring vaccination was used, but this fell to 22.3 deaths when only 5 ring vaccinations per day were available (again assuming a population of 10000, and 10 index cases). With a mass vaccination level of 5% per day, an average of 18.8 deaths occurred without ring vaccination, and 15.8 deaths occurred when only 5 ring vaccinations per day were possible. (At a mass vaccination rate of 3% per day, containment as defined above was achieved in all 100 replications for 95% of the scenarios even without ring vaccination; at a mass vaccination rate of 5% per day, containment was achieved in all replications for all scenarios.) These results show that over a wide range of simulated epidemics, even seemingly small levels of ring vaccination (coupled with follow-up) may have a substantial effect in preventing epidemic spread and reducing deaths from smallpox, even during a mass vaccination campaign. Note that many jurisdictions in the United States are planning mass vaccination campaigns which could reach 10%-20% of the population per day, far greater than the mass vaccination levels examined here; it is interesting to note that mass vaccination cam- Figure 10 Mass and ring vaccination together. Low-level mass vaccination programs are improved substantially by the addition of ring vaccination. The shaded pie segments represent the fraction of 1000 scenarios for which containment (as defined in the text) was never realized; the vertical position of the pie chart represents the fraction of the 1000 \"calibrated\" scenarios for which containment was always achieved. As the fraction of the population mass vaccinated increases or the ring vaccination capacity increases, the probability of containment increases. paigns may be effective in preventing a widespread epidemic even at much lower levels than are being planned for. Where feasible, such rapid mass vaccination rapidly eliminates smallpox transmission in our model; vaccination of contacts is still beneficial, since we are assuming that earlier vaccination yields a greater probability of preventing or ameliorating infection (results not shown).\nWe constructed a simple network model of smallpox transmission, and addressed the question of what circumstances contribute to the success of a ring vaccination campaign designed to control smallpox. Our analysis focused on the use of contact tracing/ring vaccination to prevent a widespread epidemic following a deliberate release.\nWe conducted a sensitivity analysis based on particular, but reasonable, ranges for the unknown parameters. Our results are consistent with prior vaccination models in identifying prior vaccination and ring vaccination capacity as significant factors in determining the spread of smallpox. Unsurprisingly, we also find that household size and ring vaccination speed are particularly important parameters; these results are intuitively plausible. The contact finding probability did not appear important in this analysis only because a narrow range of values was chosen.\nWe illustrated smallpox control by presenting scenarios based on control of moderately severe smallpox epidemics. We find that swift, aggressive contact tracing and ring vaccination is is usually sufficient to bring the infection under control. Provided that there is sufficient capacity, vaccination of contacts of contacts is beneficial, and results in fewer infected individuals and more rapid elimination of infection; investigating contacts of contacts allows the chain of transmission to be outrun to some extent. When ring vaccination capacity is small, diversion of crucial resources away from contacts is harmful; contacts of contacts should only be traced and vaccinated provided that no resources are diverted away from contacts of cases. The increased surveillance (or isolation) of contacts, together with improved rates of diagnosis due to community awareness, play important roles in smallpox control; we note that in some cases, lowered diagnosis rates among severe cases contributed to a small extent to loss of epidemic control, and suggest that any public awareness campaign include information to help the public be more aware of the full spectrum of the clinical features of smallpox.\nOne limitation of our analysis is that we chose not to explicitly incorporate the specific epidemiology of health care workers (or mortuary workers), who are likely to be exposed to infected individuals during any smallpox epidemic (e.g. [17, 22] ), and who may then infect further members of the community [22] (as was also seen in the recent outbreak of SARS, e.g. [48] ). Transmission to health care workers may be considered to amplify the initial attack or to be simply accounted among the exposures we considered (and thus be approximated by the behavior of our model), since health care workers and their household contacts are in all likelihood traceable contacts, and ring vaccination/contact tracing would identify and halt these chains of transmission as in our model. The disruption of smallpox control and patient care that may occur is not accounted for in our analysis, however, causing our model in this sense to err on the side of optimism. The appropriateness of pre-event vaccination of health care workers or other first responders has been addressed by other analyses [12, 49] , and is beyond the scope of our model.\nWhile we analyzed the effect of contact tracing, case and contact isolation, and ring vaccination (together with mass vaccination), in a real smallpox epidemic, in practice, control efforts are unlikely to be limited strictly to vaccinating contacts (and health care workers, as likely contacts) and isolating cases. Indeed, making vaccine available to individuals who believe they live near cases or to others on a voluntary basis occurred in smallpox control efforts in the past [22] . Vaccination of such individuals can only harm the disease control effort if it hinders or delays the diagnosis of cases or the investigation and vaccination of contacts; our results show that even relatively low levels of vaccination of the general population may have a beneficial effect in preventing the epidemic from escaping control.\nMore serious is the possibility that individuals who should be vaccinated or isolated would be missed; this could occur either because individuals or institutions did not cooperate with the disease control effort, or because the individuals simply could not be found. Our analysis suggests that ring vaccination need not be perfect to successfully contain the epidemic, and yet, under conditions where there is a high rate of infection among contacts, or a relatively high rate of casual transmission, high rates of contact finding (in excess of 90%), together with increased surveillance and contact isolation, are needed to contain the epidemic.\nFinally, the vaccination of individuals at low risk of contracting smallpox will cause harm due to adverse events of the vaccine; in our model, the assumed death rate due to vaccination was small compared to the probability of death from smallpox, and played essentially no role in the analysis. In practice, individuals suspected to be at high-risk for vaccine complications, but at relatively low risk for contracting smallpox, might simply be isolated or closely monitored even during an outbreak; while the presence of individuals in the population at higher risk for vaccine complications would increase the death rate during an outbreak, such individuals are unlikely to impair the containment of the epidemic (the primary focus of this analysis).\nOur results support ring vaccination against epidemics of smallpox (even assuming high rates of transmission to close contacts), but do note that stochastically, for severe (rapidly transmissible) smallpox, scenarios of loss of control are seen, with resulting widespread epidemics. In scenarios in which the transmission potential of smallpox is smaller, such loss-of-control scenarios occur less frequently (results not shown). Mass vaccination campaigns, when conducted quickly and with very high coverage, do not result in loss of control in our model. Nevertheless, fewer deaths due to smallpox result when ring vaccination is conducted along with mass vaccination.\nSimulated smallpox epidemics with ring vaccination suggest that aggressive, fast ring vaccination can control epidemics of smallpox. To do so, however, smallpox must be identified quickly and contacts vaccinated promptly. We also identify public awareness of smallpox -leading to prompt identification of cases -as a major factor in smallpox control; in some simulations, it may play a role as significant as ring vaccination itself [15] . However, we also found that uncertainty in (1) transmission from mild cases, (2) the household size, and (3) casual transmission contributed to the overall uncertainty in the epidemic size. Other parameters to which the number of infections were highly sensitive were the prior vaccination fraction, parameters related to infectiousness, and parameters related to transmission prior to the rash.\nBecause our model combines network structure with response logistics, our results support and complement the results of other investigators. Our results support the notion that prior vaccine protection may play an important role in slowing the epidemic [11] , despite the possibility that some vaccinated individuals may develop mild cases which are harder to identify, but which nevertheless transmit disease. Likewise, our results provide support for the view that ring vaccination should play a central part in smallpox control. If initiated, ring vaccination should be conducted without delays in vaccination, should include contacts of contacts (whenever there is sufficient capacity to cover all contacts of cases), and should be accompanied by a vigorous campaign of public awareness which can facilitate more rapid identification and isolation of cases. We assumed that ring vaccination could be fast (little delay between identification of a case and vaccination of the contacts), effective (nearly all household contacts can be found, and most of workplace and social contacts), and available (there is sufficient capacity). To be effective, ring vaccination planning must yield a system capable of meeting these benchmarks; we should not only be able to assess the number of contact vaccinations that will be possible per day, but should have a plan in place to (1) identify contacts by working with individuals, employers, schools, community representatives, and authorities or businesses who may have access to information facilitating contact tracing, (2) rapidly investigate and vaccinate such individuals, perhaps using field teams managed by central dispatch. It is important to realize that for highrisk, transient, or unstably housed populations where reliable contact tracing is impossible, the conclusions of the model we present cannot be applied. It is important to note that while our model suggests that ring vaccination together with contact tracing and isolation is likely to be successful, we found that for some scenarios (where smallpox was more transmissible, or was relatively more transmissible before the rash), epidemic containment required not only ring vaccination, but increased public awareness, the isolation of contacts, and tracing of contacts of contacts. For scenarios in which the smallpox was less transmissible, epidemic containment was possible at lower contact finding probabilities. Thus, while our simulations suggest that contact tracing/ring vaccination need not be perfect to succeed, because of uncertainties in our knowledge of the behavior of bioterrorist smallpox, it is impossible to know in advance how good it will have to be. Thus, that high contact finding rates, mass public awareness leading to early identification of cases, isolation of contacts, and investigation of contacts of contacts should all be conducted with maximum effectiveness to reduce the probability of a widespread epidemic.\nWhile the possibility of smallpox uncontrollable by ring vaccination has made mass vaccination preparations wise, and while mass vaccination may be unavoidable in the event of a deliberate release of smallpox, we believe that ring vaccination is essential in any case. This is not only because individuals recently exposed to smallpox may be protected if they are vaccinated promptly, but because each contact identified potentially lies in the immediate future of the transmission chain. From the standpoint of epidemic control, it is far more valuable to vaccinate individuals next in the transmission chain than to vaccinate other persons. Our results support the idea that ring vaccination/case isolation may in many, if not most cases, eliminate smallpox even without mass vaccination, but also support planning for mass vaccination (so that the vastly more costly and difficult policy of mass vaccination will be available in the event of an explosive epidemic). When faced with the unknown, multiple redundant prep-arations are appropriate; case investigation/isolation may control smallpox even if the vaccine does not work at all, but mass vaccination is useful in the event of an explosive epidemic for which case tracking becomes impossible." }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: Oxidative stress leading to the overproduction of free radicals in the lungs is present in many clinical situations. Such clinical settings include acute respiratory distress syndrome (ARDS), infants of prematurity going on to develop bronchopulmonary dysplasia (BPD), pathogenesis of chronic obstructive pulmonary disease (COPD), asthma, cystic fibrosis, ischemia-reperfusion injury, druginduced lung toxicity, cancer and aging [1] [2] [3] [4] . Although the use of oxygen may be clinically indicated in hypoxemic situations, one must consider the potential longterm toxic side effects. For example, we know that oxygen creates cellular damage by a variety of mechanisms. Normal cellular metabolism of oxygen involves the transfer of electrons from NADH to O 2 molecules to form water (H 2 O). At normal partial pressure, 95% of oxygen molecules (O 2 ) are reduced to H 2 O and 5% are partially reduced to toxic byproducts by normal metabolism in the mitochondria [5] . These metabolites include the superoxide anion (O 2 -), hydrogen peroxide (H 2 O 2 ), and hydroxyl radicals ( • OH) all of which make up what are known as Reactive Oxygen Species (ROS) [6] . Exposure to conditions of hyperoxia as well as ionizing radiation (IR) leads to increased amounts of these ROS and their damaging effects.\nROS are known to attack the lipids, proteins, and nucleic acids of cells and tissues [5, 7] . Lipids, including pulmonary surfactant, react with ROS to produce lipid peroxides, which cause increased membrane permeability, inactivation of surfactant, and inhibition of normal cellular enzyme processes. Proteins reacting with ROS result in decreased protein synthesis due to inhibition of ribosomal translation or destruction of formed proteins. This ultimately leads to inactivation of intracellular enzymes and transport proteins resulting in impaired cellular metabolism and accumulation of cellular waste products. Lastly, ROS cause damage to nucleic acids by leading to modified purine and pyrimidine bases, apurinic (AP) / apyrimidinic sites, and DNA protein cross-links which can lead to single strand breaks [8] .\nSeveral defense mechanisms exist to combat the damaging effects of ROS. Intracellular enzymatic systems include superoxide dismutase which eliminates the superoxide anion, catalase which catalyzes the reduction of H 2 O 2 directly to H 2 O without the production of the hydroxyl radical, and glutathione peroxidase which directly reduces H 2 O 2 and lipid peroxides. Free radical scavengers, which stop free radical chain reactions by accepting electrons, include α-tocopheral (vitamin E), ascorbic acid (vitamin C), niacin (vitamin B), riboflavin (vitamin B 2 ), vitamin A, and ceruloplasmin [1, 2, 9] . These systems usually provide enough protection against oxygen metabolism under normal conditions, but may become depleted under conditions of increased oxidative stress [7, 10] .\nThe defense mechanism of interest in this paper involves the repair of oxidative damage through the human DNA base excision repair pathway (BER). BER is the most important cellular protection mechanism that removes Base excision repair pathways for Oxidative DNA damage oxidative DNA damage [11] . Damaged bases are excised and replaced in a multi-step process. Lesion-specific DNA glycosylase repair genes initiate this process. After removal of the damaged base, the resulting AP site is cleaved by APendonuclease generating a 3'OH and 5'deoxyribose phosphate (dRP). β-polymerase, which possesses dRPase activity, cleaves the dRP residue generating a nucleotide gap and then fills in this single nucleotide gap. The final nick is sealed by DNA ligase [12] [13] [14] ( Figure 1A ).\nThe oxidative repair genes that we have analyzed in this study include 8-oxoguanine DNA glycosylase (hOgg1), human Mut Y homologue (hMYH), human Mut T homologue (hMTH), and endonuclease III (hNTH) all of which are present in human cells and involved in the protection of DNA from oxidative damage. The repair enzyme hOgg1 is a purine oxidation glycosylase that recognizes and excise 8-oxoguanine lesions (GO) paired with cytosine. GO can pair with both cytosine and adenine during DNA replication [15] . If repair of C/GO does not occur, then G:C to T:A transversions may result [5, [15] [16] [17] . The repair enzyme hMYH is an 8-oxoguanine mismatch glycosylase that removes adenines misincorporated opposite 8-oxoG lesions that arise through DNA replication errors [5, [18] [19] [20] . The repair enzyme hMTH hydrolyzes oxidized purine nucleoside triphosphates such as 8-oxo-dGTP, 8-oxo-GTP, 8-oxo-dATP, and 2-hydroxy-dATP, effectively removing them from the nucleotide pool and preventing their incorporation into DNA ( Figure 1B ) [21] . Lastly, the repair gene endonuclease III (hNTH) is a pyrimidine oxidation and hydration glycosylase that recognizes a wide range of damaged pyrimidines [22] . hNTH has also been shown to have a similar DNA glycosylase/AP lyase activity that can remove 8-oxoG from 8-oxoG/G, 8-oxoG/A, and 8-oxoG/C mispairs [23, 24] . Subsequent steps following hNTH are identical to those following hOgg1 ( Figure 1A ).\nA previous study has shown that over-expression of the DNA repair gene hOgg1 leads to reduced hyperoxiainduced DNA damage in human alveolar epithelial cells [25] . The primary goal of our present study was to compare the protective effects of the four main lesion-specific DNA glycosylase repair genes by individually overexpressing each in lung cells and determining which of these provides the greatest degree of protection under conditions of increased oxidative stress.\nThe human alveolar epithelial cell line A549 (58 year old Caucasian male), was purchased from ATCC Cat No CCL-185. The cells were grown in DMEM (Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS) (HyClone, Logan, UT) and penicillin (100 U/ml)/ streptomycin (100 µg/ml) (Gibco, Grand Island, NY).\nPassaging of cells was performed every 3-4 days with cells grown to 80% confluency in a 10 cm cell culture dish (Corning Incorporated, Corning, NY). Cells were kept at 37°C in a humidified, 5% CO2 incubator.\nThe retroviral vector pSF91.1, a gift from Dr. C. Baum from the University of Hamburg in Germany, was constructed with an internal ribosome entry site (IRES) upstream to the gene expressing enhanced green fluorescent protein (EGFP) as previously described [26] .\nFour DNA repair genes were individually ligated into the retroviral vector pSF91.1.\nhOgg1-6pcDNA3.1 was initially amplified by PCR by primers to introduce a kozak sequence at the 5' end [27] . Digestion of this product with EcoRI and SalI was performed and then hOgg1 was subcloned into digested plasmid vector pSF91.1, with T4 DNA ligase. DNA sequencing was performed to confirm integrity of the hOgg1 gene.\nhMYH/PGEX4T-1 and hMTH/PGEX4T-1 hMYH was a gift from Dr. A. McCullough (University of Texas Medical School, Galveston, TX) and hMTH was cloned in Dr. Kelley's lab. Plasmid DNA was prepared as above by digestion with EcoRI and SalI and ligated into pSF91.1 as above and sequencing was performed to confirm integrity of the genes.\nPGEX-6PI-hNTH1-wild type this gene was a gift from Dr. S. Mitra (University of Texas Medical School, Galveston, TX). Digestion with BamHI and SalI was performed and the hNTH1-wt fragment was ligated into the empty plasmid vector PUC18. The hNTH1-wt fragment was then excised with both sides flanked by EcoRI restriction sites and ligated into pSF91.1. Proper orientation of the gene was confirmed and sequencing was performed to determine the integrity of the gene. 2.5 × 10 5 A549 cells were suspended with the viral supernatant and plated in 1 well of a 6-well plate along with polybrene (Sigma, St. Louis, MO). This exposure was performed 6 hours per day for three days. At approximately five days from the beginning of the infection, the infected cells were analyzed using flow cytometry and sorted for EGFP expression.\nCell pellets of sorted cells were resuspended in NuPage buffer (Invitrogen, Carlsbad, CA) and protein concentrations were determined using the DC protein assay (Bio-Rad, Hercules, CA). 20 ug of protein were loaded into individual lanes of a NuPage Bis-Tris Gel (Invitrogen, Carlsbad, CA). The gel was then transferred to nitrocellulose paper (Osmonics Inc, Gloucester, MA). The membranes were then blocked with 1% blocking solution (Roche Diagnostics, Indianapolis, IN) for 1 hour at room temperature and then incubated overnight at 4°C with rabbit polyclonal antibodies to hOgg1 (Novus Biologicals, Littleton, CO), hMTH (Novus Biologicals, Littleton, CO), hMYH (Oncogene Research Products, Darmstadt, Germany) and hNTH (Proteintech Group Inc, Chicago, IL) all at a dilution of 1:1000 except hNTH which was diluted 1:2500. They were then washed 2 times with TBST and 2 times with 0.5% blocking solution, 10 minutes per wash. The membranes were incubated with anti-rabbit secondary antibodies at 1:1000 for 1 hour at room temperature. Lastly, the membranes were washed 4 times with TBST, 15 minutes per wash. The membranes were briefly soaked in BM chemiluminescence blotting substrate (Roche Diagnostics, Indianapolis, IN) and then exposed to high performance autoradiography film (Amersham Biosciences, Buckinghamshire, England). Kodak Digital Science 1D Image Analysis software was utilized to quantify the region of interest (ROI) band mass of individual bands on films where visualized differences were detected.\nSorted EGFP positive A549 cells infected with the above DNA repair genes were counted and seeded into 96-well plates at a density of 1000 cells/well, 6 wells per gene. Six hours after seeding, individual plates were placed into an oxygen chamber supplied by Dr. L. Haneline (Wells Center for Research, Indianapolis, IN) located in a 37°C incubator. The oxygen chamber was then infused with 95% O 2 and 5% CO 2 . Individual plates were removed after 12, 24, 48, and 72 hours of exposure. Control A549 cells were incubated in a normal 37°C humidified-5% CO 2 incubator. O 2 concentrations were monitored with a MAXO 2 analyzer (Maxtec, Salt Lake City, UT). Four days from the beginning of the exposure, cells were assessed for cell growth/survival using the sulforhodamine B assay (SRB assay).\nThe SRB assay (Sigma, St. Louis, MO), developed by the National Cancer Institute, provides a sensitive measure of drug-induced cytotoxicity through a colorimetric endpoint that is non-destructive, indefinitely stable, and visible to the naked eye. This assay was used to assess the cell growth/survival of over-expressed cells [28] . Cold 10% TCA was used to fix the cells to the plate. After incubation for 1 hour at 4°C, the individual wells were rinsed with water. After air-drying, SRB solution was added to each well and cells were allowed to stain for 20-30 minutes. 1% acetic acid wash was used to rinse off unincorporated dye. Incorporated dye was then solubilized in 100 µl per well of 10 mM Tris. Absorbance was measured by a tunable microplate reader (Molecular Devices, Sunnyvale, CA) at a wavelength of 565 nm. Background absorbance measured at 690 nm was subtracted from the measurements at 565 nm.\nSorted EGFP positive A549 cells were seeded into 96-well plates at a density of 1000 cells/well. Six hours after seeding, individual plates were then exposed to radiation at doses of 250, 500, 1000, and 1500 Rads or 0. well. All the plates were placed into a 37°C humidified-5% CO 2 incubator. Every 24 hours for 4 days, 1 plate was removed and the cells were fixed and analyzed by the SRB assay looking at cell growth under non-toxic conditions. Growth curves and exponential growth equations were determined to look at the doubling time (DT) of cells infected with each repair gene of interest compared to vector infected and uninfected wild type cells.\nAll drug exposure experiments were performed at least three times and individual drug doses included 6-8 wells for each group of infected cells. Analysis of cell growth and exponential growth equations were determined using Microsoft Excel. All experiments involving drug exposures were normalized to the zero dose. Data are expressed as means ± SE. The significance of differences were calculated using the paired Student's t test with significance being accepted for p < 0.05.\nThe DNA repair genes hOgg1, hMYH, hMTH, and hNTH were ligated into the retroviral vector pSF91.1 ( figure 2 ). This vector, derived from a murine stem cell virus backbone, along with each individual repair gene, was used for transfection of phoenix amphotropic cells. Viral supernatant was then collected and used to stably infect A549\nWestern analysis of A549 cells over-expressing individual repair genes and effect on endogenous glycosylase level contained the genes of interest integrated into their DNA (data not shown).\nWestern blot analysis was performed on sorted cells in order to verify over-expression of the four genes of interest. hOgg1, hMYH, hMTH, and hNTH were all detected at their correct position on western blots (data not shown).\nWestern analysis was also utilized to assess whether overexpression of each individual repair gene resulted in altered endogenous expression of the other repair genes under both non-toxic and toxic conditions (24 hrs of 95% O 2 and 1000 Rad). Cells over-expressing the repair genes hOgg1, hMYH, hMTH, and hNTH did not lead to altered expression of the other endogenous repair genes under the above conditions when compared to each other or pSF91.1 vector control cells ( Figure 3A ,3B,3C and 3D). hOgg1's endogenous expression was below the level of detection. The pattern of endogenous expression of hNTH was consistent for each condition when comparing cells over-expressing hOgg1, hMYH, hMTH, and pSF91.1. Reduced expression of hNTH after exposure to 95% O 2 was noted.\nLastly, we assessed endogenous expression of each individual repair gene in cells infected with pSF91.1 following non-toxic and toxic conditions (24 hrs of 95% O 2 and 1000 Rad) at 24 and 48 hrs after the onset of exposure. Endogenous hMYH and hMTH were expressed to the same degree. hOgg1's endogenous expression was below the level of detection using western analysis (results not shown). When analyzing endogenous hNTH expression, it was noted that hyperoxia at 24 hrs and 48 hrs resulted in reduced protein expression by 93% and 64% respectively. There also was a small increase in expression of hNTH noted after 1000 Rad one day post exposure that was back to baseline by two days post exposure. ROI band mass quantification demonstrated this finding ( Figure 4A and 4B). Two or more replicates were performed for each western analysis to determine consistency of the results.\nA549 cells expressing hMYH demonstrated increased survival after exposure to conditions with elevated levels of oxygen compared to cells expressing only the pSF91.1 vector ( Figure 5A ). Results were highly significant at all time points except after 12 hours O 2 where it almost reached a highly significant value. The differences between pSF91.1 and hMYH varied from 12% after 12 hours O 2 exposure to 7% after 72 hours O 2 exposure. A549 cells expressing hMYH also demonstrated increased survival after exposure to all doses of radiation in comparison to pSF91.1 ( Figure 5B ). These results were also highly significant at all doses of radiation except at 250 Rads where it almost reached a highly significant value. The differences between pSF91.1 and hMYH varied from 12%-14% for all doses of radiation. Also noted in these experiments was that vector control cells demonstrated no Experiments looking at the effects of H 2 O 2 on cells expressing the repair genes did not demonstrate increased survival for any of these repair genes when compared to vector control cells ( Figure 5C ). This data demonstrates that over-expression of hMYH has the ability to improve cellular survival under conditions of hyperoxia and radiation but may not be able to overcome the toxicity of H 2 O 2 .\nCell growth under normal conditions was ascertained to determine if over-expression of any of the repair genes caused an alteration in the growth of cells in the absence of oxidative stress. Wild type A549 cells and cells expressing pSF91.1, hNTH, hOgg1, and hMTH appeared to grow at similar rates with doubling times within the same range. A549 cells expressing hMYH did show a slower growth rate that resulted in significant differences in cell number by day 3. The calculated doubling time for the cells over expressing hMYH is > 3 hrs longer than the cells with the other repair genes and vector alone ( Figure 6 ). This slowing of growth may allow for more time to repair Cell survival analysis following O 2 , IR, and H 2 O 2 treatments \nOxidative stress to the lung leads to cellular DNA damage as evidenced by the release of specific gene products known to regulate DNA base excision repair pathways such as p53 and p21 [29] [30] [31] . Alterations in pro-inflammatory mediators, transcription factors, and other related gene products are also observed [32] . This injury has been shown to be associated with features of both cellular necrosis and apoptosis [33] [34] [35] . The resultant cellular inflammation and death from oxidative stress has a dramatic impact on the outcome of patients in the clinical setting [7, 36] .\nMost of our current clinical therapy towards oxidative stress in the lung involves both supportive measures and prevention. Research dealing with oxidative lung injury has focused mainly on enhancing antioxidant enzymatic processes and free radical scavengers [37] [38] [39] [40] . The ability to alter cellular survival by increasing specific DNA repair mechanisms may add another approach to the treatment of oxidant-mediated lung injury.\nMany investigators have used hydrogen peroxide as a substitute for hyperoxia since it is known to be one of the metabolites produced by the metabolism of oxygen. ROS such as H 2 O 2 and those produced by hyperoxia clearly lead to DNA damage but questions exist as to whether H 2 O 2 leads to the same deleterious effects upon DNA as hyperoxia. Analysis of our growth curves after exposure to H 2 O 2 in comparison to hyperoxia and IR clearly indicates that cellular protection by oxidative DNA repair genes is specific to the agent used. Because no protection was observed with over-expression of any of the repair genes following exposure to H 2 O 2 , we speculate that the damage it causes is dissimilar. It may be that its damage not only involves oxidized bases, but may also include other forms of DNA, lipid, and protein damage that are not corrected by oxidative DNA repair genes. Alternatively, the amount and type of damage evoked by H 2 O 2 could be beyond that which can be corrected by over-expressing these repair genes.\nAnother form of stress known to induce damage through the formation of ROS is IR. Radiation induced free radical damage to DNA has substantial overlap with that of oxidative damage [41] [42] [43] . The protection provided by specific oxidative DNA repair genes under conditions of IR, was notable throughout our experiments only with the repair enzyme hMYH.\nThe primary agent utilized to induce the formation of ROS was an oxygen rich environment. The use of oxygen as a stressor leading to the formation of ROS, offers a distinct advantage over IR and H 2 O 2 by mimicking the clinical situation where constant exposure to hyperoxia leads to cumulative cellular damage which further compromises repair. We determined that survival of A549 cells was also enhanced to a small degree with increased expression of the repair enzyme hMYH. This was an unexpected finding as we anticipated the repair gene hOgg1 would demonstrate the greatest protection in response to oxidative stress based on previous studies, however these experiments utilized the colony forming assay (CFA) to detect improvements in survival [25] . Additionally, the CFA may provide different results compared to the SRB assay, which allows for growth analysis over a shorter window of time. Furthermore, their study did not look at the repair enzyme hMYH and its impact on survival. Another study has investigated the repair function of hMYH in MYH-deficient murine cells. It was demonstrated that transfection of the MYH-deficient cells with a wild-type MYH expression vector increased the efficiency of A:GO repair [44] .\nAn interesting observation noted while doing our experiments lead us to look at individual growth characteristics of cells over-expressing each of the oxidative repair enzymes. Cells over-expressing the repair enzyme hMYH clearly grow at a slower rate when compared with the other enzymes. The mechanism behind this is not understood at this point in time. The repair action of Cell growth curve and associated doubling times (DT) Figure 6 Cell growth curve and associated doubling times (DT hMYH is known to remove adenines misincorporated opposite 8-oxoG lesions. This lesion occurs when a C/GO lesion is allowed to replicate before being corrected by hOgg1. Repair by hMYH is not a final corrective measure. The product of hMYH activity is the lesion C/GO, which allows hOgg1 to have another opportunity to remove 8-oxoG opposite cytosine. We know that A549 cells possess the hOgg1 gene based on a previous study demonstrating the presence of this gene after amplification by genomic PCR [45] . We also have demonstrated endogenous activity of hOgg1 in A549 cells by using an 8-oxoguanine bioactivity assay. Therefore, our explanation of these results is that the slowed growth created by hMYH may provide a wider window of opportunity for the repair process to take place, which ultimately grants endogenous hOgg1 another opportunity to remove the 8-oxoG lesion created by oxidative stress.\nAs noted in the methods section, the SRB assay provides a sensitive measure of drug-induced cytotoxicity that is used to assess cell proliferation/survival. The reduced cell proliferation of A549 cells over-expressing hMYH under nontoxic conditions may likely underestimate the magnitude of the protective effect of this particular repair enzyme. This may in fact make the results even more significant.\nRecent studies have discovered hereditary variations of the glycosylase hMYH that may predispose to familial colorectal cancer [46, 47] . Others have looked for hMYH variants in lung cancer patients and have not identified any clear pathogenic biallelic hMYH mutations or an overrepresentation of hMYH polymorphisms [47] . The A549 cell line has not demonstrated somatic mutations in hMYH, but a single nucleotide polymorphism (SNPs) has been noted [45] . The impact on function by this SNP is unknown. It would appear that the function of hMYH is very important in preventing somatic mutations leading to cancer in the gastrointestinal tract. Although studies to date have not demonstrated this same relationship with lung cancer, we do know that the lungs are subjected to large quantities of ROS under certain conditions as discussed earlier. The formation of mutations from oxidative stress does have other deleterious effects on cells including cellular death by necrosis and apoptosis. Tissue viability is dependent upon mutation correction and replication of the surviving cells to replace those that have died. The ability to enhance cellular survival, after specific oxidative exposures, is evident after increased production of the hMYH repair gene in these experiments.\nWe additionally wanted to determine the level of endogenous expression of the glycosylase repair genes in the pulmonary epithelial A549 cell line. Others have demonstrated how different stressors lead to alterations in the endogenous production of specific repair genes. For example, it has been shown that endogenous gene expression of hOgg1 was elevated following exposure to crocidolite asbestos which is known to cause an increase in 8-oxoG levels [48] . It has also previously been reported that treatment of A549 cells with sodium dichromate, a prooxidant, leads to a reduction of hOgg1 protein expression that was not observed with H 2 O 2 [49] . One additional study demonstrated a dose dependent down regulation of hOgg1 protein expression in rat lung after exposure to cadmium, a known carcinogen associated with the formation of intracellular ROS [50] . In our experiments we were able to demonstrate that both hyperoxia and IR do not appear to impact the endogenous expression of hOgg1, hMYH, and hMTH at 24 and 48 hours following exposure. It was noted that endogenous hNTH was reduced after hyperoxia at 24 and 48 hours after the onset of exposure. One would speculate that this reduction in endogenous hNTH secondary to hyperoxia is related to either decreased production or increased destruction in response to O 2 exposure. Over-expression of this repair enzyme did not result in improvements in survival after O 2 exposure based on our experiments. It may be that endogenous levels are adequate to correct this specific mutational burden for these experiments.\nFurthermore, no previous studies have determined how cells over-expressing specific repair genes may impact endogenous expression of the other oxidative BER genes under both normal and oxidative stress conditions. We were also able to demonstrate that endogenous expression of glycosylase repair genes were not altered under these conditions secondary to the over-expression of any of these genes. This is an important finding for interpretation of survival data; protection of cells is due to the overexpression of the specific gene and not due to enhancement of other endogenous repair enzyme levels, at least for the genes studied under these conditions. Some limitations may exist in using a lung carcinoma cellline, which likely differs both in proliferative properties as well as in response to oxidative stress in comparison to primary epithelial cells. The enhanced cell growth observed with cell lines may be more reflective of undifferentiated alveolar type II cells which are likely to replace terminally differentiated alveolar type I cells after injury/ death due to oxidative stress. This may not be a true reflection of growth under non-toxic conditions when very little cell division is occurring. This is an inherent problem observed when comparing cell lines with primary cells and results need to be interpreted in a way that considers this.\nIt is difficult to know how this will translate to pulmonary epithelial cells in vivo at this stage. It certainly would appear that the protection observed is modest in degree in this pulmonary epithelial cell line. Further experiments assessing the function of the repair enzyme hMYH in this model will be important to perform in order to delineate the findings of slowed growth under normal conditions and improved survivability under conditions of O 2 and IR. More research looking at the potential for combination therapy, including DNA repair mechanisms in conjunction with other antioxidant defense mechanisms may be another approach to enhancing cell survival, which may lead to better clinical outcomes. Alternatively, cell survival may not be the most important end point for hyperoxia studies. Given that 8-oxoG, if left unrepaired, leads to G:C to T:A transversions, there may be an increase in mutational burden by these cells that isn't reflected in cell survival. Further experiments studying the impact on mutation production is underway. Ultimately, experiments need to be done in animal models to determine the translation to in vivo pulmonary cells.\nIn summary, we have demonstrated that over-expression of the DNA glycosylase repair enzyme hMYH may enhance survival of a pulmonary epithelial cell line after exposure to conditions of IR and hyperoxia. We have also demonstrated that over-expression of hMYH leads to a slowing of growth of A549 cells under non-toxic conditions, which may in part play a role in this enhancement of survival by providing a wider window of opportunity for repair of oxidized lesions to occur. Lastly, we demonstrated that over-expression does not lead to altered endogenous expression of these repair genes. As the understanding of DNA repair mechanisms continues to grow and the evolution of gene therapy takes place, more treatment options may be available in the clinical setting to help with many disease processes including the damaging effects of oxygen and its metabolites." }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: The purpose of this study has been to identify domains with homology to AlkB in viral genomes, in order to get a better understanding of distribution and possible function of such domains. The AlkB protein of E. coli, and probably most of its homologues, is involved in repair of alkylation damage in DNA and RNA. It repairs 1-methyl-adenine and 3-methylcytosine by oxidative demethylation and direct reversal of the methylated base back to its unmethylated form. Recently the protein was identified as a member of the 2-oxoglutarate (2OG)-and Fe(II)dependent oxygenase superfamily [1] [2] [3] . The catalytic reaction requires molecular oxygen, Fe 2+ and 2-oxoglutar-ate, which is subsequently converted into succinate, CO 2 and formaldehyde [4] .\nThe 2OG-FeII oxygenase superfamily is widespread in Eukaryotes and bacteria [1] , and is currently the largest known family of oxidising enzymes without a heme group [5] . The 3D structure of several of these oxygenases is known, and they share a common fold with a structurally conserved jelly roll β-sheet core with flanking α-helices. Very few residues are totally conserved across these structures, basically just the residues involved in coordination of the Fe(II) ion and the 2-oxoglutarate.\nAlkB-like genes are widespread in most types of organisms except Archaea. However, whereas bacteria normally have just one or at most two AlkB homologues [6] , multicellular Eukaryotes tend to have several homologues. In the human genome at least 8 different AlkB homologues (ABHs) have been identified [7] . These homologues seem to have slightly different properties with respect to substrate preference and subcellular localisation, and this may be a reason for the proliferation of ABHs e.g. in humans. However, a detailed functional mapping of all ABHs has not yet been carried out.\nA sequence alignment of known ABHs identifies very few residues as totally conserved, basically just a HxD motif, a H and a RxxxxxR motif. These residues are also conserved in the more general 2OG-FeII oxygenase superfamily as described above, except for the final R. The first three residues (HxD and H) are involved in Fe(II)-coordination, whereas the first R is involved in 2OG-coordination. The final R is most likely involved in AlkB-specific substrate binding.\nIn addition to DNA repair, it has been shown that E. coli AlkB and the human AlkB homologue hABH3 may be involved in RNA repair. When expressed in E. coli both AlkB and hABH3 reactivate methylated RNA bacteriophage MS2 in vivo. This illustrates that direct repair may be an important mechanism for maintenance of RNA in living cells [4] . RNA repair proceeds by the same mechanism as DNA repair. Repair of damaged RNA was previously considered very unlikely, due to the natural redundancy of RNAs in a cell [8] . However, RNA is essential for cell function: unrepaired RNA can lead to miscoded or truncated proteins, and alkylated RNA could signal cell cycle checkpointing or apoptosis [9] . Consequently the occurrence of RNA repair does not come as a great surprise. The mechanism of direct reversal of methylation as used by AlkB homologues is particularly important for RNA repair, as it means that single-stranded regions may be repaired without introducing strand breaks. Repair of alkylation damage in DNA and RNA has recently been reviewed [10] .\nAlkB homologues have also been found in plant viruses. It has been suggested that methylation may be used in host-mediated inactivation of viral RNAs, and that AlkB homologues in some plant viruses may be used to counteract such defence mechanisms [1] . However, no detailed study of this has been published.\nThe research project reported here has focused on a better understanding of the distribution and potential function of putative AlkB homology domains by using in silico mapping of viruses in which such domains have been found, as well as related viruses.\nThe general mapping strategy of the project was to identify viral genomes with AlkB homology domains, identify common features of these genomes, and subsequently find additional genomes with similar features, but without AlkB homology domains. This data set could then be used to analyse the properties and distribution of AlkBlike domains in viruses, as a basis for generating hypotheses about the evolution and function of these domains.\nThe PSI-Blast search for viruses in the NCBI nr protein sequence database was initiated with ALKB_ECOLI (NCBI gi113638), restricted to residues 110 to 210 and using the default inclusion threshold of 0.005 on E-values. The [11] .\nIn all of these viruses the AlkB domain is a part of the replicase polyprotein, which normally consists of a viral Other Pfam domains -Peptidase_C21, C23, C33, C34, C35 and C41, A1pp and OTU -were also identified in subsets of sequences. A1pp is a member of the Appr-1-p processing enzyme family, and the domain is found in a number of otherwise unrelated proteins, including non-structural proteins of several types of ssRNA viruses. OTU is a mem-ber of a family of cysteine proteases that are homologous to the ovarian tumour (otu) gene in Drosophila. Members of this family are found in Eukaryotes, viruses and pathogenic bacteria.\nThe MT, HEL and RdRp domains identified by Pfam as described above were extracted from the library sequences, aligned by ClustalX, and combined into a new alignment consisting of only these domain regions. This turned out to be necessary in order to get robust alignments. The intervening regions between the conserved domains are extremely variable in these sequences, and this tended to confuse alignment programs in the sense that conserved regions were not correctly aligned. The combined sequence alignment of domains from Closteroviridae, Flexiviridae and Tymoviridae was then used as input for building a phylogenetic tree with MEGA2. The final tree is shown in Figure 2 , with polyproteins containing AlkB-like domains indicated.\nA second alignment was generated from all sequences with AlkB-like domains, using only the regions corresponding to MT, AlkB, HEL and RdRp Pfam domains. The domains were aligned individually, and the combined alignment was used as input for MEGA2. However, this data set did not give a reliable phylogeny (data not shown), and the separate domains of this alignment were therefore analysed individually and compared. This analysis is summarised in Tymoviridae measures (including SJA) for comparison of random trees [12] . The SJA values shown in Table 2 for comparisons between MT, HEL and RdRp NJ trees were 14.2 -17.1 standard deviations from the expectation value of 0.665 for a tree with 22 nodes, whereas the corresponding values for the AlkB NJ tree were 4.4 -5.4 standard deviations from the expectation value. Similar ranges were observed for the ML trees as well as for alternative distance measures, e.g. the Symmetric Difference (SD) measure (data not shown). Although this means that the SJA value for comparing AlkB trees to MT, HEL and RdRp trees were significantly better than for random trees, it also shows that the MT, HEL and RdRp trees were clearly more similar to each other than to the AlkB tree.\nThe alignment of the AlkB domain seemed to be of comparable quality to the other alignments. In fact the AlkB domain had the highest average pairwise sequence identity, as seen in Table 2 (see Figure 3 for the actual alignment). In other words, these AlkB domains were as similar to each other as the other three domains with respect to sequence identity, but they did not represent a consistent evolutionary history when compared to the other domains of this polyprotein. This may indicate that the AlkB domains have evolved separately from the other domains, and possibly as several independent instances.\nThe degree of co-evolution was analysed by computing pairwise distances between sequence regions in the alignment of MT, AlkB, HEL and RdRp domains described above. In Figure 4 selected results are shown as scatter plots, where the Blosum 50 score value between e.g. the MT domains in a pair of sequences is plotted against the score value for AlkB domains in the same pair of sequences. Plots for the MT, HEL and RdRp domains show that they are strongly correlated for MT vs. RdRp (r 2 = 0.95), MT vs. HEL (r 2 = 0.87) and HEL vs. RdRp (r 2 = 0.81). The plot of the AlkB domain vs. these three domains for the same set of sequences shows a very low degree of correlation for AlkB vs. RdRp (r 2 = 0.10), AlkB vs. MT (r 2 = 0.12) and AlkB vs. HEL (r 2 = 0.16).\nAs mentioned above the genome organisation of these replicase polyprotein sequences seems to be very flexible. In order to analyse domain organisation the location of identified Pfam domains were plotted for a number of sequences, as shown in Figure 5 .\nThe results described above may indicate that the AlkB domains have been integrated into the replicase polyprotein relatively recently (see Discussion). In order to test for potential sources selected AlkB domains were compared to non-viral sequences. PSI-Blast was used to search the NCBI nr database, removing all viral hits in the final search report. Most of the remaining top-scoring hits were from bacteria. This included two different strains of Xanthomonas, X. axonopodis pv citri and X. campestris pv campestris. Xanthomonas attacks plants such as citrus, beans, grapevine, rice and cotton [13] . The search also returned high-scoring hits from another plant pathogen, Xylella fastidiosa. This bacterium infects a great variety of plants, including grapevine, citrus, periwinkle, almond, oleander and coffee [14] .\nPfam searches obviously will only identify known domain types in protein sequences. In order to identify potential similarities in regions that were not recognised by Pfam, systematic PSI-Blast searches were performed, using the polyprotein regions between the MT and HEL domains and searching against the NCBI database of reference sequences [15] , excluding all viral entries. A maximum of 5 PSI-Blast iterations were allowed, with an inclusion threshold of 0.005. The expected homologues of the AlkBdomain were found with high confidence, as most of the E-values were < 1 × 10 -50 . Homologues of typical viral domains like the viral peptidases were obviously not found, as all viral database entries were excluded. Very few Multiple alignment of sequence regions corresponding to the AlkB domains Figure 3 Multiple alignment of sequence regions corresponding to the AlkB domains. The alignment was generated with ClustalX. The residues involved in coordination of the essential Fe 2+ ion are completely conserved, except in one of the Vitivirus sequences. These residues are the HxD motif, a single H, and the first R in the RxxxxxR motif. The function of the remaining conserved residues is unclear, but at least some of them may be involved in coordination of the substrate [10] . Pairwise distances between sequence regions corresponding to methyltransferase (MT), RdRp and AlkB domains. Each data point corresponds to e.g. RP-RP and MT-MT distances for the same pair of sequences, and sequences showing similar evolutionary distance in these two regions will fall on the diagonal. The pairwise distances were estimated from multiple alignments using the Blosum50 score matrix [47] . Trend lines were estimated with Excel. The trend line for AlkB vs. RdRp is heavily influenced by the point at (675, 670). It represents two Foveavirus sequences (NCBI gi3702789 and gi9630738), they are 98% identical over the full polyprotein sequence. Alignment score (AlkB) Alignment score (RdRp) r 2 = 0.10 new similarities were found by these searches. Pepper ringspot virus (Tobravirus, NCBI gi20178599) showed significant similarity to site-specific DNA-methyltransferase from Nostoc sp (E = 1 × 10 -74 ), as well as other cytosine 5Cspecific DNA methylases. Bamboo mosaic virus (Potexvirus, NCBI gi9627984) showed similarity to aggregation substance Asa1 from Enterococcus faecalis (E = 6 × 10 -34 ). A small number of additional similarities seemed to be caused by biased sequence properties (e.g. proline-rich regions), and were probably not significant. This included matches against mucin and cadherin-like proteins from Homo sapiens and multidomain presynaptic cytomatrix protein (piccolo) from Rattus norvegicus. In general the variable regions seemed to be truly variable, with very little similarity to other proteins, except for the Pfam domains already identified.\nAs seen in Figures 2 and 5 , some closely related sequences are lacking specific domains in the sense that HMMER does not find a significant similarity to the Pfam entries for these domains. In order to understand the degree of sequence variation associated with this domain loss, as well as the general sequence variation in conserved vs. non-conserved regions of typical polyproteins, several dot plots were generated. The dot plot for two Carlavirus sequences, Potato virus M (NCBI gi9626090) and Aconitum latent virus (NCBI gi14251191), is shown in Figure 6 . The dot plot confirms that these two sequences are closely related in the MT, HEL and RdRp domains. However, there are significant differences in the region between MT and HEL. Potato virus M is lacking the AlkB domain whereas Aconitum latent virus is lacking the OTU domain. As seen from the dot plot, short regions of similarity close to the diagonal shows that both domains may have been present in an ancestral sequence. However, this region shows a high degree of sequence variation, and as indicated by the dot plot they are almost exclusively mutations. Non-essential or non-functional domains are probably rapidly lost. In this particular case, none of the typical AlkB motifs seem to be conserved in Potato virus M, indicating that this indeed is a non-functional AlkB domain.\nThe N-terminal domains of Flexiviridae and Tymoviridae are methyltransferases As described above the Pfam methyltransferase motif (Vmethyltransf) did not match any of the putative methyltransferase domains of Flexiviridae and Tymoviridae, despite the fact that they had been identified via PSI-Blast searches starting with known methyltransferases. Therefore an additional Pfam-type profile was generated. It is obviously a possibility that these domains in Flexiviridae and Tymoviridae are not methyltransferases, and that they are false positives from PSI-Blast. However, the essential residues of a typical viral methyltransferase motif are conserved in the alignment of these domains (data not shown) [16] . In Bamboo mosaic virus, which belongs to Flexiviridae, the residues H68, D122, R125 and Y213 have been identified as putative active site residues with similarity to the Sindbis virus-like methyltransferase [17] , and it has been demonstrated that this region of the Bamboo mosaic virus has methyltransferase activity, as it catalyses the transfer of a methyl group from S-adenosylmethionine (AdoMet) to GTP or guanylylimidodiphosphate (GIDP). The corresponding sequence positions are almost completely conserved in the alignment of Flexiviridae and Tymoviridae N-terminal domains. This is most likely significant, as only 7 positions in total are completely conserved in this alignment, which means that the majority of the conserved positions are known to be essential for methyltransferase activity. Work e.g. by Hataya et al. seems to support the assumption that this sequence region is a methyltransferase domain [18] . It therefore seems likely that all the sequences with AlkB domains also contain functional MT, HEL and RdRp domains. The MT Location of Pfam domains in the variable region of Flexiviridae 2 sequences Figure 5 Location of Pfam domains in the variable region of Flexiviridae 2 sequences. The regions have been extracted directly from Pfam output, and sequences and regions are drawn to scale. The black bar at each end of a motif indicates that a fulllength motif has been found, for partial motifs the bar at the truncated end would be missing. domains are probably involved in capping of genomic and subgenomic RNA [19] .\nBased on the bioinformatic evidence generated here, it seems reasonable to assume that the viral AlkB domains identified by Pfam are functional. All the essential residues found in 2-oxoglutarate-and Fe(II)-dependent oxygenases are conserved, in particular the putative Fe 2+ coordinating H, D and H residues at alignment positions 19, 21 and 91 of Figure 3 , and the 2-oxoglutarate coordinating R at position 100. The conserved R at position 106 is also very characteristic of AlkB homologues [10] . The fact that all AlkB-like domains identified in these viral genomes are full-length, compared to the Pfam profile, also seems to support the hypothesis that these domains are functional.\nThe Pfam searches show that AlkB domains are found only in a subset of the viral genomes. This subset is phylogenetically consistent (see Figure 2 ), as it is mainly restricted to the Flexiviridae, and in particular to a subset of the Flexiviridae consisting of Viti, Capillo, Tricho, Fovea and Carlavirus. This subset is well separated from the remaining Flexiviridae in the phylogenetic analysis. The split seems to be robust from bootstrap analysis, therefore this family will be discussed here as two subfamilies, Flexiviridae 1 and 2. The same split was observed by Adams et al. in their recent analysis of the Flexiviridae family [20] . Most of the AlkB domains (15) are found in Flexiviridae 2. The remaining AlkB domains are found in Flexiviridae 1 (5) and Closteroviridae (2) . In general, all the Flexiviridae 2 sequences have at least one extra domain in addition to MT, HEL and RdRp: either AlkB, OTU-like cysteine protease or a peptidase. Most other plant viruses that are included in this survey do not have additional domains, except for Tymoviridae where a peptidase domain seems to be common. For the remaining plant virus families included here (excluding Tymoviridae and Flexiviridae 2), only 14% seem to have additional domains.\nThe observed distribution of AlkB domains could most easily be explained by assuming that an ancestral AlkB domain was integrated into the genome of the last common ancestor of the Flexiviridae 2 subfamily. Subsequent Figure 6 Dot plots for Potato virus M (NCBI gi9626090) and Aconitum latent virus (NCBI gi14251191). To the left the full sequences are shown, using the program default for similarity threshold, and to the right the region with AlkB, OTU and peptidase integration, using a slightly lower (more sensitive) threshold for sequence similarity. The Pfam regions corresponding to MT (magenta), AlkB (red), OTU (green), peptidase (blue), HEL (yellow) and RdRp (cyan) domains are indicated. virus generations derived from this common ancestor would then also contain an AlkB domain, except in those cases where the domain was lost again. This scenario could also include subsequent transfer to a small number of other virus families e.g. by recombination.\nIf this scenario was correct, then one would expect the different domains of the polyprotein to have a similar evolutionary history. From the phylogenetic analysis (Table 2) this seems to be confirmed for the MT, HEL and RdRp domains, but not for the AlkB domain. This indicates that the AlkB domain may not have co-evolved with the other domains, at least until relatively recently. This seems to be confirmed by looking at the degree of co-evolution, which was analysed by computing pairwise distances between alignment regions representing the relevant domains ( Figure 4 ). In the case of perfect co-evolution all points should fall on a diagonal. This seems to be the case for the MT, HEL and RdRp domains. However, the plot of the AlkB domain vs. these three domains for the same set of sequences does not show a similar correlation. Only some of the closely related sequence pairs in the upper right quadrant of the plot in Figure 4 show some degree of correlation for AlkB vs. RdRp. The most likely explanation seems to be that most of the AlkB domains have not coevolved with the other domains for any significant period of time. This seems to rule out the possibility of ancient integration of the AlkB domain, except if we assume that an ancient viral AlkB domain has frequently recombined with other AlkB domains. However, it is difficult to distinguish a scenario with frequent recombination of AlkB domains from de novo integration, and the net effect on the properties observed here would be the same.\nAs seen in Figure 4 , the range of score values is generally smaller for the AlkB domains than e.g. the RdRp domains, particularly if we exclude a couple of very high-scoring cases (see figure caption) . On the other hand, the degree of sequence variation within the collection of AlkB domains is significant, average sequence identity for pairwise alignments is 38%, and only 10% of the positions are totally conserved. This can be consistent with a recent integration if we assume that several different AlkB-type vectors have been used for integration (see below for details). An increased mutation rate after integration could also have contributed to sequence diversity in this region. Moving the AlkB domain into a novel structural and functional context would have removed many of the original evolutionarily constraints, as well as introduced some new ones. This could have created a \"punctuated equilibrium\" type of situation, potentially leading to a very rapid evolution that could have introduced significant differences between the AlkB domains, independent of the evolution in the other domains. A high mutation rate seems to be the case for this region in general, as indi-cated in Figure 6 . Although the MT, HEL and RdRp domains seem to be well conserved from the dot plot, there are very large sequence variations in the intervening region. One sequence in Figure 6 has a well conserved AlkB domain, the other an OTU domain. The fact that there are very weak sequence similarities in these two domains in the dot plot indicates that both sequences originally had both domains. However, the fact that this similarity now is very weak and without any of the typical AlkB active site motifs also indicates a high mutation rate where non-essential domains are rapidly lost. Therefore the conservation of AlkB domains is a strong indication that they are functional, as already mentioned.\nIf we assume that AlkB domains have been integrated relatively recently, then either de novo integration or recombination (horizontal gene transfer) may have been the main driving force for spreading the AlkB domain to new genomes. In the first case a large number of individual integrations could have lead to the present situation. If horizontal gene transfer was the main driving force, the initial number of integrations might have been quite small. It is not easy to differentiate between these two situations.\nThe map of Pfam motifs in the variable region between the MT and HEL domains in Flexiviridae 2 polyproteins ( Figure 5) shows that they have a very similar domain organisation, basically an AlkB domain followed by an OTU domain and a peptidase domain, located towards the C-terminal part of the sub-sequence. The relatively constant domain organisation seems to be consistent with a small number of initial integrations that were subsequently diffused to related genomes e.g. by homologous recombination. However, this is not fully consistent with the fact that the viruses with AlkB domains have been collected from hosts at very different locations, e.g. Canada, USA, Russia, Italy, Germany, France, India, Taiwan, China and Japan. Although import of virus-infected species or transmission by insects may transport viruses over significant distances, it is not obvious that this is enough to explain the observed distribution of AlkB-like domains. Therefore several independent integrations, mainly from closely related hosts, have to be considered as an alternative explanation. This explanation seems to be supported by the apparent lack of any consistent evolutionary relationships between the various AlkB domains, as seen in Table 2 . It is not easy to see how this model can be consistent with the observed similarities in domain organisation in Flexiviridae. Assuming that this region has a high degree of variability, one would expect the variability to affect localisation of integrated domains as well. However, it is possible that conserved regions e.g. in the polyprotein play a significant role in integration of novel domains. It may be relevant in this context that preliminary simulations indicate that e.g. the AlkB domains tend to form independent folding domains in the folded RNA structure of the polyprotein RNA (F. Drabløs, unpublished data). This property may possibly facilitate the insertion of such domains into the viral genome.\nThere are many groups of organisms that can act as vectors and spread viruses, including bacteria, fungi, nematodes, arthropods and arachnids. The plant viruses may have acquired the AlkB domain either from the vector or from the host itself. As already mentioned, searching with viral AlkB domains in protein sequence databases resulted mainly in bacterial sequences, including the plant pathogens X. fastidiosa and campestris. It is therefore a reasonable possibility that AlkB domains in plant viruses have originated from bacterial mRNA. It is also possible that the mRNA originated from other vectors or from the host itself, but at the present time this is not easily verified or disproved because of the limited number of insect and plant genomes that have been sequenced.\nIt has previously been suggested that the viral AlkB domain may be involved in protecting the virus against the post-transcriptional gene silencing (PTGS) system of the host [1] . PTGS is known as one of a plant's intrinsic defence mechanisms against viruses [21] . Gene silencing can occur either through repression of transcription (transcriptional gene silencing -TGS) or through mRNA degradation, PTGS. The PTGS-mechanism in plants shows similarities to RNA interference (RNAi) in animals [22] . This mechanism results in the specific degradation of RNA. Degradation can be activated by introduction of transgenes, RNA viruses or DNA sequences homologous to expressed genes [23] . Many viruses have developed mechanisms to counteract PTGS in order to successfully infect plants [24] . Two of these suppressors of PTGS have been identified as Hc-Protease and the 2b protein of Cucumber mosaic virus [25] . Although both proteins suppress PTGS, it is likely that they do so via different mechanisms. Could the AlkB-like domain found in some of the plant viruses also be a suppressor of PTGS? Previously reported research indicates that methylation of transcribed sequences is somehow connected with PTGS, and the methylation can be mediated by a direct RNA-DNA interaction [26] . This RNA-directed DNA methylation has been described in plants, and leads to de novo methylation of nearly all cytosine residues within the region of sequence identity between RNA and DNA [27] . Both RNA methylation and methylation of host proteins that are essential for viral replication would be detrimental to the virus. It has already been mentioned that AlkB repairs 1methyladenine and 3-methylcytosine by oxidative demethylation. It is therefore possible that AlkB demethylates the nucleotides methylated by the PTGS mechanism, helping the virus to overcome one of the major defence mechanisms of the plant.\nAs shown here, only a subset of plant viruses have the AlkB domain. However, other viruses may be utilising naturally occurring AlkB proteins in the host. Viruses have to rely on a number of host proteins in order to replicate [28] . In some cases it is probably beneficial for the virus to integrate such genes into their own genome in order to ensure that they are accessible, although there will be a trade off between this advantage and the increased cost of maintaining a larger genome [29] .\nHowever, there is an alternative hypothesis with respect to the AlkB integration that also has to be considered. As discussed above, the AlkB domain seems to have been integrated relatively recently in viruses found at very different geographical locations, and the only obvious connection seems to be that most viruses belong to a subset of the Flexiviridae. However, the source of these viruses points at another common feature. As seen from the table given in Additional file 1, AlkB domains are often found in viruses associated with grapevine, apple, cherry, citrus and blueberry -crops where the usage of pesticides is common. It is known that several common pesticides (e.g. methyl bromide and some organophosphorus compounds) may cause methylation of DNA and RNA [30] [31] [32] [33] . An integrated repair domain for methylation damage as part of the viral replication complex would therefore give the virus a competitive advantage in a highly methylating environment. The application of such pesticides would probably also stimulate AlkB production e.g. in co-infecting bacteria, giving these viruses easy access to AlkB mRNA for integration into their RNA genome.\nIt could be argued that a more active PTGS system in these plants would give a similar effect. However, in that case we would expect to see more ancient integrations of AlkB domains. It could also be argued that the presence of AlkB domains may be an artefact caused by promiscuous viral domains picking up available mRNA sequences during cultivation of viruses in the laboratory. However, given the large number of different laboratories involved, and the number of different hosts used (data not shown), this seems to be a very unlikely explanation.\nThe hypothesis that environmental compounds, in particular pesticides, may have provoked the integration of AlkB domains into the viral genomes depends upon a high mutation rate and frequent integrations of non-viral domains. The integrations have to be recent, not only in relative terms, compared to other domains in the same genome, but also in absolute terms, compared to the progress of modern agriculture. The integrations also have to be frequent, in the sense that it is likely that integration could have happened several times, in different biotopes.\nIt is difficult to estimate mutation rates in RNA viruses. They evolve very rapidly, and it is often difficult to assign reliable phylogenies. However, recent studies indicate that most ssRNA viruses have a mutation rate close to 10 -3 substitutions per site per year [34] , e.g. the SARS virus has 1.16-3.30 × 10 -3 non-synonymous substitutions per site per year, which is considered to be a \"moderate\" ssRNA mutation rate [34] . If we assume that most ssRNA viruses have effective mutation rates within the same order of magnitude, a realistic mutation rate for the viruses included here might be something like 2.0 × 10 -3 . In that case, the MT, HEL and RdRp trees shown in Additional file 2 represent approximately between 325 and 750 years of evolution. In general the NJ trees estimate a slightly shorter evolutionary history (between 325 and 450 years) compared to the ML trees (between 550 and 750 years). In this estimate the Ampelovirus sequences have not been included, as they seem to have diverged from the remaining AlkB-containing viruses at a much earlier stage. If we believe that the AlkB integrations happened after the divergence of most sequence included here, as indicated by the lack of co-evolution in Figure 4 , it does not seem unrealistic to assume that most of these integrations happened within the last 50 -100 years or so. This estimate is of course very approximate, in particular since we do not know the true mutation rate of these viruses. However, it shows that a likely time span for AlkB integration is compatible with the evolution of modern agriculture. Unfortunately, because of the lack of any robust phylogeny for the viral AlkB sequences it does not make sense to do a similar estimate for that domain.\nAlthough it is generally accepted that viruses frequently use recombination to acquire functionality [35] , it is less well known how often this includes nonviral sequences. However, there are some well-documented examples, and in particular the properties of the ssRNA positive-strand Pestivirus may be relevant in this context. There are two biotopes of the pestiviruses, cytopathogenic (cp) and noncytopatogenic (noncp). The host is infected by the noncp form which is converted into the cp form by integration of a fragment of a cellular gene into the viral genome [36] . This introduces a protease cleavage site in the polyprotein. However, the important point here is that this happens as part of the normal infection process. It has been suggested that the integration is facilitated by the viral polymerase undergoing two subsequent template switches during minus-strand synthesis [37] , although nonreplicative RNA recombination also may be a possibility [38] . Inte-gration of cellular sequences have also been observed in other viruses, e.g. in influenza virus [39] . This shows that at least some viruses do have efficient mechanisms for recruitment of host genes into the viral genome. Therefore a recent and rapid integration of AlkB domains into selected plant virus genomes does not seem to be an unlikely scenario.\nThis study has focused on the AlkB domain, mainly as an attempt to get a better understanding of potential functions associated with this domain. However, it is likely that additional information about integration patterns and the relative importance of de novo integration vs. recombination can be achieved by a closer investigation of the other variable domains, e.g. by looking at how they correlate with the evolution of the AlkB domains.\nWe believe that the viral AlkB-like domains are conventional repair domains targeted towards the viral RNA. The integration of AlkB domains into viral genomes may have been provoked by environmental methylating agents, e.g. the introduction of DNA/RNA-methylating pesticides in farming. The hypothesis [1] that the domain interferes with the PTGS system of plants can not be excluded, but seems to be less consistent with observed features of the AlkB integration.\nand Tymoviridae was generated from a ClustalX alignment, using hmmbuild and hmmcalibrate from the HMMER package. Visualisation of motif positions in viral sequences was generated directly from the HMMER output files using a local tool as an interface to the GNU [50] groff software. Systematic large scale searches with polyprotein subsequences were done locally with PSI-Blast and the NCBI reference sequence database [15] . Dot plots for comparison of viral protein sequences were generated with Dotter version 3.0 [51] . " }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: The management of infectious diseases in an increasingly complex world of mass international travel, globalization and terrorism heightens challenges for Federal, State and Territory Governments in ensuring that Australia's laws are sufficiently flexible to address the types of problems that may emerge.\nIn the 1980's and 1990's HIV/AIDS was the latest \"emerging infectious disease\". Considerable thought was put into the legislative response by a number of Australian jurisdictions. Particular attention had to be given to the unique features of the disease such as the method of transmission, the kinds of people who were at risk, and the protections needed by the community and the infected population to best manage the care of those infected and to minimize new infections. Health workers and researchers began to find that \"the most effective strategies that we have so far found to help promote reduction of the spread of HIV involve the adoption of laws and policies which protect the rights of people most at risk of infection\" [1] . A good example of a legislative response which adopts this approach is found in section 119 and 120 of the Victorian Health Act 1958. These sections emphasize the need to protect the privacy of the infected individual and to undertake a staged response which is proportional to the risk presented by the infected individual. The legislation has been very effective with HIV and has been praised for its progressive approach [2] .\nIn 2003 the community has been faced with the emergence of two new infectious diseases, SARS and Anthrax. Whilst there were no cases of either disease in Australia, the threat of a possible outbreak had to be acknowledged and a response planned. Anthrax is not a new infectious disease. Humans can become infected with anthrax by handling products from infected animals or by breathing in anthrax spores from infected animal products (like wool, for example). People also can become infected with gastrointestinal anthrax by eating undercooked meat from infected animals. However, its manufacture and use as a weapon for bioterrorism forces us to rethink its management in a new context. These two infectious diseases have very different features from HIV which spreads only via transmission of infected bodily fluids such as blood or semen. SARS, by contrast is transmitted via droplets from infected cases which, as a result of coughing, carry the virus to close contacts [3] Thus, the infection profile of SARS requires planning for the possible overrun of Intensive Care Units and the likely infection of a number of ICU staff affecting both morale and capacity to cope. Anthrax raised different problems. These include the possible investigation of terrorist suspects alongside investigation of the outbreak of the infectious disease. Difficulties are also raised by likelihood of public panic, and the flooding of public health officials with reports of suspicious white powder.\nIn early 2004 the media reported the spread of avian influenza across South East Asia. This disease has different features from HIV/AIDS and SARS and an approach to an Australian outbreak would also be different. The main difference is in the source of transmission of the virus, that is, from infected birds to humans. There is very little difference [from ordinary influenza] in the symptoms (though these may vary in severity) or treatment of the virus [4] It is too early to predict whether this may be the next \"emerging infectious disease\", but its current spread has given rise to concern about such a possibility [5] Australia is a federal system. There are two parallel sets of laws in operation. The Commonwealth Constitution sets out the legislative powers of the Commonwealth. Specific powers are listed in the Commonwealth constitution but State constitutions have broad powers covering matters such as peace, order and good governance. As the Commonwealth has no specific power to legislate with respect to health, other than the quarantine power, national legislative schemes in public health which rely upon a cooperative approach from all States and Territories are cumbersome and difficult.\nWithout a specific head of power, the Commonwealth has limited ability to legislate with respect to health. \"That is, the legislative powers of the Commonwealth are specified in the Constitution and do not include expressly most of the activities that together comprise the field of public health\" [6] For this reason, there are no Commonwealth emergency health powers except quarantine powers. Quarantine powers are currently restricted to isolation at the border of the country of people, plants, and animals to prevent the spread of disease. There is a real possibility that quarantine laws could have a broader scope. It depends on how widely the High Court would interpret section 51(x) of the Commonwealth Constitution. A quarantine law could override state laws as long as it remained a law \"with respect to quarantine\". However, \"the power is potentially a colossus so far as the expansion of legislative authority in the fields of public health is concerned\". [6] The quarantine power would be the most likely candidate for a head of power on which to base development of commonwealth laws for the management of public health emergencies. Another possibility may be the external affairs power, if there was a relevant treaty or international agreement which could be given effect to in domestic law. However the legislation would have to be limited to laws giving effect to the treaty.\nStates and territories have a range of emergency powers available to them in their existing public health legislation. Some are relatively old. For example, the Health Act 1911 (WA), Public Health Act 1952 (NT) based on an 1898 Ordinance (Both these Acts are currently under review). Health emergency powers vary from one jurisdiction to another, but include powers to support disease surveillance, contact tracing and orders to restrict behavior or movement of individuals with an infectious disease in certain circumstances. There are also powers to recall food, search premises and seize property, close buildings and a range of other substantial and intrusive powers.\nIt is suggested that it is time to consider whether state and territory public health legislation contains sufficient measures to manage the outbreak of an infectious disease in a modern environment which includes mass travel, swift spread of infection and additional complexity raised by fears of bioterrorism.\nCurrently, in a public health emergency caused by the spread of an emerging infectious disease, Australia could need to rely on a patchwork of legislative measures to assist it to cope. Commonwealth quarantine laws and State and Territory powers in public health legislation may all be needed to address the problem. If an outbreak occurred on a border, or in some area where jurisdiction may be in doubt such as airspace or offshore and a state or territory response was required in addition to any quarantine measures, there could be confusion over jurisdiction for the application of State and Territory powers. State and Territory public health acts do not adequately provide for interjurisdictional communication and cooperation. There could also be difficulties if an infectious disease caused overseas deaths of people from more than one State or Territory in circumstances where an Australian coronial investigation was considered desirable. In such a situation, the jurisdiction of more than one Australian coroner would be triggered. Several State and Territory coronial laws could apply and there could be different inquests under different laws undertaken by different coroners into the same incident.\nIt is suggested that it is time to look at the efficiency of the emergency powers laws of Australia as a whole: to map the laws in each jurisdiction and the Commonwealth quarantine laws and to consider their effectiveness in the face of the outbreak of a fast moving, easily spread infectious disease. The efficacy of Australia's laws should also be considered in relation to bioterrorism. While there were no infections from anthrax in 2003 despite a great deal of media coverage and infections and deaths in the US, a responsible legislature ought to acknowledge the possibility and ensure that the law is ready to support a swift and effective response.\nIt is not enough to consider whether the individual pieces of legislation are up to the task of managing outbreaks of newly emerging infectious diseases. Indeed many of the jurisdictions are currently reviewing their public health legislation and will no doubt give proper consideration to this issue as part of the review. But who is thinking about how the legislation of all jurisdictions and the Commonwealth quarantine fits together? What powers enable communication and cooperation between jurisdictions about the outbreak of infectious disease? What kind of opportunity is there for a coordinated response? Can public health orders made in one jurisdiction travel to another jurisdiction when the infected individual travels? What arrangements can be made if an outbreak occurs on or close to a interstate border? What if there is an outbreak on a bus carrying passengers from Victoria, through South Australia to the Northern Territory?\nIt is encouraging to note that, even without specific legislation, there has been a mechanism to achieve communication and cooperation between jurisdictions through the Communicable Disease Network of Australia (CDNA). This Network has in fact been quite successful in fostering regular communication between the Communicable Disease Units across the country and has been involved in coordinated actions during a number of multistate outbreaks.\nDespite the existence of this network and other good working relationships between government officials and various agencies in different jurisdictions, a serious outbreak of communicable disease would require the existence of legislative powers. Public health emergencies generate confusion, even panic. Clarity of powers and the way those powers interact with each other would be crucial in an emergency. It became apparent after the Bali tragedy in 2002 that coroner's jurisdiction was triggered differently in different jurisdictions and some acts did not support communication and cooperation when inquests might be needed for deaths of people ordinarily resident in several jurisdictions. The time to find the shortcomings in the legislation is well before the crisis.\nA review of the efficacy of how these laws work together to protect the public health of all Australians should be undertaken. It has been possible to overcome the hangovers of federation for the betterment of all Australians in relation to corporations law. When doubts were recently raised about the constitutional basis of the corporations law scheme, the States and Territories were able to cooperate and refer the necessary powers to the Commonwealth to provide certainty about the laws which govern our corporations. Is our public health any less important than governance of our corporations? Could we cooperate to give ourselves certainty, flexibility and a consistent approach which protects the rights of those subject to some very broad powers?\nThe States and Territories are generally reluctant to refer powers to the Commonwealth. It may be time to seriously discuss referral of powers in the context of health emergency powers. At the very least, it is time that the Commonwealth, States and Territories recognised the need for the laws to work as a set of laws to protect the whole country, not simply individual laws to protect individual jurisdictions.\nThere has been work done internationally in this area. A model State Emergency Health Powers Act has been developed in the US in 2001 [7] In the preamble to this Act a rationale for its development is set out: \"In the wake of the tragic events of September 11, 2001, our nation realizes that the Government's foremost responsibility is to protect the health, safety and wellbeing of its citizens. New and emerging dangers including emergent and resurgent infectious diseases and incidents of civilian mass casualties -pose serious and immediate threats to the population. A renewed focus on the prevention, detection, management and containment of public health emergencies is thus called for.\" The US, like Australia, is a Federal system. The model was intended to be taken up by those US states which wished to do so. To date, it has been passed in over half the US states. This bill would be an excellent starting point for development of an Australian model. There are a number of legislative mechanisms which could be used to support a nationally uniform approach to health emergency powers legislation in Australia.\nThe development and adoption of the model food legislation provides a useful model of a cooperative uniform approach. A model act was developed in consultation with all jurisdictions. It covered areas agreed to be core areas of the Act which ought to be the subject of a national approach and other provisions which were considered to be administrative and were to be adopted at the discretion of each jurisdiction. An intergovernmental agreement was signed as a mechanism to protect the uniformity of the legislation. The agreement sets up a Ministerial Council, supported by a Food Regulation Standing Committee. The Council has responsibility for deciding on proposals to amend the model [8] If a decision is made in favor of amendment, States and Territories will use their best endeavors to submit to their respective Parliaments, legislation which gives effect to the amendment.\nThe law is an important tool in supporting the management of the outbreak of infectious diseases. The existence of our Federal system has meant that we have a different approach in each State and Territory together with Commonwealth control of quarantine. Newly emerging infectious diseases creating real threats to public health in an era of easy mass travel, and the present threat of bioterrorism mean that it is time Australia examined all laws to contain and manage infectious disease outbreak. The laws should be examined both for their effectiveness in the areas they cover, and as part of a whole which ought enable a response which protects the health of all Australians, and crosses borders as easily as SARS or avian influenza." }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: Lactic Acid Bacteria (LAB) are anaerobic Gram positive bacteria with a GRAS (Generally Regarded As Safe) status. They are also food grade bacteria, and therefore, they can be used for the delivery of proteins of interest in foodstuff or in the digestive tract. A last advantage compared to other well-known protein producers is that L. lactis does not produce LPS or any proteases as Escherichia coli or Bacillus subtilis do, respectively.\nIn the last two decades, genetic tools for the model LAB, Lactococcus lactis, were developed: transformation protocols, cloning-or screening-vectors [1, 2] , and mutagenesis systems [3] are now available. Moreover L. lactis genome is entirely sequenced [4] . Many protein expression-and targeting-systems have also been designed for L. lactis [5] [6] [7] . These systems have been used to engineer L. lactis for the intra-or extra-cellular production of numerous proteins of viral, bacterial or eukaryotic origins (Table 1) . To produce a protein of interest in fermentors, secretion is generally preferred to cytoplasmic production because it allows continuous culture and simplifies purification. To use L. lactis as a protein delivery vehicle in the digestive tract of humans or animals, secretion is also preferable because it facilitates interaction between the protein (e.g. enzyme or antigen) and its target (substrate or immune system).\nIn LAB, like in other Gram positive bacteria, secreted proteins are synthesized as a precursor containing an N-terminal extension called the signal peptide (SP) and the mature moiety of the protein. Precursors are recognized by the host secretion machinery and translocated across the cytoplasmic membrane (early steps). The SP is then cleaved and degraded, and the mature protein is released in the culture supernatant (late steps). Sometimes, secreted proteins require subsequent folding and maturation steps to acquire their active conformation [8] .\nIn most of the works describing heterologous protein production by recombinant lactococci, only one cellularlocation (i.e. cytoplasm, external media or surface anchored) is described. Only a few works report the production of a given protein in different locations using the same backbone vector, the same induction level and or promoter strength, allowing thus a rigorous comparison of the production yields of cytoplasmic and secreted forms.\nHere, six examples of different heterologous proteins produced in L. lactis in both secreted and cytoplasmic forms are reviewed and discussed. Our major conclusion is that the best production yields are observed in most of these cases with secretion (up to five-fold higher than with cytoplasmic production). Moreover, engineering the expres-sion cassette to enhance the secretion efficiency (SE, proportion of the total protein detected as mature form in the supernatant) resulted in increased overall amounts of the protein. L. lactis is able to secrete proteins ranging from low-(< 10 kDa) to high-(> 160 kDa) molecular mass through a Sec-dependant pathway. Altogether, these observations suggest that i) heterologous proteins produced in L. lactis are prone to intracellular degradation whereas secretion allows the precursor to escape proteolysis, and ii) conformation rather than protein size is the predominant feature that can impair SE. New perspectives are now opened in the studies of heterologous protein production in L. lactis. Indeed, there is a need for food grade systems and for a better understanding of the host factors influencing heterologous protein secretion in L. lactis . For example, HtrA-mediated proteolysis (HtrA is the unique housekeeping protease at the cell surface) is now well-characterized in L. lactis [9] and can be overcome by use of a htrA L. lactis strain designed for stable heterologous protein secretion [10] . However, intracellular proteolysis (involving Clp complex -the major cytoplasmic housekeeping protease-, and probably other cellular components) remains poorly understood and is also discussed here.\nGenetic tools to target a given protein in different cellular compartments were developed using several reporter proteins [6, [11] [12] [13] (Table 1 ). The staphylococcal nuclease (Nuc) is a well-characterized secreted protein whose activity is readily detectable by petri plate assay and it has been used as a reporter protein for secretion studies in several Gram positive hosts [14] [15] [16] . In L. lactis, Nuc was used to develop protein targeting- [6] and SP screening-systems [1, 2] . Nuc was chosen to develop the pCYT and pSEC vectors for controlled production in L. lactis of cytoplasmic or secreted forms of a protein of interest, respectively ( Fig. 1 ) [5] . The pCYT and pSEC plasmids, where expression is controlled by a nisin inducible promoter, should be used in L. lactis NZ9000 (hereafter referred to as NZ) strain bearing a nisR,K chromosomal cassette, required for the nisin signal transduction [17] . In each case described below, protein sample concentration was adjusted to the cell density of the producing culture (for details see [18] ). At similar induction levels in lactococcal strains containing pCYT:Nuc and pSEC:Nuc vectors, the highest production yields were observed with the secreted Nuc form ( Table 2) . Similar results were obtained with constitutive nuc expression cassettes for cytoplasmic and secreted forms. Nuc was the first heterologous protein where highest protein yields were obtained with the secreted form.\nSimilar results were obtained for the production of a Brucella abortus ribosomal protein. B. abortus is a facultative intracellular Gram negative bacterial pathogen that infects Unpublished results Bacteriocins human and animals by entry through the digestive tract. The immunogenic B. abortus ribosomal protein L7/L12 is a promising candidate for the development of oral live vaccines against brucellosis using L. lactis as a delivery vector. L7/L12 was produced in L. lactis using pCYT and pSEC vectors [19] . Similarly to Nuc production, the production yield of secreted L7/L12 was reproducibly and significantly higher than that of the cytoplasmic form (Table 2) .\nAnother example of higher protein yields in secreted vs cytoplasmic form is the production the human papillomavirus type 16 (HPV-16) E7 antigen, a good candidate for the development of therapeutic vaccines against HPV-16 induced cervical cancer. The E7 protein is constitutively produced in cervical carcinomas and interacts with several cell compounds. E7 was produced in a cytoplasmic and a secreted form in L. lactis [20] . Using similar induction level in exponential phase cultures, E7 production 1: protein samples were adjusted to the cell density and protein quantification was performed as described in the references either by western blot or by ELISA. *: E7 was not quantified but ratio was calculated by scanning the western blot signals and comparing their intensity as described in the corresponding reference. nd: not determined was higher for the secreted form than for the cytoplasmic form (Table 2) . This difference was even higher when induction occurred in late-exponential phase, where intracellular E7 was detected at only trace amount whereas secreted E7 was accumulated in NZ(pSEC:E7) culture supernatant (see below). Thus, production of E7 clearly illustrates the fact that secretion results in higher yields in L. lactis.\nProduction of ovine interferon omega (IFN-ω) further illustrates this observation. In the case of poorly immunogenic antigens, co-delivery of an immuno-stimulator protein can enhance the immune response of the host. In order to optimize the use of lactococci as live vaccines, the production of cytokines was investigated in L. lactis [5, 21, 22] . IFN-ω is a cytokine able to confer resistance to enteric viruses in the digestive tract by reduction of viral penetration and by inhibition of intracellular multiplication of the viruses. Delivery of ovine IFN-ω in the digestive tract by recombinant L. lactis strains could therefore induce anti-viral resistance and could protect the enterocytes. Ovine IFN-ω cDNA was cloned into pCYT and pSEC plasmids for intracellular (pCYT:IFN) and secreted (pSEC:IFN) production respectively [5] . Induction of recombinant NZ(pCYT:IFN) and NZ(pSEC:IFN) strains were performed at equal level and IFN-ω production was measured. The levels of IFN-ω activity showed that i) an active form of IFN-ω was produced in both strains, and ii) the activity of IFN-ω found in the supernatant and cell fractions of NZ(pSEC:IFN) strain was about two-fold higher than that observed for the cytoplasmic form (Table 2) . Similarly to what was observed for Nuc and E7, secretion leads to higher heterologous protein yields.\nL. lactis has been engineered to secrete of a wide variety of heterologous proteins from bacterial, viral or eukaryotic origins (Table 1) . There are reports about secretion bottlenecks and biotechnological tools for heterologous secretion in model bacteria such as Escherichia coli and Bacillus subtilis [23, 24] , but only few data are available concerning this aspect in L. lactis. Protein size, nature of the SP and presence of a propeptide are parameters that may interfere with protein secretion. Some data available about these features are compiled here.\nTo optimize secretion and thus production yields, the nature of the SP was the first parameter to modify on heterologous precursor as previously shown using Nuc as a reporter protein. The replacement of the native staphylococcal SP Nuc by the homologous lactococcal SP Usp45 to direct the secretion of Nuc in L. lactis led to an increased SE [25] (Table 3) . On the other hand, the replacement of SP Nuc by SP Usp45 did not enhance the SE of NucT (a truncated mature moiety of Nuc devoid of N-terminal propeptide) suggesting the importance of the propeptide in the SE for Nuc [25] (Table 3) . However, in several cases, the use of a homologous SP (and especially SP Usp45 ) allows a better SE compared to a heterologous one. Screening vectors were thus developed to search for new homologous secretion signals in L. lactis [1, 2] . These screening works offer now a panel of SPs that are suitable for heterologous secretion. However, when compared to SP Usp45, the newly described SPs were less efficient to direct secretion of Nuc [1] . Even after a direct mutagenesis on SP310, one of these new SPs identified using a screening strategy [1] , the enhanced SE was still lower than the one measured with SP Usp45 [26] . However, a recent study by Lindholm et al. showed that a Lactobacillus brevis SP (originated from a Slayer protein) drove the secretion of the E. coli FedF Schematic representation of Nuc cassettes for controlled and targeted production in L. lactis adhesin more efficiently than SP Usp45 [27] . High SE might thus result, at least in part, from good adequacy between the mature protein and the SP used to direct secretion.\nThe fusion of a short synthetic propeptide between the SP and the mature moiety is another innovative biotechnological tool to enhance protein secretion. One such propeptide (composed of nine amino acid residues, LEISSTCDA) was developed and was shown to enhance the SE of several heterologous proteins in L. lactis: NucB, NucT, (Table 3 ) [18] , the B. abortus L7/L12 antigen (Table 3 ) [19] , and the α-amylase of Geobacillus stearothermophilus (Table 3 ) [18] . Directed mutagenesis experiments demonstrated that the positive effect of LEISSTCDA on protein secretion was due to the insertion of negatively charged residues in the N-terminus of the mature moiety [25] . Furthermore, the enhancement effect does not depend on the nature of the SP, since the secretion of NucB fused to either SP Nuc or SP Usp45 was enhanced by LEISSTCDA insertion [25] . Strikingly, the enhancement of SE was reproducibly accompanied by an overall increase of protein yields as determined in Western blot experiments. This observation suggests that heterologous precursors are degraded by intracellular proteases when they are not efficiently secreted and that a higher secretion could be a way to escape proteolysis.\nProteins with molecular mass ranging from 165 kDa (size of DsrD, the Leuconostoc mesenteroides dextransucrase, [28] ) to 9.8 kDa (size of Afp1, a Streptomyces tendae antifungal protein; Freitas et al., submitted) have been successfully secreted in L. lactis. This suggests that protein size is not a serious bottleneck for heterologous protein secretion in L. lactis. In contrast to protein size, conformation may be a major problem for heterologous secretion in L.\nlactis as illustrated by some recent examples. The first example is the production of the non-structural protein 4 (NSP4) of the bovine rotavirus, the major etiologic agent of severe diarrhea in young cattle. In order to develop live vaccines against this virus, the NSP4 antigen was successfully produced in L. lactis [29] . Derivatives of pCYT and pSEC plasmids were constructed to target NSP4 into cytoplasmic or extracellular location. The highest level of production was obtained with the secreted form. However, no secreted NSP4 was detected in the supernatant and both SP Usp45 -NSP4 precursor and NSP4 mature protein were detected in the cell fraction. Two degradation products were detected in addition to the NSP4 precursor and mature protein. These results suggest that the cytoplasmic form of NSP4 was probably totally degraded inside the cell whereas fusion to the SP Usp45 protected NSP4 protein against intracellular proteolysis.\nSimilar results were obtained when pCYT and pSEC vectors were used to produce the B. abortus GroEL chaperone protein: only pSEC:GroEL plasmid was obtained and subsequently the fusion SP Usp45 :GroEL was detected in Western blot experiments (V. Azevedo, unpublished data). In this case, B. abortus GroEL is likely to interact with lactococcal cytoplasmic proteins leading to severe cellular defects and thus to a lethal phenotype. On the other hand, fusion of SP Usp to GroEL might keep the chimeric protein in an unfolded and/or inactive state allowing thus its heterologous production.\nAnother example is the production of the bovine β-lactoglobulin (BLG) in L. lactis [30, 31] . BLG, a 162 amino acid residues globular protein, is the dominant allergen in cow's milk and was produced in L. lactis to test the immunomodulation of the allergenic response in mice when BLG is delivered by a bacterial vector [30] . Western blot and ELISA showed that BLG production was significantly higher when BLG was fused to SP Usp45 although the SE was very low, with no detectable BLG in the supernatant of pSEC:BLG strains [30] . Further studies revealed that a fusion between the LEISS propeptide and BLG could not enhance the SE of BLG above ~5%, as determined by ELISA [31] .\nFor rotavirus NSP4, B. abortus GroEL, and BLG (which are medium-sized compared to DsrD or Afp1), either very low secretion yields or absence of secretion was observed in L. lactis. In all cases, fusion to a SP stabilizes heterologous protein production even though they are not efficiently secreted. These results could be due either to the SP itself that reportedly acts as an intramolecular chaperone or to the protection of the chimeric precursor from intracellular proteolysis by the cytoplasmic chaperones of the Sec-machinery. GroEL (a cytoplasmic chaperone), NSP4 (a structural protein), and BLG (a globular protein) have dramatically different primary sequences. A higher affinity of intracellular housekeeping proteases for these particular sequences cannot be hypothesized since the fusion of a SP leads to the stabilization of the protein. Change of conformation is therefore the predominant criterion involved in the stabilization of the precursors and the higher yields observed. On the other hand, these proteins might undergo rapid folding right after their synthesis, which interferes with (or hampers) the secretion process. Such interferences between protein conformation and SE were previously shown in E. coli and B. subtilis [32, 33] . Altogether, these results suggest that protein conformation rather than protein size is a major problem for heterologous protein secretion in L. lactis as well.\nIt was clearly demonstrated that the secreted form of E7, a reportedly labile protein, can be stabilized by fusion to Nuc [20, 34] . Nuc is reportedly a stable protein and its use, as a fusion partner, does not affect its enzymatic activity. The production of the resulting chimerical protein is thus easy to follow. The cytoplasmic form of E7 was stabilized by the fusion to Nuc even when the production was induced in stationary phase ( Fig. 2A) , whereas cytoplasmic E7 alone was degraded (see below; Fig. 3 ). Thus, fusion to the stable Nuc could rescue E7 production in L. lactis and allowed higher protein yields compared to E7 alone [20] . Stabilization by fusion to Nuc was observed for several secreted proteins as well. First, a Nuc-E7 fusion on a pSEC backbone resulted in higher production yield although the SE was altered (Fig. 2B) . Fusion to the synthetic propeptide LEISSTCDA in a pSEC:LEISS:Nuc:E7 construction restored an efficient secretion yield [34] . Second, in an attempt to increase the protein yield of the secreted L7/L12, a fusion to Nuc (pSEC:Nuc:L7/L12) resulted in a 2.5-fold increase in production yield (Fig. 2B ) [19] . Recent results concerning the production of BLG provide a third example of yield enhancement by fusion to Nuc. A pSEC:Nuc:BLG construction allowed a 2-fold increase in BLG yields compared to pSEC:BLG [31] . These results show that Nuc is a stable carrier protein and has a protective effect on labile heterologous chimerical proteins by reducing its sensitivity to intracellular proteolysis. To our knowledge, Nuc is the fusion partner most commonly tested so far for stabilization in L. lactis. Bernasconi et al (2002) fused the Lactobacillus bulgaricus proteinase PrtB to BLG, which was subsequently stabilized by the PrtB carrier [13] . It is thus difficult to postulate any rule concerning the stabilization effect. Different results (i.e. no stabilization) could perhaps be observed with a different partner and thus could help to determine the mechanism of the stabilization effect. In biotechnological use of recombinant L. lactis strains for protein production, fusions can also facilitate purification (e.g. His-tag strategy). Protein fusion has also been successfully used to optimize the production of the two subunits of heterodimeric complexes as demonstrated with murine interleukin-12 in L. lactis [22] or with heterodimeric enzymes in E. coli [35] . In both cases, the resulting fusion had the expected properties. In other cases however, such fusions might dramatically interfere with the conformation of one or both of the proteins, which might be deleterious for the expected activity. Nevertheless, when L. lactis is used as an antigen delivery vector, fusions can be envisioned since it was demonstrated that both moieties of the chimerical protein are still recognized by the corresponding antiserum [10, 20, 34] and are immunogenic [36] .\nSeveral of the results mentioned above suggest that secretion could be an efficient way to escape intracellular proteolysis. This hypothesis was particularly tested in E7 production [20] . E7 was indeed degraded when intracellular production was induced in late exponential or early stationary growth phase (Fig. 3) . E7 production was then tested in a clpP deficient strain (ClpP is reportedly the major house keeping protease in L. lactis; [37] ) and in a dnaK deficient strain (DnaK is an intracellular chaperone that may promote proteolysis by maintaining the protein in an unfolded state; [38] ). In exponential or stationary phase cultures, no significant difference in E7 patterns was observed between wild type and clpP - (Fig. 3 ) or dnaK -(not shown) strains: E7 was equally degraded in the cytoplasm and remained unchanged in supernatants samples. Altogether, these results indicate that E7 intracellular proteolysis is ClpP-and DnaK-independent. Until recently, only two cytoplasmic proteases, ClpP and FtsH [39] , have been identified in L. lactis. The existence of a third, as yet unidentified protease was postulated by studies of a clpP mutant suppressor [40] . E7 may thus be a useful screening target to identify a putative L. lactis protease that, as suggested by our data, is activated in stationary phase.\nBesides the features of the precursor itself, these results also rise that host factors are involved in protein stability and SE (Fig. 4) . Research efforts are now focusing on the analysis of host factors involved in protein production and secretion by either directed or random mutagenesis in L. lactis [41] .\nAlthough L. lactis possesses a wide range of enzymes (peptidases, housekeeping proteases) dedicated to intracellular proteolysis, it possesses only one extracellular housekeeping protease (HtrA) [9] and its major extracel-lular scavenger protease, PrtP, is plasmid encoded [42] . Thus, a plasmidless strain does not present any protease activity in the medium. Better production yields could then be expected when secretion is used versus cytoplasmic production. These results give clues and provide the research workers with target proteins to study intracellular proteolysis and protein stability inside and outside the host strain. Such studies already led to the development of htrA deficient L. lactis strains. Heterologous protein secretion and anchoring in a htrA deficient strain allowed Fusion to Nuc rescue E7 in intracellular production and increase protein yields for the secreted forms of E7 and L7/L12 \nNative E7 production in wt L. lactis depends on growth phase Figure 3 Native E7 production in wt L. lactis depends on growth phase. E7 production and secretion were analyzed by Western blot from cultures induced at different times so that, 1 hour after nisin induction, the samples are harvested at exponential (OD 600 = 0.5-0.6, upper panels) or stationary phase (OD 600 = 1.5, lower panels). wt/pCYT-E7, NZ(pCYT-E7) strain (encoding native E7, cytoplasmic form). wt/pSEC-E7 NZ(pSEC-E7) strain (encoding the precursor preE7). Positions of E7 mature and precursor forms are given by arrows. C, cell lysates; S, supernatant fraction. ClpP is not involved in the intracellular degradation of E7 in L. lactis. Analysis by western blot shows that a strain of L. lactis deficient in the intracellular protease ClpP cannot rescue cytoplasmic E7 production. Induced cultures samples of wt L. lactis or L. lactis clpP mutant strain containing pCYT-E7 (clpP/pCYT-E7) or pSEC-E7 (clpP/pSEC-E7) taken at exponential-(upper panel) or stationary-(lower panel) phase.\nStationary-phase \nhigher protein stability at the cell surface for several heterologous proteins [10] .\nCurrent research works are now focusing on other host factors that affect protein production and secretion in L. lactis. L. lactis complete genome sequence analysis revealed indeed that the Sec machinery comprises fewer components than the well-characterized B. subtilis Sec machinery. Notably, L. lactis does not possess any SecDF equivalent and complementation of the lactococcal Sec machinery with B. subtilis SecDF results in better secretion yields as determined for Nuc reporter protein (Nouaille et al., submitted) . Random mutagenesis approaches also revealed that features of some cell compartment, such as the cell wall, play an important role in the secretion process [41] . Similar approaches allowed the identification and characterization of genes of unknown functions specifically involved in production yields of the secreted proteins in L. lactis (Nouaille et al., in preparation) .\nMany molecular tools are now available to direct heterologous protein secretion in L. lactis and the list of heterologous proteins produced in this bacterium is regularly increased. The reports where cytoplasmic and secretion production can be compared mostly show that secretion allows better protein yields compared to intracellular Schematic presentation of the molecular tools and the cellular events that can affect the production yields of heterologous pro-tein in L. lactis Figure 4 Schematic presentation of the molecular tools and the cellular events that can affect the production yields of heterologous protein in L. lactis. Thicknesses of the arrows are proportional to the final production yields. All the host factors involved in the cellular events are not identified and or characterized yet. SP, signal peptide (encoded in pSEC constructions), +Nuc, fusion between the protein of interest and the stable Nuc protein. production; and allow a better understanding of the protein production and secretion process in L. lactis.\nFuture works should investigate the L. lactis capacities for protein modifications. For example, we showed that proteins that require a disulfide bond (DSB) to acquire their native conformation can be efficiently produced and secreted in L. lactis [5, 22, 27] . However, no equivalent of E. coli dsb or B. subtilis bdb, the genes involved in DSB formation, was found by sequence comparison in L. lactis. Similarly, other folding elements (i.e. PPIases, so-called maturases...) are still to be identified and the L. lactis capacities for post-translational modifications are still to be investigated.\nAltogether, these works will contribute to the development and the improvement of new food-grade systems for L. lactis [43] and should lead, in a near future, to the construction of lactococcal strains dedicated to high-level production of proteins of interest. The GRAS status of L. lactis and LAB in general, is a clear advantage for their use in production and secretion of therapeutic or vaccinal proteins." }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: It is now widely admitted that actual genomes have a common ancestor (LUCA, Last Universal Common Ancestor). Their current diversity results from events that have modified genomes during evolution. While some of these events happen at the nucleotide level (point mutation, indel of few nucleotides), others [strand inversion, duplications, repetitions, transpositions and horizontal transfers (HTs)] may concern significant parts of the genome. It has been postulated that HTs (exchange of genetic material between two different species) were very frequent during the first stages of evolution and are essentially subsisting nowadays in prokaryotes (1) (2) (3) (4) . As a consequence, the detection of HTs appears crucial to the understanding of the evolutionary processes and to the qualitative and quantitative evaluation of exchange rate between species (5) (6) (7) (8) (9) .\nThe recent complete sequencing of several genomes allows to systematically search for the presence of DNA transfers in species, especially in prokaryotes where the probability of occurrence is higher (10) (11) (12) (13) (14) . It has been reported in particular that (i) HTs in bacteria account for up to 25% of the genome (8, (14) (15) (16) ; (ii) archaebacteria and non-pathogenic bacteria are more prone to transfers than pathogenic bacteria (15, 16) ; and (iii) operational genes are more likely transferred than genes dealing with information management (15) (16) (17) .\nThe HT concept has been originally coined to explain the dramatic homologies between genes of unrelated species (18, 19) . An 'unusual' match is subsequently the criteria for the detection of HTs (20, 21) . While this approach allows detection of gene transfers with only a partial knowledge of genomes, it requires the sequencing of homologous genes in a number of species and consequently cannot be used for HT screening.\nGenes from a given species are very similar to one another with respect to base composition, codon biases and short oligonucleotide composition (15, 16, (22) (23) (24) . As a general rule, usage of oligonucleotides varies less along genomes than among genomes (24) (25) (26) (27) . In addition, it has been observed that transferred DNA retains (at least for some time) characteristics from its species of origin (8, 14) . These particularities are used alone or in conjunction to detect DNA transfers between species (8, 12, 13) . Transferred DNA is consequently detected on the basis of some of its singularities with respect to the sequence characteristics of the recipient species. However, these techniques suffer several drawbacks and weaknesses (28) (29) (30) that led us to consider generalizing the above approach for the screening of atypical regions in sequences. In fact, the genomic signature that accounts for all possible biases in DNA sequences has been shown to be speciesspecific (26, 27, 31, 32) . The signature is approximately invariant along the genome in such a way that the species of origin of DNA segments as small as 1 kb could be identified with a surprisingly high efficiency by means of their signatures (25, 27) . As a consequence, the sequence signature may be most often (at least in bacteria) considered a valuable estimation of the genomic signature. Assuming that (i) transferred DNA fragments exhibit signature of the species they come from and (ii) recipient and donor signatures are different, the screening of local variations of signature along genomes is expected to reveal regions of interest where HTs might be located. In addition, the status of HT is strongly suggested if the signatures of these regions of interest are found close to the signature of other species.\nThe sequence signature is defined as the frequencies of the whole set of short oligonucleotides observed in a sequence (26, 31) . It can be easily obtained thanks to a very fast algorithm derived from the Chaos Game Representation (CGR) (33) , which allows coping with a 1 Mb sequence in a few seconds on a laptop computer. Signatures may be visualized as square images where the color (or gray level) of each pixel represents the frequency of a given oligonucleotide (called word thereafter) (31) (for examples of signatures, see Supplementary Materials 2, 4 and 6).\nDNA sequences are gathered from GenBank. The genomes of 22 prokaryotes are scanned for HTs, B.subtilis, E.coli and H.influenzae genomes being given a special attention to illustrate our approach. In particular, B.subtilis and E.coli provide valuable benchmark thanks to the set of previous works addressing that very issue (12, 14, 16, (34) (35) (36) (37) . Signatures of about 12 000 species are obtained from genomic sequences longer than 1.5 kb. Sequences derived from the same species are concatenated for accuracy purposes. Species from the three domains of life, archaea ($260 species), bacteria ($3950 species) and eukarya ($6750 species) as well as viruses ($1300 species), are represented for a total amount of 1.0 Gb.\nThe detection of atypical regions is based on the observation of deviation of local signatures (i.e. signature of small fragments of DNA) from the genomic signature of the recipient species. Genomes are consequently sampled by means of a sliding window with an appropriate size. In fact, it would be interesting to have windows the smallest as possible for highest sampling accuracy. However, intra-genomic variability of signature increases for small windows. In addition, variability depends on species and word length. Base composition (1-letter word), 2-and 3-letter words are poorly speciesspecific: they do not allow a good discrimination between species (25, 27) . As a general rule, the longer the words (up to 9-letter long), the higher the specificity of the signature (25, 27, 31) . However, counts of long words in small windows are too low to allow a reliable estimation of the parameters. In our hands, the analysis of 4-letter words in a sliding window of 5 kb (with a 0.5 kb step) offers a good trade-off between reliability of count, file size and computational charge, whatever the species. In addition, a double-strand signature (called local signature thereafter) is computed for each window to get rid of variations induced by strand asymmetry (38) (39) (40) (41) (42) .\nFor illustration purposes, local signatures are developed as vertical vectors and stacked together in genome order to give an overall picture of word usage variations along each genome. In such plots, horizontal lines show the variation in frequency of words along the genome, whereas local changes in word usage appear as vertical breaks ( Figure 1 ). Figure 1 . Signatures (4-letter words and 5 kb windows) along genome for Clostridium acetobutylicum, Deinococcus radiodurans and Mycobacterium tuberculosis. In this kind of displays, lines represent the frequency of words along genome, columns represent signature of windows.\nConsidering that the greatest part of the genome is speciestypical, the signature of the recipient species might have been estimated from the analysis of the whole sequence. Although the vast majority of local signatures look mostly the same (believed to be instances of the recipient species signature), some of them may greatly differ. In order to avoid potential biases linked to these outliers, it has been subsequently decided to select typical local signatures on the basis of their similarities, observed after clustering. The underlying idea is that typical local signatures aggregate in few large groups, whereas outliers are found in small complementary groups at a great distance from the recipient genome signature. Groups were consequently determined with the K-means clustering tool, using every scheme of clusters between 3 and 8 for each species. Finally, the best scheme of clusters was obtained by a decision tree-based partition [CART algorithm (43) ]. The purpose of the CART algorithm is to predict values of a categorical dependent variable (clusters of local signatures in this work, each signature being characterized by its distance to the estimated genomic signature) from one or more continuous and/or categorical predictor variables [the different clustering schemes (3-8 clusters) in this work]. The CART algorithm thus provides an optimal split between groups collecting signatures close to the estimated recipient genome signature and the others groups. For each species, a clustering scheme is selected (e.g. the 5-group clustering) and a partition offered (continued example: group 2 and 3 on one side; 1, 4 and 5 on the other). The recipient species signature is subsequently calculated as the mean of the signatures of the groups belonging to the partition with the smallest distance to the estimated genomic signature.\nComparison of signatures is made possible, thanks to an Euclidian metric, accounting for differences in word usage. It must be pointed out that distances between signatures are calculated for high dimensional data (256 dimensions corresponding to the 256 different 4-letter words) and are consequently subjected to the so-called 'concentration of measure phenomenon' (44) . All distances in a high dimension space seem to be comparable since they increase with the square root of the dimension of the space, whereas the variance of their distribution remains unchanged. In fact, the radius of the hyper sphere holding 99% of the signatures of our database is only seven times the nearest neighbor distance (smallest distance between two species). Small differences in distance may consequently be considered highly significant.\nFor each species, a set of recipient-specific distances is obtained, every local signature belonging to the large clusters being given a distance to the host signature. In order to select outlying signatures, a cut-off distance is chosen on the basis of the distribution of distances observed for each species. It appears that the 99% percentile offered a good trade-off between sensibility and specificity for outlier detection (for impact of the threshold on detection of atypical regions, see Results). Most signatures from minority clusters are detected in this way. Isolated signatures are detected as well, while very few signatures from the recipient species clusters are selected (1%). Outliers together with the flanking regions on the genome are later on reanalyzed with smaller window and step (1/10 th of the original size typically) in order to more accurately determine their limits, when signal-to-noise ratio allows it.\nFinally, the gene content of all detected regions is analyzed with the help of species dedicated databases [Genome Information Broker, http://gib.genes.nig.ac.jp/]. A BlastN search (GenBank, default settings) is carried out for each atypical region in order to identify the origin of potential HTs if homology is high enough.\nSearch for the origin of atypical regions About 12 000 species (including chromosomal, plasmidic, mitochondrial and chloroplastic DNA) from GenBank are found eligible for a genomic signature. Given the signature of an atypical DNA fragment, species with a close signature might be considered as potential donors. Such a screening is performed for every atypical region of the 22 species under consideration. The first five nearby species are retained when their distance to the outlier was donor-compatible.\nA total of 22 genomes are screened for atypical regions (Table 1 and Supplementary Material 1). On the average, the 6-cluster scheme offers the best partition. However, in a single case (Aeropyrum pernix), nine clusters are required. In general, a single cluster is devoted to rRNA. The mean distance of windows to host varies over species from 121 to 145 (mean = 132, coefficient of variation = 3%). It is tightly correlated (P-value for the Pearson correlation coefficient <10 À4 ) with the cut-off distance that varies from 178 to 289 (mean = 234, coefficient of variation = 14%). Such large variations can hardly be explained on the mere basis of statistical fluctuations. As already observed (31, 45, 46) , variation of oligonucleotides usage along genome depends on species and can consequently be considered as a species property.\nSegmentation quality of atypical regions can be tested using rRNA genes. About 94% of rRNA is detected as atypical ( Table 1) . Borders of rRNA genes are accurate to within 130 nt (0.5 kb window and 50 bp step, threshold 99%). Meanwhile, adjacent tRNAs are identified as well. As a general rule, it can be concluded that rRNA has a specific signature that is consistently at variance with the host signature. In this context, it is worth noticing that rRNA and the remaining outliers lie at comparable distances from the species they belong to, but they are clearly different from one another, rRNAs being consistently found in their own cluster.\nThe percentage of RNA-free outliers (at the nucleotide level) varies from 1.3 to 13% as a function of species (threshold 99%, Table 1 ). B.subtilis shows the highest percentage of atypical regions, whereas Pyrococcus abyssi has the lowest. Percentages among species are found correlated with the cut-off distance: the higher the cut-off distance, the lower the percentage of outliers (P = 0.007). In fact, a high cutoff distance takes place in species that display a high intragenomic variability, also expressed by a high mean distance to the host (Table 1) . Whether the actual percentage of atypical DNA is an intrinsic property of the species or a mere consequence of the resolution power of nucleotide biases-based methods remains consequently an open question. In addition, as already observed (13, 14) , the percentage of outliers is significantly higher for longer genomes (P = 0.004), whereas the cut-off distance is not related to the length of the genome (P = 0.69).\nThe mean cut-off distance for the 22 species is 234 (Table 1) . This value is chosen to select credible donors. About 50% of atypical regions are subsequently given credible donors (Supplementary Material 1). Each species has it own set of (Table 1) . Many plasmids and viruses are also found in agreement with the known molecular mechanisms of horizontal transfer (Table 1 and Supplementary Material 1).\nA clustering with three classes allows assessing the signature of B.subtilis. The most populated class (collecting 84% of the segments) is chosen to represent B.subtilis. For this subpopulation, the mean distance (arbitrary unit) to the recipient (centroid of the class) and the cut-off distance are 126 and 204, respectively ( Table 1 ). Runs of contiguous outlying windows sharing the same cluster are considered as single transfer events. As a consequence, 58 regions (Figure 2a and Supplementary Material 2) fall beyond the cut-off distance and are thus potential candidates for hosting foreign DNA (for a segmentation of the B.Subtilis genome in terms of genes, see Supplementary Material 3). Figure 2b illustrates the accuracy of segmentation of an atypical region obtained by using a sliding window of 0.5 kb with a 50 bp step. rRNA genes make up $1.1% of B.subtilis genome ( Table 1) . All rRNA genes are found in the outlier population. In addition, all windows containing rRNA are assigned to a specific cluster. In fact, it is known that rRNA has its own signature, which is at variance from the host signature (12) . rRNA genes account for 7% of the outliers (tRNAs are not considered in this study, because their size is too small to generate a significant deviation from the host signature if they are isolated).\nA total of 86% of the B.subtilis genome should be considered as B.subtilis typical (Table 1) . When looking for the origin of B.subtilis segments in the 12 000 signature database, B.subtilis appears in the 10 first potential donors for 84% of the whole set of 5 kb sequences that can be derived from its genome. This result confirms that segments having signatures belonging to the predominant clusters are good representatives of the recipient species signature.\nThe 49 rRNA-free atypical regions vary in size from 1.5 to 135 kb and make up 13% of the total genome (Table 1) . About 50% of atypical regions are less than (or around) 6 kb long. Distances of outlier from first potential donor often fall within the intra-genomic range ( However, in some instances, the outlier-to-donor distance is too great to consider the 'closest' species as potential donor. In contrast, unusual small values deserve a specific attention. In particular, the very small distance between bacteriophage SPBc2 and '2150751-2285750' atypical region (d = 2) allows to spot the part of B.subtilis genome where bacteriophage SPBc2 is incorporated (12, 47) . Other regions in the genome are also found similar (in terms of signature) to bacteriophage SPBc2. Most of them correspond to bacteriophages, imbedded in B.subtilis genome, whose free forms are not sequenced (12, 47) . Observed similarities with SPBC2 are, however, expected since signatures of phages usually share some characteristics with the species they infect (48) . The SPBc2 sequence is the only foreign sequence identified in B.subtilis, using homology as criterion (BlastN, with parameters set to default). In fact, Blast analysis of B.subtilis outliers leads to contrasted results. Besides SPBc2 and 7 out of 9 prophages imbedded in the genome, the only atypical regions identified are those containing the 30 rRNA genes coded in B.subtilis genome. The only few genes that are homologous to parts of atypical regions are found in species belonging to the Bacillus genus. It is interesting to note that no house-keeping genes (except rRNA) are detected in atypical regions. In fact, a great number of genes in atypical regions (except bacteriophage genes and rRNA) have no known function.\nA clustering with five classes is required to determine the recipient species signature of H.influenzae. The three most populated classes (collecting 94% of the segments) are chosen to calculate the H.influenzae signature. Mean distance to host and cut-off distance is subsequently found equal to 130 and 239, respectively (Table 1) . Similarly to B.subtilis, one cluster (1.5% of H.influenzae genome) is devoted to the 18 rRNA gene copies (Table 1) . A total of 91% of rRNA is labeled atypical and account for 29% of the outliers.\nAnalysis of Table 1 shows that 95% of the H.influenzae genome should be considered as H.influenzae typical. In fact, H.influenzae is one of the 10 first potential donors for 92% of all 5 kb sequences that can be derived from its genome. As already observed for B.subtilis, the concordance of these two percentages corroborates the partition procedure used for the selection of typical/atypical fragments.\nThe 13 rRNA-free atypical regions vary in size from 1.5 to 19.5 kb and make up 3.3% of the genome (Table 1 , Annex 4 and Figure 3 , see Annex 5 for a segmentation of the H.influenzae genome in terms of genes). About 50% of atypical regions are less than (or around) 2.5 kb long. Numbers for H.influenzae are clearly at variance with those for B.subtilis: a smaller percentage of the genome qualifies as atypical and the average size of atypical regions is also smaller. This result is examined below in the context of intra-species signature variability (see Discussion).\nA clustering with six classes is required to determine the recipient species signature of E.coli. The main features are summarized in Table 1 . The potential donors of the 84 RNAfree atypical regions are given in Annex 6 (for a segmentation of the E.coli genome in terms of genes, see Annex 7). It is worth noticing that 56% of E.coli potential donors belong to the Enterobacteriales family. Segmentation in terms of genes is displayed in Annex 7. The analysis of this genome is particularly useful for the comparison with literature (see below).\nNumerous approaches for detecting horizontal gene transfers have been proposed in the last 2 decades. Phylogenetic trees of protein or DNA sequences, unusual distribution of genes, nucleotide composition (including codon biases) are some of the HT features that are considered within the framework of these models (16, 34) , Hidden Markov Models (HMMs) (12, 14, 35) and Factorial Correspondence Analysis (FCA) (37) are some criteria that are currently employed. Each of the resulting models has its own advantages and caveats (28) (29) (30) . As it has been recently pointed out by Ragan (49) and Lawrence and Ochman (50) , each approach deals with a particular subset of HTs, being for example more efficient for detecting recent transfers, or more effective for the detection of ancient HTs. Our approach, which is clearly based on oligonucleotide composition, assumes that different species have different signatures but does not rely on any other assumption. It is not surprising, therefore, that the genomic signature approach provides results (in terms of % of DNA transferred) in reasonable agreement with those proposed by Garcia-Vallve (16) and Nakamura et al. (14) for the 22 species that were analyzed in common. Correlations between percentages of HTs found by these three methods are highly significant Two species are extensively studied for HT content: B.subtilis (five methods including ours) and E.coli (six methods including ours). H.influenzae is also analyzed by Garcia-Vallve (16) and Nakamura (14) . Comparisons of methods are presented in Tables 2-4 and detailed in Supplementary Materials 3, 5 and 7. A voting procedure (majority rule) has been implemented to determine the status of genes with respect to atypicality. For that task, our initial analysis is converted in terms of genes (Supplementary Materials 3, 5 and 7). Degree of agreement between methods is subsequently observed using the statistical Kappa coefficient (51) . Kappa measures the degree of agreement on a scale from minus infinity to 1. A Kappa of one indicates full agreement, a Kappa of zero indicates that there is no more agreement than expected by chance and negative values are observed if agreement is weaker than expected by chance (a very rare situation). (14, 13, 11, 13 and 15%, respectively). The number of detected genes per method is close, ranging from 457 for Nakamura (14) to 599 for this work (median 537). Detailed votes are given in Table 2 . Among the 4100 genes of B.subtilis genome, 1011 genes are detected by at least one method (about 25% of B.subtilis genes). The number of 'single vote' genes ranges from 116 for Garcia-Vallve (16) to 47 for Nicolas (12) . A total of 470 genes make up the majority consensus set and we detected 453 of them, which is the best score of the five methods. The best agreement with the majority consensus (in terms of Kappas) is reached by Nicolas (12), followed by our method and Moszer (36) ( Table 2 ). Our method gets the best agreement with Nicolas (12) and the worst with the other HMM method used by Nakamura (14) (pairwise Kappa comparison, Table 2 and Supplementary Material 3). In fact, Nakamura approach is at variance with every other approach (14) . It gets the lowest Kappa with the Garcia-Vallve (16) Hayes (35) Lawrence (34) Nakamura (14) Medigue (49) This work majority consensus or with whatever other methods. From Table 2 , the probable number of HT genes in B.subtilis would range from 230 to 1011 with a 'reasonable' estimation around 470 corresponding to the majority consensus. It is to be noted that our method is unable to find two genes that are detected by every other methods (Supplementary Material 3) . These genes are 338 and 236 nt long, respectively, as compared with 2500 nt, the median size of atypical regions detected by our method (Table 1) . Clearly, our method is not appropriate for detecting short isolated atypical genes.\nH.influenzae. Garcia-Vallve (16), Nakamura et al. (14) and we are the voters concerned with the analysis of the H.influenzae genome (Supplementary Material 5 and Table 3 , H.influenzae). The originality of results obtained by Nakamura (14) is the salient feature of this comparison. The number of detected HT genes is more than twice higher for Nakamura et al., whereas the part belonging to the majority consensus is the smallest ( Table 3) . Eleven genes are detected both by Garcia-Vallve and Nakamura (14, 16) but not by our method; however, the small number of voters precludes any specific comment in this respect. The probable number of HT genes in H.influenzae would range between 11 and 273, with a 'reasonable' estimation around 60 (majority consensus of 57) ( Table 4 ).\nThe results obtained by Hayes and Borodovsky (35) are clearly at variance with the others (Table 4 ). Although the proportion of claimed outliers is within the range of published numbers for E.coli (14, 16, 24, 34, 35, 37) , 37% of them are method-specific, and the agreement with other methods is weak (Table 4 ). Hayes and Borodovsky have obviously developed an approach based on HMM dealing with specific outliers. Lawrence and Ochman (34) also get a poor rating especially because they detect about twice as many genes as the other authors do (Table 4) .\nIt is worth noting that if the cut-off distance for our method is lowered, i.e. 95% instead of 99% for instance, some of the 'single vote' genes are dug out (for details about the impact of the cut-off distance, see Supplementary Material 7). Meanwhile, the percentage of outliers as reported by our approach rises to 20% and the percentage of 'single vote' genes reaches 24%. As expected, a high cut-off distance provides few single vote genes at the risk of missing some potentially transferred genes. Lowering the cut-off increases the proportion of single vote genes with the advantage of detecting most of the potential transfers (Supplementary Material 7) . There is obviously a continuous grading in gene 'atypicality'. It is suggested to first consider most 'consensual' genes as potential HTs and then apply amelioration models to explain the grading.\nIt is difficult to assess the relevancy of proposed donors, because genes detected as potential HT have generally undergone amelioration (8) . The comparison of recently diverged genomes (species or strains) provides the opportunity to find recent HTs, for which corresponding homologous genes in the donor species may be detected (52) . Such a study is performed for five E.coli strains (two K12 strains: E.coli MG1655, E.coli W3110, one uropathogenic strain: E.coli CFT073, two enterohaemorrhagic strains: E.coli O157-H7 RIMD 0509952, E.coli O157-H7_EDL933) and two Shigella flexneri strains (S.flexneri 2a 2457T, S.flexneri 2a 301). These seven strains/ species have recently diverged, genome sizes are different and the proportion of horizontally transferred genes varies from one strain/species to another (14, 52) . For instance, only $40% of the non-redundant set of proteins is common to E.coli strains CFT073, 0157-H7 EDL 9333 and MG1655 (53) . These strains/species can be clustered in four groups with respect to phylogeny (Table 5) .\nTwo criteria are used to searching for 'recent horizontally transferred genes': atypical regions (window size 1 kb, step 0.5 kb) (i) must have a signature that differs greatly from that of the host [distance to host must be at least >325, 2.5 times the E.coli intrinsic mean distance (Table 1) ] and (ii) must be present in a limited number of strains/species to ascertain their recentness. In fact, outliers meeting the first criterion generally aggregate into several heterogeneous clusters (K-means clustering) that usually include samples from each strain/species. In some instances, however, some strains/species were absent from the cluster. It was subsequently considered that the corresponding regions might have been recently acquired by the relevant strains/ species. Table 5 shows a selection of potential recently transferred genes. Each cluster of atypical regions contains genes present in a specific set of strains. Some atypical genes are strainspecific, some are only absent in the non-pathogenic K12 strains and intermediate situations are also encountered.\nFASTA and Blast searches confirm that these genes are absent from some of the tested strains as already observed in the analysis complete genomes (53) (54) (55) . In a large number of cases, we are able to find a well-conserved homologous gene in another species (Table 5) . It is interesting to note that some of the suggested donors using our 12 000 signature database are in agreement with the species found by alignment methods. When no homologous gene is found, the proposed donors give credit to the known mechanisms of gene transfer (bacteriophages or plasmids) ( Table 5) .\nIt is worth noticing that most of the selected genes that are absent in K12 strains are involved in the pathogenicity of the other strains (52) . E.coli 0157-H7 is the strain exhibiting the greatest number of genes absent in K12 strains [about 1400 (54) ]. It has the greatest number of genes for which no homolog can be found (Table 5) . Moreover, we are unable to propose a donor for a great part of these genes (Table 5) . Many selected genes for E.coli 0157-H7 lie in the Ter region of the genome (between positions 2 000 000 and 2 500 000) in agreement with the published results (56). \nWe have observed that most genomic regions are typical of the genome they belong to, using the signature as endpoint.\nConsidering that the genomic signature is species-specific, atypicality of a region in terms of oligonucleotide usage has been promoted as a criterion for the detection of HTs.\nHowever, atypicality-based methods suffer several caveats that reduce their effectiveness in such a way that only a part of HTs can be detected. In fact, transfers between species with close signatures cannot be detected: significant differences between characteristics of transferred DNA and recipient species DNA are required. For similar reasons, HTs that were drastically ameliorated following their introduction cannot be detected either (8, 14) . The most stringent constraint, however, results from the size of the screening window. On the one hand, ideally, the best signal-to-noise ratio would be obtained when windows and HTs have a comparable size.\nOn the other hand, the window size must be large enough to provide significant word counts, a requirement that strengthens with the size of the words under consideration and the intrinsic variability of the genomic signature along the genome. All together, the trade-off that has been implemented in this paper allows detecting atypical regions as small as 1 kb. In fact, rRNA regions sharing this characteristic were consistently detected. It must be pointed out that smaller fragments can be eventually detected if their signatures are radically atypical. G+C% atypicality has often been considered as criterion for detecting HTs (8, 24) , but this approach suffered several drawbacks (28) (29) (30) . It is to be noted that our signature-based method detects regions for which the G+C% lies within one standard deviation from the mean G+C% of the species (for instance, regions 2675251-2676250 in B.subtilis or 534751-535250 in H.Influenzae, see also Supplementary Materials 2 and 4).\nAs already observed by Nicolas et al. (12) for B.subtilis, rRNA has definitely an atypical signature. It is systematically classified as outlier, whatever the species (Table 1) . Although transfer of rRNA from one species to another is unlikely (11, 57) , it cannot be firmly ruled out. However, it is clear that the atypical signature of rRNA does not imply that they are horizontally transferred.\nThe signature approach has an interesting property (that it shares with HMM) (7, 12, 28) : detection is not bound to any specific function in the genome. In contrast with most other methods, the signature approach not only detects genes, but whole transferred regions as well, in agreement with the described mechanisms of DNA exchange between species. It is to be noticed that the method allows detecting several atypical non-coding regions (Supplementary Materials 3, 5 and 7). One major difference between HMM and signature method lies beyond the time required for the learning process, in the few resources that HMM can mobilize to deal with a short 'one of its kind' HT. On the other hand, HTs shorter than 1 kb can hardly be detected by a signature-based approach. An innovative HT detector is likely to result from an adequate fusion of both methods.\nSeveral factors contribute to the efficiency of the search for donors. Of course, distance between putative HT and donor signatures is essential. Accuracy of signatures, linked to the length of available sequences, density of signatures in the 'vicinity' of HT, amount of amelioration sustained by HT during its presence in the host are also of importance [P. Deschavanne, S. Lespinats and B. Fertil, unpublished results; (25, 27, 31) ]. Distance between the signature of a putative HT and the closest species varies to a large extent, but usually the shortest ones fall within the intra-genomic range ( Table 1 , Supplementary Materials 1, 2, 4 and 6) . In some cases, the distance between the closest donor signature and the atypical segment signature is so great that no potential donor can be proposed (Supplementary Materials 1, 2, 4 and 6) .\nWhen strong similarities between a given DNA sequence and a foreign species are observed, the hypothesis for an underlying transfer is highly strengthen. However, the 'true' donor has to be previously sequenced and included in our bank of signatures to allow such a situation to occur. Moreover, we must take into account the intrinsic variability of short DNA segment signature (which is a function of their size, but also species-specific) when compared with the signature of a complete genome or any other large species sample (25, 27, 31) . In the present state, our signature database is in no way representative of the diversity and richness of life. However, it must be noticed that there is already an obvious structure (in terms of distances between signatures) expressing taxonomy relationships between species in our signature database (31, (58) (59) (60) (61) . Related species are often found close to one another. Clusters of potential donors may consequently provide pertinent information about the origin of HTs.\nThe diversity of signatures of putative HTs that can be observed for most of the species analyzed in this paper reveals the multiplicity of transfer events and donors (Supplementary Materials 2, 4 and 6). However, several outliers, not necessarily neighbors in the genome, are given the same set of potential donors (Table 1 , Supplementary Materials 1, 2, 4 and 6). In general, the potential donors belong to few sets of taxonomically close species (Table 1 ) and share the biotope of the host (Supplementary Materials 1, 2, 4 and 6). For instance, B.subtilis, H.Influenzae and E.coli live in distinct biotopes; their potential donors do so as well. It is particularly encouraging to find that most of the potential donors that our approach has pointed out have had the opportunity to exchange DNA material with the recipient species.\nNumerous viruses and plasmids qualify as potential donors (Tables 1 and 5 , Supplementary Materials 1, 2, 4 and 6 ). It is not really surprising since they are known as HT vectors. They are often totally or partially inserted together with transferred genes in the host genome (14) .\nSome atypical DNA segments are particularly peculiar. They are isolated, have a specific signature (distances from neighbors are great), so that they cannot be given a credible set of donors (Supplementary Materials 1, 2, 4 and 6) . Lack of data in the search domain, shift of signature features after a substantial amelioration process, structural constraints serving special functions or roles (14,62) (as it is for rRNA coding regions) are some of the tracks that remain to explore in these circumstances.\nIt would be interesting to localize the region the transfer may come from when the complete genome of the donor is available. However, homology (at the DNA level) is not a pertinent criterion for the comparison of sequences as soon as amelioration has taken place (8, 14) . In fact, homology is sometimes weak, e.g. between genes of Escherichia and Salmonella although these species have 'recently' diverged (34) . It is clear that a more powerful search for the origin of putative HTs would have to embody models of amelioration [such as the one designed by Lawrence and Ochman (8) ].\nWhen searching for very recent horizontally transferred genes, in different strains of a species for instance, it was possible to find a great homology between detected genes and some genes from other species (Table 5 ). In numerous cases, the selection of donors is consistent with FASTA results ( Table 5 ). This confirms the pertinence, beyond the similarity of signature between putative HTs and donors, of the proposed method to retrieve the species of origin of a transferred region. It seems that the search for origin of HTs on the basis of genomic signature is a powerful approach to understand some of the mechanisms of evolution (13, 63) .\nOligonucleotide usage is known to be species-specific and to suffer only minor variations along the genome (25, 27) . Considered together, these properties allow searching for atypical local signatures that may point out DNA transfers. Results obtained with the 22 genomes analyzed in this paper are found in good agreement with literature (Tables 2-4 , Supplementary Materials 3, 5 and 7) (12, (14) (15) (16) 24, 34, 35) .\nThe species specificity of signature allows searching for donor species. Quite often, sets of donor species with common taxonomic features are obtained. With the help of environmental considerations, it is subsequently possible to identify (or collect clues about) potential donors. The search for donor makes use of non-homologous sequences. Partially sequenced species become consequently eligible, inasmuch 1.5 kb of the genome is available (25, 27) . Thanks to the exponentially growing rate of nucleotide databanks, the search for donor species by means of the sequence signature will turn more and more pertinent and fruitful in the future. In this context, it is worth noticing that computational power is clearly not an issue since the CGR algorithm described in this paper is fast and of 0 order (calculation time is proportional to the number of nucleotides).\nSeveral methods are proposed to look for HTs. The signature method, based on different hypotheses, is complementary to those already described. It seems that each method detects preferentially certain types of HTs (49, 50) . In agreement with many authors (1, 16, 49, 50, 64) , it appears that the conjunction of several methods is required to obtain an overview of HT extent in a genome.\nThe signature method described in this paper generalized many approaches that ground the detection of outliers on the basis of the bias in oligonucleodides. The strong species specificity of the signature not only allows detecting various kinds of outliers but also provides clues about their possible origin. Obviously, the detection of HTs remains an open question; a consensus has still to emerge.\nAdditional materials and experimentation with the genomic signature are available from the GENSTYLE site (http:// genstyle.imed.jussieu.fr)." }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: Oligonucleotide microarrays are a powerful technological platform for large-scale screens of common genetic variation and disease-causing mutations (1) (2) (3) (4) (5) . In most published studies (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) , oligonucleotide microarrays are designed to screen specific sequence tracts, up to megabases in length (11, 15, 22, 23) , for all possible single nucleotide substitutions. With some exceptions (24) (25) (26) (27) (28) (29) (30) (31) , the same emphasis was not placed on identifying all possible small insertions and deletions in the heterozygous state. Nevertheless, it is crucial to detect such small insertions and deletions since they can play a major role in inactivating or altering gene function by disrupting functional elements (e.g. splice junctions, cis-acting elements and open reading frames) and also represent another class of common genetic variation.\nTwo fundamental approaches are commonly used to analyze data sets from oligonucleotide microarrays tailored to identify genetic variation in specific DNA segments purely by hybridization (1, (3) (4) (5) 9) . One approach involves identifying statistically significant gains of target hybridization signal to oligonucleotide probes complementary to specific sequence variants (9) . In theory, the gain of signal approach has the advantage of both detecting the presence of genetic variation and identifying the nature of the sequence change in the target. However, it is not feasible to screen for virtually all possible insertions and deletions due to the overwhelming The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact [email protected] number of mutation-specific probes needed for this analysis. Furthermore, little effort has been made to systematically access the hybridization properties of probes complementary to these small insertions and deletions. The second approach involves identifying losses of hybridization signal to perfect match (PM) probes that are fully complementary to the DNA segment of interest (8, 25, 27, 30, 31) . In theory, the loss of signal approach allows one to screen for all possible sequence changes, including insertions and deletions, that cause a given target nucleic acid sequence to contain mismatches with specific PM probes. However, this necessitates the sequencing of specific DNA regions to identify the nature of the sequence changes (8, 25, 27, 30, 31) . Thus, a combination of the gain and loss of hybridization signal analysis could provide the most robust means of identifying and characterizing mutations using non-enzymatic oligonucleotide microarray assays.\nHere, we analyze the specificity and reproducibility of nucleic acid hybridization to oligonucleotide microarrays used in the large-scale mutational analysis of the ATM (ataxia telangiectasia mutated) gene that is responsible for autosomal recessive disorder involving cerebellar degeneration, immunodeficiency, radiation sensitivity and cancer predisposition and is also commonly mutated in certain lymphoid malignancies (32, 33) . These microarrays include 25mer oligonucleotide probes complementary to all possible single base substitutions and insertions as well as one and two base deletions on both strands of the ATM coding region. This provides the first comparative analysis of the hybridization properties of substitution, insertion and deletion probes in an oligonucleotide microarray-based mutational analysis of a large gene.\nA series of 120 DNA samples derived from biopsies of lymphoma patients were previously screened for all possible ATM mutations using oligonucleotide microarrays (30) . Here, we have selected a total of 68 samples that showed robust amplification signals in all 62 coding exons for further analysis (30) . A total of 17 unique mutations, each in a one-to-one mixture with wild-type sequence, occurred once in these samples. The impact of any given mutation in a single sample is minimal given that 67 other samples with wild-type sequences in the region encompassing a given mutation are included in this analysis. Several single nucleotide polymorphisms (SNPs) were present multiple times: 735 C/T, 2572 T/C and 4258 C/T in two samples; 3161 C/G in four samples; and 5557 G/A in five samples. Likewise, these SNPs have a minimal effect on our global analyses given the large number of samples and bases interrogated in this study.\nAs previously described (30) , individual ATM coding exons were amplified from genomic DNA using primers containing T3 and T7 RNA polymerase tails, pooled, and then in vitro transcribed using T3 or T7 RNA polymerase to create biotinlabeled sense and antisense strand targets, respectively. Fluorescein-labeled reference target was made using genomic DNA from an unaffected individual. Reference and test sample targets were fragmented, diluted in hybridization buffer [3 M TMA-Cl (tetramethylammonium chloride), 1· TE, pH 7.4, 0.001% Triton X-100] and hybridized to the ATM microarrays as described previously (30) . Afterwards, the microarray was stained with a phycoerythrinstreptavidin conjugate and digitized hybridization images from both reference and test targets were acquired using the Gene Array Scanner (Hewlett Packard, Palo Alto, CA) equipped with the appropriate emission filters.\nCustom software was used to quantify hybridization signals for each probe and subtract background hybridization signals. We exclusively focused on raw data from the biotin-labeled test targets since they provide approximately seven times the hybridization signal of the fluorescein-labeled wild-type reference target in this system (28) . This enhanced signal provides greater sensitivity toward detecting weak hybridization.\nFor each sample, for each base and for each potential type of mutation (i.e. substitution, one or two base deletion or one base insertion), the specificity was calculated as the ratio of the PM probe hybridization signal of the wild-type target to their cognate insertion, deletion or single base substitution probes on each strand. The logarithm of these ratios was plotted as a function of the position within the gene. To illustrate the special patterns and to smooth out random variation, running averages of data from 10 bases were used. To capture the variability, at each base, the sample-to-sample standard deviation was again calculated using data derived from a running average of 10 bases for each sample.\nTo estimate the mean hybridization specificity for each type of mutation, the geometric mean (i.e. the antilog of the average of the logged ratios) over all bases and over all specimens was calculated (Table 1) . To further examine the variability of the specificity ratios, the coefficient of variation (cv) was calculated in two ways. The cv is the ratio of the standard deviation divided by the mean; it is useful for understanding the amount of variability relative to the magnitude of the mean or typical value. For the intra-sample cv, the cv was calculated for each of the 68 samples (using the running average of 10 at each ATM base) and the average of the 68 coefficient of variations was taken. For the inter-sample cv, at each of the bases, the cv a Hybridization specificity ratio is defined as the ratio of PM probe hybridization signal to that of the brightest mismatch probe within a given category. The global average of all hybridization specificity ratios for each base in all samples for a given probe type is provided. b Determined for hybridization specificity ratios averaged across windows of 10 bases either within (intra) or across (inter) samples.\nwas calculated using the 68 samples, and the average of the coefficient of variations was taken. For both calculations, the moving average of 10 was used, instead of the original value, since the goal was to understand how the specificity varied over bases and across samples, rather than to estimate the experimental (or measurement) error.\nIn order to determine the relative specificity of the hybridization of complex nucleic acid targets to oligonucleotide probes complementary to single base substitutions, insertions and deletions, we analyzed data generated from oligonucleotide microarray-based mutational analysis of the 9168 bp ATM coding region (30) . These studies used a pair of oligonucleotide microarrays (Affymetrix, Santa Clara, CA) containing over 250 000 probes (25 nt in length) specifically designed to screen the sense and antisense strands of the ATM coding region for genetic variation (27, 30) . Collectively, the ATM sense and antisense microarrays contain 55 008 probes complementary to all possible single base substitutions, 73 344 probes complementary to all possible one base insertions, and 18 336 probes complementary to all possible one base deletions and 18 336 probes complementary to all possible two base deletions in the ATM coding sequence (Figures 1 and 2 ). These microarrays have been used to screen for sequence variation in the ATM gene in over 100 DNA samples (30) . SNPs and gene inactivating mutations were uncovered by screening for localized losses of hybridization signal to PM probes complementary to every 25 nt segment of the ATM coding region (8, 25, 27, 30) . However, hybridization data from deletion and insertion probes were not relied upon in this analysis. Therefore, this data set provides a unique opportunity to examine the relative hybridization specificity of nucleic acid targets to each of these classes of mismatch probes.\nIn order to gain a global overview of hybridization specificity, we determined the average ratio of PM probe hybridization signal of wild-type target (see Materials and Methods) to their cognate insertion, deletion and single base substitution probes on each strand (Table 1 ). In these calculations, we considered data for all 9168 interrogated bases in all 68 DNA samples (see Materials and Methods). For example, we report the ratio of the PM probe signal to the signal from its cognate 1 or 2 bp deletion probe. However, for single base substitutions, we report the ratio of the PM probe signal to that of the cognate substitution probe with the highest hybridization signal. This provides the most rigorous assessment of cross-hybridization to single base substitution probes. Likewise, for single base insertion probes, we report the ratio of the PM probe signal to that of the cognate insertion probe with the highest hybridization signal.\nFor both sense and antisense strands, we found that the two base deletion probes had the highest average PM to cognate MM hybridization specificity ratio (3.26-fold sense and (Table 1) .\nTo provide a finer-scale analysis of hybridization specificity, we determined the relative frequencies of hybridization specificity ratios in defined bins. There was a similar distribution of specificity ratios for single base substitution and two base deletion probes on both strands ( Figure 3 ). The overall lower hybridization specificities of single base deletion and insertion probes are reflected by the increased frequencies of probes within the lower specificity bins (i.e. <2-fold ratio) and decreased frequencies of probes within higher specificity bins (i.e. >3-fold ratio) on both strands.\nNext, we sought to uncover underlying trends in the hybridization specificity of different classes of mismatch probes across the entire ATM coding region within a given sample (intra-sample variation). This provides insights into sequence context effects that may influence the hybridization specificity of each class of mismatch probe. To approach this problem, we plotted the average hybridization specificity ratios of substitution, deletion and insertion probes for all 1168 bases across the 68 samples (Figure 4 and Supplementary Figure 1) . We analyzed data determined over running averages of 10 bases in order to maximize our ability to detect trends and minimize the effect of randomly dispersed confounding factors (e.g. intra-or intermolecular secondary structure) that may skew data for any given base.\nAs expected from Table 1 and Figure 3 , the two base deletion probes consistently showed a higher average hybridization specificity ratio followed by single base substitution, single base deletion and single base insertion probes on both strands of exon 50 ( Figure 4) . Nevertheless, the hybridization specificity ratios for all classes of mismatch probes fluctuate across the exon 50 sequence (Figure 4 ). For example, two base deletion probes showed a peak value of 6.76 (unlogged) centered at base 7071 and a trough value of 1.90 (unlogged) centered at base 7002 on the sense strand. We also found similar fluctuations in specificity ratios for all mismatch probe types in the remaining 61 ATM coding exons (Supplementary Figure 1 ).\nTo assess intra-sample variability in hybridization specificity by a different means, we determined the average cv for substitution, deletion and insertion probes within a given experiment (Table 1) . Again, we analyzed data from running average of 10 bases in order to maximize our ability to detect trends and maintain consistency in our data analysis. Substitution probes had the lowest average intra-sample cv, 0.31 and 0.23 for sense and antisense strands, respectively. One base deletion, two base deletion and insertion probes showed comparable intra-sample coefficients of variation on the sense strand, 0.37, 0.39, and 0.38, respectively. However, insertion probes showed relatively higher variability than the deletion probes on the antisense strand. Coupled with plots shown in Supplementary Figure 1 , it is evident that of all the mismatch probe types, the hybridization specificities of base substitution probes were least affected by target sequence context. Intrigued by the above observations, we next searched for specific target sequence tracts that produced the lowest hybridization specificity among and between the different classes of mismatch probes. To approach this problem, we determined how many mismatch probes within running windows of 10 bases gave poor hybridization specificity, previously defined as a hybridization specificity ratio <1.2 (26). In Table 2 , we report nucleotide tracts where at least 8 probes within a given 10 base window showed poor hybridization specificity ratios. A comprehensive listing of probes with poor hybridization specificity is provided in Supplementary Table 1 .\nRepetitive sequence tracts, including homopolymer, homopurine and homopyrimidine, are highly represented in Table 2 . Upon closer inspection, it became apparent why the cross-hybridization is strong for probes in homopolymeric regions. In these sequence contexts, substitution and deletion probes can form duplexes with wild-type target that are longer than 12 bp in length. For example, the probe designed to detect a single base deletion at position 633 is designed to form one 12 bp and one 13 bp duplex with wild-type target. However, this probe can form duplexes that range from 12 to 18 bp in length with wild-type sense strand target due to slippage ( Figure 5 ). This type of ambiguity leads to increased stability of these DNA-RNA heteroduplexes (34) .\nIn principle, the homopurine and homopyrimidine tracts uncovered have the capacity to form higher order structures, such as triple helices (35) . These tracts are known to alter the conformation and stabilities of RNA-DNA heteroduplexes (36, 37) , such as those formed between RNA targets and DNA probes in our system. Finally, we expect the ATM target to be especially rich in such sequence tracts given that both strands of the 3 0 -splice acceptor sequences, typically containing homopyrimidine tracts, for all 62 coding exons are included in the ATM target. This increases the likelihood that highly related sequence tracts in the ATM target can cross-hybridize to probes interrogating a particular homopurine or homopyrimidine sequence tract and reduce the overall hybridization specificity in this region.\nNext, we screened for potential structures that can form in the PM probes listed in Table 2 or their targets that could explain their poor hybridization specificity. To do this, we used Mfold (38) to calculate Gibbs free energies for intramolecular structures that can form in these PM probes and targets. Based on these Gibbs free energy values, we classified the probes and targets as having strong (S) [DG < (À3 kcal/mmol)], medium (M) [(À1 kcal/mmol) > DG > (À3 kcal/mmol)] and weak (W) [G > (À1 kcal/mmol)] potential for secondary structure. We found that several target and probe sequences could form substantial secondary structures, as displayed in Figure 6 . This could artificially lower the affinity of target to PM probes and thus lower the hybridization specificity. It is more difficult to model intermolecular structure in the solution-phase complex target and in the solidphase oligonucleotide probes. However, it appears likely that such structures could also have a similar negative impact on hybridization specificity.\nThe relative variability in hybridization specificity ratios across samples (inter-sample variability) represents another important issue that should be considered in resequencing analysis (9) . To uncover general trends in inter-sample variability for each type of mismatch probe, we calculated an average cv for mismatch probe hybridization specificity ratios determined over running windows of 10 bases (Table 1) . Interestingly, on both strands, the single base substitution probes showed the lowest inter-sample cv. The one and two base deletion probes showed at least 2-fold higher coefficients of variation on both strands, relative to the substitution probes. Surprisingly, the one base insertion probes showed significantly higher coefficient of variations than any of the other classes of mismatch probes across samples. In fact, they are 3.5-fold higher than the corresponding substitution probes on each strand.\nThe relative levels of inter-sample variation for all mismatch probes across exon 50 are displayed graphically in Figure 4 . The error bars represent one standard deviation from the mean of the hybridization specificity ratio determined over a running window of 10 bases in each of the 68 samples. Note that the substitution probes show lower inter-sample variability than one base deletion, two base deletion and . Hybridization specificities of mismatch probes. A 10-base running window of the log 10 hybridization specificity ratios of substitution (red), one base deletion (green), two base deletion (blue) and one base insertion (black) was plotted for the sense (A) and antisense (B) strands of ATM exon 50. The light red, light green, light blue and gray shaded areas represent -1 SD of the log 10 hybridization specificity ratios for the substitution, one base deletion, two base deletion and one base insertion probes, respectively. one base insertion probes, in agreement with Table 1 . The variability in hybridization specificity measurements is consistent across all 62 ATM coding exons (Supplementary Figure 1) .\nOverall, our analyses indicate that, on average, single base insertion probes show substantially lower reproducibility across experiments than base substitution, one base deletion and two base deletion probes. The increased inter-and intrasample variability in hybridization specificity of single base insertion and deletion probes relative to single base substitution and two base deletion probes should be considered when designing and interpreting microarray-based screens for genetic variation. For a given microarray design, substantially more control hybridization experiments may be needed to determine baseline fluctuations in the hybridization specificities of insertion and deletion probes relative to those of substitution probes.\nIn contrast to single nucleotide mismatches, detailed thermodynamic analyses of double helical nucleic acids with bulged nucleotides have only recently been conducted (34, (39) (40) (41) . In such cases, the bulged nucleotide is unpaired on only one of the nucleic acid strands. These studies are relevant to understanding the properties of the deletion and insertion probes since they can form duplexes containing bulges with target nucleic acid. For deletion probes, the bulged nucleotide is located on the target strand ( Figure 7) . Conversely, the insertion probes contain the bulged nucleotide in duplexes with wild-type target (Figure 7) .\nAlthough subject to sequence context effects, duplexes containing a single base bulge are predicted to be more stable than those containing single nucleotide mismatches (34, (39) (40) (41) . This is reflected in the lower average hybridization specificity of single base deletion and insertion probes relative to that of substitution probes (Table 1 and Figure 4) . Conversely, duplexes containing two base bulges are predicted to be generally less stable than those containing a single base mismatch (40, 41) . In part, this is due to the assumption that helical stacking is interrupted by bulges of two or greater bases in length while it is preserved for one base bulges (40, 41) . The higher average hybridization specificity ratios of two base (38) was used to predict the intramolecular structures with the lowest Gibbs free energy (DG) for either the 25-30 base stretches that encompass each listed sequence tract in the target or for the PM probes complementary to each sequence tract. We use these DG values to predict the stability of these structures. DG > (À1 kcal/mmol) = weak (W); (À1 kcal/mmol) > DG > (À3 kcal/mmol) = medium (M); and DG < (À3 kcal/mmol) = strong (S). c Type of mismatch probe that provided poor hybridization specificity ratios. d Low hybridization specificity found on both sense and antisense strands. e Immediately following the 3 0 end of this segment is a (T) 5 sequence tract.\ndeletion probes relative to substitution probes are in agreement with the predicted properties of these probes ( Table 1) . The considerably lower average inter-sample variability of substitution probes relative to deletion and insertion probes was unexpected given that the same target was hybridized to all mismatch probes simultaneously in the same experiment. The sources of inter-sample variation include sample preparation, hybridization conditions and the microarrays themselves. It is reasonable to assume that the microarrays themselves are not the major source of variability since the combinatorial manufacturing processes should lead to roughly equivalent synthesis quality for all the arrayed probes (42, 43) . It seems more likely that the insertion and deletion probes are more sensitive to subtle changes in target preparation (e.g. amount of fragmentation and dye incorporation) and hybridization conditions (e.g. target concentration, temperature and wash conditions) than the substitution probes. However, a definitive explanation for our observations will require further investigations (44) (45) (46) (47) (48) (49) (50) (51) (52) .\nIn addition to their potential value, it is important to note some of the caveats when relying upon mismatch probes for mutation detection. For example, it is important to screen for all possible sequence changes, including multiple base insertions and deletions, in mutational analyses of disease-related loci, such as the ATM, BRCA1 and BRCA2 genes. Given that 4 N probes per base per strand are needed to screen for insertions of length N in a mixed sequence, it is unlikely that oligonucleotides complementary to insertions of two or more base pairs will be represented on microarrays screening large sequence tracts for mutations in the near future. Deletions represent a more tenable situation since only one probe per base per strand is needed to screen for a deletion of a given length in a mixed sequence. Nevertheless, there will still be limitations as to the number of deletion probes that can be realistically represented in a given microarray.\nFinally, it is often critical to precisely determine the nature of a sequence change within a given sample in order to properly assess its functional significance. Thus, it is important to consider error rates when assigning the identity of a mutation based on mismatch probe data. When dealing with clinical samples, it will be especially important to confirm the identity " }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: Sialic acids are a diverse family of acidic nine-carbon sugars that are frequently found as terminal units of oligosaccharide chains on different glycoconjugates in higher invertebrates and vertebrates [1, 2] . As a part of determinants in many glycoproteins [3, 4] , sialic acids play an important role in intercellular and/or intermolecular recognition [5] . The 9-O-acetylation and de-Oacetylation are the most common modifications of sialic acids found in mammalian cell surface sialoglycoconjugates, which can alter its size, hydrophobicity, net charge, and antigenicity [2, 6, 7] . These modifications can regulate a variety of biological phenomena, including endogenous lectin recognition, tumor antigenicity, virus binding, and complement activation [8, 9] .\nEnzymes specifically capable of removing O-acetyl esters from the 9-position of sialic acids are sialic-acidspecific 9-O-acetylesterase. The enzymes in mammals have two forms, one is cytosolic sialic-acid-specific 9-O-acetylesterase (Cse) in the cytosolic fraction and another is lysosome sialic-acid-specific 9-O-acetylesterase (Lse) in the lysosomal/endosomal compartment [10] . Lse is likely to participate in the terminal lysosomal degradation of 9-O-acetylated sialoglycoconjugates, while Cse is likely to salvage any 9-O-acetylated molecules that escape the initial action of the Lse enzyme. The process of de-O-acetylation of sialic acid, which is catalyzed by sialicacid-specific 9-O-acetylesterase, has been implicated in organogenesis and cellular differentiation [2, 5] .\nSpermatogenesis is a complicated process of germ cell differentiation in adult testis, which is established during testicular development. There are five types of germ cells, each at a specific developmental stage, found in the seminiferous tubules: spermatogonia, primary spermatocytes, secondary spermatocytes, spermatids and sperms. They can be divided into three groups according to their DNA content: 4N DNA content cells (4C cells), 2N DNA content cells (2C cells), and 1N DNA content cells (1C cells). The separation of these cells enables researchers to investigate the molecular mechanisms underlying testicular development and/or spermatogenesis. In the present study, we separated the 2C and 4C cells of seminiferous tubules in human adult testis by flow cytometry, and identified human H-Lse by differential display RT-PCR. The expression pattern of H-Lse was found to be developmentally regulated and stage-specific, indicating its possible role in testicular development and/or germ cell differentiation.\nHuman testes were obtained from Donation Center of Nanjing Medical University with consent of relatives. The seminiferous tubules were collected in DMEM/F12, which contained collagenase, and washed to remove the Leydig cells as well as interstitial cells. Trypsin treatment and a brief treatment with DNase I were used to release the spermatogenic cells from seminiferous tubules. The suspension of cells was filtered with nylon mesh.\nDisaggregated spermatogenic cells were suspended at 1 × 10 6 cells/mL in 0.5 M sodium citrate solution (PH 2.35) with fresh 0.1% DEPC overnight at room temperature and at 4 • C for two days; they were centrifuged and resuspended in 0.5 M sodium citrate solution (PH 4.5) with fresh 0.1% DEPC for at least 1 day. The day before use, the cells were centrifuged and resuspended in PBS with 10 mM HEPES (PH 7.0), 0.1% BSA, and fresh 0.1% DEPC. Then the cells were spun down and resuspended in PBS with 100 µg/mL PI (propidium iodide) and fresh 0.1% DEPC. The cells were stained overnight at 4 • C [11] .\nThe flow cytometry (FCM) used in this research was FACSVantage SE (Becton Dickinson, Calif) equipped with argon laser (power: 200 mW, wavelength: 488 nm); a 585 nm/42 nm filter set was used before the FL2 detector. Cellquest (Becton Dickinson) was used for sorting and the sorting mode was Normal-R. Drops per sort were 3 and drop delay was 13.6. The density of cells for sorting was about 1 × 10 6 cells/mL.\nIsolation of total RNA from 2C cells and 4C cells was performed with Trizol Reagent (Gibco BRL, Ontario). One hundred nanograms of total RNA was used for differential display RT-PCR [12] . The first chain cDNA was synthesized by using T12G, T12C, and T12A oligo (dT) primers, and then was used as template in PCR. PCR was performed as follows: 94 • C, 1 minute; 37 • C, 1 minute; 72 • C, 2 minutes for 40 cycles. Ten microlitres of the PCR products from the two cells were run on a 1.5% agarose gel. The fragments highly or specifically displayed in 4C cells were excised and purified. This DNA was reamplified with the same combination of primers and then subcloned into Pinpoint Xa1-T vector (Promega, USA).\nThe colonies of full-length cDNA were screened by PCR. Human Testis Large-Insert cDNA Library (Clontech, Calif) was first converted into plasmid cDNA Library, and then an arrayed cDNA library in 96-well plates was made according to the method of Munroe [13, 14] . In this arrayed cDNA library 1.54 × 10 6 colonies were screened by PCR.\nMultiple tissue northern (MTN) blots (Clontech) were hybridized with the 32 P-labeled probes. The probe corresponding to 1378-1634 bp of H-Lse was used for hybridization. After stringent wash, the blot was placed on the storage phosphor screen (Packard, USA) and exposed for 3 hours in the dark. The signal was detected at the Cyclone storage phosphor system (Packard).\nThe Stanford TNG Radiation Hybrid Panel (Research Genetics, Huntsville, Ala) was used to map the chromosomal localization of HSE with primers HSEmapF (5 -ATGAACACCGTCTCCACC-3 ) and HSEmapR (5 -AAATCTGAAGGACCCATC-3 ), according to the manufacturer's instructions. After 35 cycles of amplification, the reaction products were separated on a 1.5% agarose gel. The positive amplification was labeled as 1 and the negative one was labeled as 0. The results were analyzed through the Stanford genome center web server to determine the probable chromosomal location.\nRNA DIG-labeled probes were made by in vitro transcription. T7 and SP6 promoter sequences were incorporated into the two sides of the templates (195-553 bp of H-Lse) by PCR, sense and antisense probes were made using DIG-RNA labeling mix (Roche, USA) according to the manufacturer's instructions. After fixation, paraffin embedding, mounting, and sectioning, sections of human embryonic and adult testes were prehybridized in hybridization buffer (DIG Easy Hyb, Roche, Germany) at 42 • C for 2 hours. Hybridization was carried in hybridization buffer containing appropriate probes at 65 • C for 16 hours in humidity chamber.\nSubcellular localization of HSEI and HSEII was performed by the method of green fluorescent protein. pEGFP-C2-HSEI AND pEGFP-C2-HSEII were constructed using two sets of primers (HSEI: 5 -GGGGAATT CAATGATATGGTGCTGCAG-3 and 5 -GGGGTCGACAT TTAGCAACATTGCTCTG-3 ; HSEII: 5 -GGGGAATTCA TGGTCGCGCCGGGGCTTG-3 and 5 -GGGGTCGACA TTTAGCAACATTGCTCTG-3 ) and EcoRI/SalI restriction sites of pEGFP-C2. Recombinant vectors were transfected into BxPC-3 cells (BxPC-3 cell is a cell lineage of adenocarcinoma from pancreas) by Lipofectin reagent (Gibco BRL). Cells were imaged 40 hours after transfection on the fluorescence microscope.\nAfter being stained with PI and measured by the FCM, three groups of cells in seminiferous tubules of human adult testis were detected (Figure 1 ), 2C and 4C cells were subsequently sorted. A clone was identified by differential display RT-PCR, which was highly expressed in the 4C cells ( Figure 2 ) and with high homology (86%) to a mouse lysosome sialic acid 9-O-acetylesterase. The clone was named H-Lse.\nIn the two rounds of screening in the arrayed cDNA library, the plasmid containing full-length H-Lse (GenBank accession number: AF303378) was found. H-Lse is 2750 bp in length, encoding a putative protein of 523 amino acids with a molecular weight of 58 kd. Its isoelectric point is 7.19. The N terminus (1-18 aa) of the protein is a region containing hydrophobic amino acid residues, which may be a signal peptide. By comparison of the protein sequences (Figure 3 ), we hypothesized that H-Lse is the human counterpart of mouse lysosome sialic acid 9-O-acetylesterase.\nAfter PCR amplification, the results can be shown as a pattern (00000000100010100000011000001000000011 000001000001000001001000000010000000000100100100 0001). Retrieving results from the Stanford genome center web server shows that HSE is localized in the human 11q24 ( Figure 4) .\nThe distribution of H-Lse in various human tissues was analyzed by Northern blot ( Figure 5 ) and the results showed the presence of three distinct mRNA species at approximately 2.7 kb, 6.0 kb, and 7.5 kb. The expected transcript of H-Lse was approximately 2.7 kb and it was consistently expressed in all the tissues examined with high expression found in the testis, prostate, and colon. The transcript of approximately 7.5 kb was exclusively expressed in the colon. The transcript of approximately 6.0 kb was distributed in the testis, colon, small intestine, prostate, and thymus, with the highest level of expression found in the testis.\nTo examine a possible role of H-Lse in testicular development and/or spermatogenesis, in situ hybridization experiments were conducted to compare H-Lse expression in human embryonic and adult testes since spermatogenesis is not initiated in the embryo and there is no meiosis in embryonic seminiferous tubules. The results showed that no signal was detected in the embryonic testis, while positive signals were detected in spermatocytes but not spermatogonia in the seminiferous tubules of adult testis. Signals were associated with germ cells but not other somatic cells in the testis, that is, Sertoli and Leydig cells. Negative control of sense probes confirmed the specificity of the results ( Figure 6 ).\nThe subcellular localization of H-Lse fusion proteins was visualized by transiently transfecting H-Lse gene fused with GFP into BXPC-3 cells. As shown in Figure 7 , the control cells transfected with GFP protein alone exhibited fluorescence evenly distributed throughout the cytoplasm, while GFP-H-Lse fusion protein was compartmentalized in numerous large dense-core vesicles in the cytoplasm.\nSpermatogenesis is a developmental program that occurs in mitotic, meiotic, and postmeiotic phases. In the mitotic phase, spermatogonia proliferate to expand the quantity of germ cells; in the meiotic phase, spermatocytes accomplish chromosomal synapsis and genetic recombination before two meiotic divisions; and in the postmeiotic phase, haploid spermatids are remodeled into spermatozoa by the processes of acrosome formation, nuclear condensation, flagellar development, and loss of the majority of cytoplasm. Under the control of intrinsic and extrinsic factors, spermatogenesis is characterized by the expression of a spectrum of genes that are celltype-specific or stage-specific. They are thought to play an essential role in spermatogenesis at particular stages. For example, MutS homologue 5 is required for chromosome pairing, CPEB and SCP3 are required for synaptonemal complex assembly and chromosome synapsis in primary spermatocytes [15, 16, 17] .\nIn the present study, we have identified a gene, H-Lse, from human adult testis with high homology to m-Cse 1 [19] . Similarly, it can inhibit binding of sialoadhesin, a macrophage-restricted and sialic-acid-dependent adhesion molecule [20] . On the other hand, 9-O-acetylation of sialic acids can form novel epitopes. Influenza virus C haemagglutinin specifically requires 9-O-acetylated sialic acids for binding to host cells [21] . Incubation of red blood cells with sialate 9-Oacetylesterase rendered the erythrocytes resistant against agglutination by influenza C virus [22] . O-acetylation of disialoganglioside GD3 by human melanoma cells has been reported to create a unique antigenic determinant [23] . Modifications of sialic acids may be an important mechanism underlying the interaction/cross-talk between different types of cells. The essential role of sialic acids modification in cellular communications may explain the presently observed wide distribution of H-Lse in all examined tissues. The present study suggests that the expression of H-Lse is developmentally regulated and spermatogenic stage-specific. The evidence for this includes: (1) lack of expression in embryonic testis; (2) association of high level of mRNA detected by DD-RT-PCR with the 4C but not 2C cells in adult testes; and (3) detection of in situ hybridization signal in spermatocytes but not spermatogonia or other somatic cells. In the absence of spermatogenesis, embryonic testis contains only two distinct cell types, spermatogonia and Sertoli cells, while the seminiferous epithelium of adult testis consists of germ cells at different stages of spermatogensis. The 4C cells found in adult testis include the primary spermatocytes and spermatogonia of G 2 /M stage, while 2C cells include spermatogonia of G0/G1 stage, secondary spermatocytes, and Sertoli cells. The absence of H-Lse mRNA in embryonic testis and the high level of its mRNA in the 4C cells of adult testis suggest that its expression is restricted to spermatocytes, particularly the primary spermatocytes. Together with the in situ hybridization results showing mRNA of H-Lse restricted to spermatocytes, but not spermatogonia, Sertoli cells or interstitial cells, these data suggest that H-Lse is likely to be involved in the process of spermatogenesis, although its role in testicular development cannot be entirely ruled out. Unfortunately, due to the deformation of the available human testes, we were not able to make further distinction between primary and secondary spermatocytes. What has been clearly shown by the present data is that H-Lse is only present at a stage beyond spermatogonia, suggesting its possible role in the differentiation of germ cells.\nG N F T Y M S A V C W L F G R Y L Y D T L Q Y P I G L V S S S W G G T Y I E V W S S R R T L K A C G V P N T 143 m-Lse 181 A G N L G H G N F T Y M S A V C W L F G R Y L Y D T L Q Y P I G L V S S S W G G T Y I E V W S S R R T L K A C G V P N T 240 h-Lse 181 S E N L G H G Y F K Y M S A V C W L F G R H L Y D T L Q Y P I G L I A S S W G G T P I E A W S S G R S L K A C G V P K Q 240 m-Cse 144 R D E R V G Q P E I K P M R N E C N S E E S S C P F R V V P S V R V T G P T R H S V L W N A M I H P L Q N M T L K G V V 203 m-Lse 241 R D E R V G Q P E I K P M R N E C N S E E S S C P F R V V P S V R V T G P T R H S V L W N A M I H P L Q N M T L K G V V 300 h-Lse 241 G S _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ I P Y D S V T G P S K H S V L W N A M I H P L O N M T L K G V V 274 m-Cse 204 W Y Q G E S N A D Y N R D L Y T C M F P E L I E D W R Q T F H Y G S Q G Q T D R F F P F G F V Q L S S Y M L K N S S D Y 263 m-Lse 301 W Y Q G E S N A D Y N R D L Y T C M F P E L I E D W R Q T F H Y G S Q G Q T D R F F P F G F V Q L S S Y M L K N S S D Y 360 h-Lse 275 W Y Q G E S N I N Y N T D\nInterestingly, the processes of 9-O-acetylation and de-O-acetylation of sialic acid have been implicated in organogenesis and cellular differentiation, since alteration of these processes could lead to interruption of cellular development such as embryogenesis. Transgenic mice constitutively overexpressed the 9-O-acetyl-sialicacid-specific esterase of influenza C that has been found to arrest embryo development at the two-cells stage. It has also been reported that in vitro development of embryonic stem cells shows that the expression level of Lse is low at the initiation of the development, and followed by an increase at later stages [24] . In transgenic mice with selective expression of 9-O-acetyl-sialic-acid-specific esterase in retina and the adrenal gland, these organs showed various abnormalities in organization, while all other tissues appeared normal [25] . Lse has also been considered to play a key role in the differentiation of B lymphocyte [2] , since it is expressed in late but not early B lymphocyte. The presently observed developmentdependent pattern of H-Lse expression is consistent with that found in other cell types: absence or low expression at early stage of differentiation but high at later stages. Taken together, 9-O-acetyl esters in sialic acids appear to be important for development or cellular differentiation.\nSpermatogenesis is a multiple-staged continuous progress of cellular differentiation. It has been reported that some cell surface glycoconjugates are modified during the early steps of spermatogenesis, and influence the differentiation of spermatogenic cells [26] . As ninecarbon sugars commonly found in many glycoproteins of spermatogenic cells, sialic acids represent a target for cell surface modification, that is, removal of 9-O-acetyl esters by enzymes such as Lse. Modification of sialic acids may result in alteration in cell-cell communication, that is, Sertoli cells and germ cells interaction, thereby influencing the differentiation of spermatogenic cells. Thus, future studies on the presently identified H-Lse may provide insight into molecular mechanisms underlying testicular development and/or germ cell differentiation during spermatogenesis in humans." }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: Although pain is the presenting symptom of most patients with chronic pancreatitis, its neurobiological basis remains poorly understood [1] . In the past, investigators have focused on the role of anatomical abnormalities such as a strictured pancreatic duct or narrowed intraparenchymal ducts. However, mechanical decompression techniques such as endoscopic stent placement or even surgical pancreatojejunostomy frequently do not provide a permanent solution to the pain [1] . More recently, investigators have begun focusing on the role of neurotransmitters and neurotrophins such as substance P and nerve growth factor with known or suspected roles in nociceptive signaling and/or sensitization and have reported an increased expression of several of them in the pancreas of patients with painful chronic pancreatitis [2] . Mast cells are also increased in both acute and chronic pancreatitis [3, 4] but their role in the generation of pain in pancreatitis has not been investigated.\nWe hypothesized that mast cells are involved in the pathogenesis of pain in chronic pancreatitis. This hypothesis is based on the following observations. First, mast cells have been associated with human conditions in which pain is a predominant symptom. Interstitial cystitis and irritable bowel syndrome are both conditions in which pain is out of proportion to the objective pathological findings [5, 6] . In both conditions, an increase in the number of mast cells has been described in the bladder and the colon, respectively [5, 6] . Further, mast cells are frequently found in close proximity to nerves in the intestinal mucosa and the bladder [7] [8] [9] . This has also been observed in the pancreas -the total number of mast cells was significantly higher in pancreatic tissue from patients with chronic pancreatitis than in the normal pancreatic controls [3] . One of the preferential locations of mast cells was around and within the perineurium of nerve fibers in tissue samples of patients with chronic pancreatitis, suggesting the potential for interactions between mast cells and the nervous system. Lastly, there is evidence for bi-directional functional communication between mast cells and nerves [10] [11] [12] . Mast cells can not only release mediators that increase excitability of neurons but in turn, neurotransmitters such as substance P can trigger mast cell degranulation [10] . Mast cells may therefore contribute to the pathogenesis of pain in pancreatitis through degranulation products that can sensitize pancreatic afferent neurons in an ongoing vicious circle of neuronally mediated mast cell degranulation.\nOur first aim was to analyze the presence and distribution of mast cells in autopsy specimens of chronic pancreatitis and study the correlation, if any, with historical documentation of pain. We then explored our hypothesis further using an experimental model of trinitrobenzene sulfonic acid (TNBS)-induced chronic pancreatitis in both wild type and Kit W /Kit W-v mice, a strain deficient in mast cells (MCDM).\nAutopsy records from the University of Texas Medical Branch from the years 1993 to 2000 were searched electronically for the term \"pancreatitis.\" One-hundred sixtysix patients were identified of which 26 patients carried an autopsy diagnosis of chronic pancreatitis and 140 patients carried a diagnosis of acute pancreatitis. The medical charts from patients with an autopsy diagnosis of chronic pancreatitis were reviewed for documentation of a medical history of chronic pancreatitis. If no such documentation was present in the chart, patients were excluded from the study (12/26) . Thus, 14/26 patients with both a documented history and an autopsy based diagnosis of chronic pancreatitis, were included in the study. Patients were categorized as painful chronic pancreatitis (8/26) when they fulfilled one of the following criteria: a documented history of chronic abdominal pain clinically attributed to chronic pancreatitis that required the use of narcotics, and/or frequent admissions for recurrent abdominal pain clinically attributed to chronic pancreatitis, and/or a surgical or endoscopic procedure for refractory abdominal pain clinically attributed to chronic pancreatitis. Patients were categorized as non-painful chronic pancreatitis (6/ 26) if patients did not fit any of the criteria listed under painful chronic pancreatitis. In addition, the following data were collected: demographic factors (age and race), cause of death, comorbidities, clinical history of pancreatitis, etiology of pancreatitis, diagnostic studies supporting a diagnosis of pancreatitis (amylase, lipase, calcifications on abdominal plain film, CT-scan, ultrasound or ERCP). Human pancreatic control tissue was obtained from 8 arbitrarily chosen patients of whom the autopsy records recorded acute myocardial infarction as the cause of death. Their medical records were reviewed to ensure that they did not have a clinical history of pancreatitis. Therefore there were three categories of patients: one with painful chronic pancreatitis, one with non-painful chronic pancreatitis and non-pancreatitis controls. A pathologist, blinded to the group assignment, verified all histological diagnoses and counted mast cells on a Giemsa stained tissue section (average of 10 high-power randomly chosen (40X) fields per specimen). The protocol was approved by the Institutional Review Board of the University of Texas Medical Branch.\nAll mice were purchased from the Jackson Laboratory (Bar Harbor, ME). Male mice were used from the following strain: WBB6F1/j-Kit W /Kit W-v (MCDM) and the respective littermate control mouse strain, Kit W-v -+/+ (WT). The mice were 3 months of age at the onset of the experiment with body weights of 25-30 gram.\nExperimental protocols involving mice were approved by our Institutional Animal Care and Use Committee (IACUC) in accordance with the guidelines provided by the National Institutes of Health.\nMice were anesthetized with sodium Nembutal (50 mg/kg body weigh, i.p.) Following a midabdominal laparotomy, a canula was introduced into the common pancreato-biliary duct; the duct was ligated proximally and distally to ensure perfusion into the pancreas and prevent entry of the injected substance into the liver or duodenum. 0.1 ml of 1% TNBS in phosphate buffered saline (PBS)-10% ethanol, pH 8, was infused into the pancreas (modified after Puig-Divi [13] ). The canula was removed and the abdomen closed. Control mice were treated in the exact same fashion but were perfused with saline instead (Figure 1 ). Mice were sacrificed 8 weeks after surgery.\nVFF hairs consist of a series of filaments of increasing diameter that produce increasing sensations of touch when applied to the skin. When the tip of a fiber of given length and diameter is pressed against the skin, the force of application increases until the fiber bends. After the fiber bends, continued advance creates more bend, but not more force of application. This principle makes it possible to apply a reproducible force to the skin surface. VFF testing is an established behavioral pain assay used to determine mechanical pain thresholds in somatic pain.\nMore recently, VFF testing has been used as a surrogate marker for visceral pain [14, 15] .\nMice were placed in a cage with a mesh floor and habituated to the environment for 30-60 minutes. Measurements were taken from the abdomen and the plantar surface of both hindpaws over a period of three weeks prior to the surgery and for a total of three weeks after the surgery ( Figure 1 ). VFF filaments of various caliber were applied to the mid-abdomen in ascending order 10 times, each for 1-2 seconds with a 10 second interval. A response was defined as: a) sharp retraction of the abdomen; b) immediate licking or scratching of site of application of hair; or c) jumping. The response frequency was defined as the total number of responses out of 10 applications (expressed as a percentage) to the skin per filament. An investigator blinded to the different treatment groups performed the behavioral testing.\nFresh specimens of the mouse pancreas were fixed in 10% formaldehyde in PBS pH 7.4 containing 1 mM MgCl 2 at 4°C overnight. Sections from paraffin-embedded specimens were stained with hematoxyline and eosin and observed under a light microscope. Pathological changes were scored based on a scale described by Tito et al. by a pathologist blinded to the different treatment groups [16] .\nComparisons of the number of mast cells in autopsy specimen were analyzed using the Mann-Whitney U test.\nFor each behavioral experiment (see figure 1), the average response frequency was calculated as the mean of the mean response frequencies for each mouse (across four measures). The \"post-pre response frequency\" was calculated by subtracting the pre-surgical average response frequency from the post-surgical average response frequency. To assess the independent effect of TNBS on VFF response (ie. to control for the effect of the surgery itself), the postpre response frequency for TNBS infusion was compared with the post-pre response frequency for saline infusion. This comparison was performed using analysis of variance for a two-factor experiment with repeated measures on time at each level of force for each type of mice (WT and MCDM). The two factors were induction of pancreatitis or not (TNBS or saline, respectively) and time (pre-surgical or post-surgical). TNBS infusion was considered to have had an independent effect on the VFF response if the postpre response frequency was greater for TNBS than for saline infusion.\nFisher's least significant difference procedure was used for multiple comparisons of least squares means with Experimental design Figure 1 Experimental design All mice underwent pre and post surgical VFF testing. For the VFF testing, 4 measures were taken for each mouse. WT and MCDM were randomized to either saline or TNBS perfusion into the pancreatic duct. \nPatient demographics are summarized in Table 1 . Alcohol abuse was the most common cause for pancreatitis in both groups. Analysis of our results, using the Mann-Whitney U test, revealed significantly more mast cells in patients with a history of painful chronic pancreatitis (n = 8) when compared to patients with either non-painful chronic pancreatitis (n = 6) (33.8 vs 9.4 average mast cell count/10 high power fields; p < 0.01) or controls (n = 8),\n(33.8 vs 6.1 average mast cell count/10 high power fields; p < 0.01) ( Figure 2 ). The increased number of mast cells in patients with painful pancreatitis was noted predominantly in interstitial areas and, to a lesser degree, in the periacinar space. Figure 3 shows the post-pre surgical response frequency for both WT and MCDM. TNBS had a significant independent effect on abdominal VFF response in WT mice at the force levels 4 and 8 mN (p = 0.007 and 0.037, respectively) ( Figure 3A ). There was a trend towards a significant effect at the force level of 16 mN (p = 0.066). In contrast, for MCDM, TNBS had no significant effect on abdominal VFF response at any force level ( Figure 3B ). There was no significant TNBS effect on VFF response in the left A g e \nPancreatic histology confirmed the presence of chronic pancreatitis in both WT and MCDM with marked fibrosis, inflammatory infiltrates and ductular proliferation mimicking changes seen in human chronic pancreatitis ( Figure 5A ). The pancreas of saline treated controls was normal. There was no significant difference in the overall inflammatory scores between the WT and MCDM ( Figure 5B ). An increased number of mast cells were counted in WT mice with chronic pancreatitis compared to saline Histology (Giemsa) of mice with chronic pancreatitis (Figure 6 ). As to be expected, no mast cells were present in pancreas of MCDM.\nChronic pancreatitis has been defined as a continuing inflammatory disease of the pancreas characterized by irreversible morphologic changes that typically cause pain and/or permanent loss of function [17] . The pathogenesis of pain in this condition remains to be satisfactorily established. We examined the association, if any, of pain with mast cells as quantified in autopsy specimens of patients with a history of painful and non-painful chronic pancreatitis and normal controls. Significantly more mast cells were present in pancreatic tissue from patients with a history of painful chronic pancreatitis, indicating an association with this condition and a potential role for these cells in the pathogenesis of pain in painful chronic pancreatitis.\nThere are clearly limitations to a retrospective, autopsybased study such as the one we report here. For instance, we do not know whether pain was present at the time of death and there was incomplete information on the different patterns of pain. Also, our findings pertain mainly to patients with a history of alcoholic pancreatitis. Nevertheless, our findings do suggest an association of painful chronic pancreatitis with an increased number of mast cells. This observation provided the rationale for further experimental testing, which we performed in mice. We first developed a model of chronic pancreatitis in mice following a modified protocol first described by Puig-Divi et al. [13] . Histological changes consisted of periductal and lobular fibrosis, duct stenosis, chronic inflammatory cell infiltrates, and gland atrophy, mimicking features of chronic pancreatitis in humans. Significantly more mast cells were present in WT mice with chronic pancreatitis, adding to the validity of this model for use in studies on the role of mast cells in pancreatitis. Both WT and MCDM developed histological changes consistent with chronic pancreatitis, indicating that the elimination of mast cells did not modulate the animals' ability to mount an inflammatory response. Therefore, any changes observed in pain behavior are unlikely to stem from differences in underlying inflammation.\nNext we determined whether this mouse model could be used to study behavioral differences associated with chronic pancreatitis. The assessment of spontaneous pain in a visceral organ presents significant difficulties. We have used a behavioral method to assess this, which relies on the association of visceral pain with sensitization of somatic regions of the body that share segmental innervation at the level of the spinal cord (referred pain). This somatic sensitization can be quantified using VFF to stim-ulate the somatotopically appropriate abdominal region and measuring the abdominal withdrawal response. Thus, VFF testing of the anterior abdominal wall can be used as a surrogate marker for visceral pain. Although this is the first time that this technique has been used for the measurement of referred visceral hyperalgesia in a mouse model of chronic pancreatitis, this method has previously been described and validated to assess the severity of referred visceral pain for models of colonic hypersensitivity [14] as well as rat models of acute necrotizing pancreatitis [15] and chronic pancreatitis [18] . The abdominal VFF response was compared to the hind paw response to assess the specificity of the interventions to the pancreas. TNBS treated mice, but not the saline control, developed increased abdominal wall withdrawal responses to VFF testing when compared to baseline, suggesting the development of force-dependent referred hyperalgesia of the abdominal wall in WT mice. There was no evidence of referred hyperalgesia in the hindpaws, suggesting that the measured effect on abdominal withdrawal is specific for an intra-abdominal origin of the pain. Vera-Portocarrero et al. previously described similar findings, increased withdrawal frequency after VFF stimulation to the abdominal area, in a rat model of chronic pancreatitis [18] . These behavioral changes were abrogated by morphine. Rats that demonstrated behavioral changes also expressed increased substance P expression in the nociceptive layers of the spinal cord, suggestive of central nociceptive changes.\nMast cells produce a variety of degranulation products in the setting of inflammation that may activate and/or sensitize primary nociceptive neurons. The neurotrophin growth factor (NGF) is one such product [19] [20] [21] [22] . NGF is released in the setting of inflammation and can not only function as a chemoattractant for other mast cells, but it can also trigger mast cell degranulation [23] . We are speculating that NGF production in the inflamed pancreas is responsible for plastic changes in the sensory neurons by activating proalgesic receptors and channels such as the NGF receptor tyrosine kinase A (TrkA) and Transient Receptor Potential Family V receptor 1 (TRPV1; previously known as VR1) thereby contributing to the generation of pain [24] [25] [26] . Similarly, other mast cell degranulation products such as tryptase and histamine are capable of modulating neuronal function [27] [28] [29] [30] [31] [32] . Tryptase may directly activate the proteinase-activated receptor-2 (PAR-2), a G-protein coupled receptor expressed by pancreatic nerves, important in the pathogenesis of pain in pancreatitis [33, 34] . Although the role for mast cells in the mediation of visceral nociceptive signaling needs to be explored further, we speculate that mast cell products released in pancreatitis, contribute to the development of pain by direct effects on nociceptors located on pancreatic afferent neurons (Figure 7 ).\nHowever, before concluding a definite role for mast cells from our experimental data, it should be noted that MCDM carry a spontaneous mutation for tyrosine kinase receptor c-kit which not only produces a deficiency of mast cells but may have an independent effect on the function of sensory neurons, which are known to express it [35] . Therefore, it remains to be determined whether the detected differences in nociceptive responses is due to the absence of mast cells per se or a yet unknown change in the responsiveness of sensory neurons due to a congenital lack of the c-kit receptor. Reconstitution of mast cells into the MCDM mice should restore their nociceptive responses close to the wild type phenotype.\nOur data should increase awareness of the importance of mast cells in the pathogenesis of painful inflammatory\nProposed involvement of mast cells in nociceptive signaling in pancreatitis Figure 7 Proposed involvement of mast cells in nociceptive signaling in pancreatitis In pancreatitis, mast cells may migrate to sites of inflammation, in response to release of mast cell chemoattractants. Mast cell degranulation products may modulate neurotransmission directly by activating proalgesic receptors and channels such as trka (NGF), TRPV1 (NGF) and PAR-2 (tryptase and trypsin). \nThe pre-publication history for this paper can be accessed here:\nhttp://www.biomedcentral.com/1471-230X/5/8/prepub" }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: As increasing numbers of full-length viral sequences become available, recombinant or mosaic viruses are being recognized more frequently [1, 2, 3] . Recombination events have been demonstrated to be associated with viruses expanding their host range [4, 5, 6, 7] or increasing their virulence [8, 9] , thus accompanying, or perhaps even being at the origin of, major changes during virus adaptation. It remains unclear, however, whether recombination events represent a highly frequent and significant phenomenon in the everyday life of these viruses.\nViruses can exchange genetic material when at least two different viral genomes co-infect the same host cell. Progeny can then become hybrid through different mechanisms, such as reassortment of segments when the parental genomes are fragmented [10] , intra-molecular recombination when polymerases switch templates (in RNA viruses) [11] , or homologous or non-homologous recombination (in both RNA and DNA viruses). Quantification of viral recombination in multicellular organisms has been attempted under two distinct experimental approaches: in vitro (in cell cultures) [12, 13, 14, 15] , and in vivo (in live hosts) [16, 17, 18] . The in vitro approach, which has so far been applied only to animal viruses, allows the establishment of the ''intrinsic'' recombination rate in experimentally co-infected cells in cell cultures [14, 15, 19] . However, it does not necessarily reflect the situation in entire, living hosts, where the frequency of coinfected cells is poorly known and depends on many factors such as the size of the pathogen population, the relative frequency and distribution of the different variants, and host defense mechanisms preventing secondary infection of cells. The in vivo experimental approach is closer to biological conditions and may thus be more informative of what actually happens in ''the real world.'' However, as discussed below, numerous experimental constraints have so far precluded an actual quantification of the baseline rate of recombination. First, many experimental designs have used extreme positive selection, where only recombinant genomes were viable (e.g., [13, 20, 21] ). Other studies did not use complementation techniques but detected recombinants by PCR within infected hosts or tissues [18, 22, 23, 24, 25] , which provides information on their presence but not on their frequency in the viral population. So far, no quantitative PCR or other quantitative method has been applied to evaluate the number of recombinants appearing in an experimentally infected live host. Finally, recent methods based on sequence analysis inferred the population recombination rate, rather than the individual recombination rate [1, 26, 27] . While results from these methods certainly take in vivo recombination into account, there are other caveats: isolates have often been collected in different hosts-sometimes in different geographical regions-and sometimes the selective neutrality of sequence variation on which these estimates are based is not clearly established. Estimates from such studies by essence address the estimation of the recombination rate at a different evolutionary scale.\nTaken together, the currently available information indicates that no viral recombination rate has ever been estimated directly at time and space scales corresponding to a single multi-cellular host infection, although this level is most significant for the biology and evolution of viruses. This study intends to fill this gap by evaluating the recombination frequency of the cauliflower mosaic virus (CaMV) during a single passage in one of its host plants (the turnip Brassica rapa).\nCaMV is a pararetrovirus, which is a major grouping containing hepadnaviruses (e.g., hepatitis B virus), badnaviruses (e.g., banana streak virus), and caulimoviruses (e.g., CaMV). Pararetroviruses are characterized by a non-segmented double-stranded DNA genome. After entering the host cell nucleus, the viral DNA accumulates as a minichromosome [28] whose transcription is ensured by the host RNA polymerase II [29] . The CaMV genome consists in approximately 8,000 bp and encodes six viral gene products that have been detected in planta ( Figure 1 ) [30] . Viral proteins P1 to P6 are expressed from two major transcripts, namely a 19S RNA, encoding P6, and a 35S RNA corresponding to the entire genome and serving as mRNA for proteins P1-P5 [31] . Using the pre-genomic 35S RNA as a matrix, the protein P5 (product of gene V) reverse-transcribes the genome into genomic DNA that is concomitantly encapsidated [30] .\nThe detection of CaMV recombinants in turnip hosts has been reported numerous times. Some studies have demonstrated the appearance of infectious recombinant viral genomes after inoculation (i) of a host plant with two infectious or non-infectious parental clones [21, 32, 33, 34, 35] or (ii) of a transgenic plant containing one CaMV transgene with a CaMV genome missing the corresponding genomic region [36] . While the former revealed inter-genomic viral recombination, the latter demonstrated that CaMV can also recombine with transgenes within the host's genome. Another study based on phylogenetic analyses of various CaMV strains has clearly suggested different origins for different genomic regions and, hence, multiple recombination events during the evolution of this virus [37] . Indirect experimental evidence has indicated that, in some cases, CaMV recombination could occur within the host nucleus, between different viral minichromosomes, presumably through the action of the DNA repair cellular machinery [21, 35] . Nevertheless, the mechanism of ''template switching'' during reverse transcription, predominant in all retroviruses, most certainly also applies to pararetroviruses. For this reason, and on the basis of numerous experimental data, CaMV is generally believed to recombine mostly in the cytoplasm of the host cell, by ''legal'' template switching between two pre-genomic RNA molecules [21, 35, 36, 38, 39] , or ''illegal'' template switching between the 19S and the 35S RNA [36, 40] . Under this hypothesis, recombination in CaMV could therefore be considered as operating on a linear template during reverse transcription, with the 59 and 39 extremities later ligated to circularize the genomic DNA (position 0 in Figure 1 ). The above cited studies clearly demonstrate that CaMV is able to recombine. However, since these studies are based on complementation techniques, non-quantitative detection, or phylogenetically based inferences of recombination, they do not inform us on whether recombination is an exceptional event or an ''everyday'' process shaping the genetic composition of CaMV populations.\nIn the present work, we aimed at answering this question. To this end, we have constructed a CaMV genome with four genetic markers, demonstrated to be neutral in competition experiments. By co-inoculating host plants with equal amounts of wild-type and marker-containing CaMV particles, we have generated mixed populations in which impressive proportions of recombinants-distributed in several different classes corresponding to exchange of different genomic regions-have been detected and quantified. Altogether, the recombinant genomes averaged over 50% of the population. Further analysis of these data, assuming a number of viral replications during the infection period ranging from five to 20, indicates that the per nucleotide per replication cycle [44] ) indicates the origin of replication via reverse transcription, which occurs in the direction indicated by the dotted outermost circle-like arrow. Reverse transcription is accomplished by the viral reverse transcriptase, using the 35S RNA as template [49] . DOI: 10.1371/journal.pbio.0030089.g001 recombination rate of CaMV is of the same order of magnitude, i.e., on the order of a few 10 À5 , across the entire genome. We thereby provide the first quantification, to our knowledge, of the recombination rate in a virus population during a single passage in a single host.\nFrom Figure 1 , and supposing that all marker-containing genomic regions can recombine, we could predict the detection and quantification of seven classes of recombinant genotypes: þbcd/aþþþ, aþcd/þbþþ, abþd/þþcþ, abcþ/þþþd, þþcd/abþþ, aþþd/þbcþ, and aþcþ/þbþd. Indeed, all classes were detected, and their frequencies in the ten CaMV populations analyzed are summarized in Table 1 .\nAltogether, the proportion of recombinant genomes found in the mixed viral populations was astonishingly high and very similar in the ten co-infected plants analyzed (Table 1 , last column), ranging between 44% (plant 5) to 60% (plants 7, 12, and 20), with a mean frequency (6 standard error) of 53.8% 6 2.0%. This result indicates that recombination events are very frequent during the invasion of the host plant by CaMV and represents, to our knowledge, the first direct quantification of viral recombination during the infection of a live multi-cellular host.\nThe inferred per generation recombination and interference rates, assuming that CaMV undergoes ten replication cycles during the 21 d between infection and sampling, are given for each of the ten plants in Table 2 . Recombination rates between adjacent markers are large, on the order of 0.05 to 0.1. Taking the distance in nucleotides between markers into account yields an average recombination rate per nucleotide and generation on the order of 4 3 10 À5 .\nInterestingly, this recombination rate does not vary throughout the genome (Kruskal-Wallis test, p = 0.16).\nTo relax the assumption of the number of replications during the 21 d, we calculated the recombination parameters assuming five or 20 generations. The effect of the number of generations on the estimates is linear: doubling the number of generations results in a halving of the recombination rate (detailed results not shown). For example, the average recombination rates r 1 , r 2 , and r 3 assuming 20 generations were equal to 0.05, 0.04, and 0.025, respectively (compare with values in Table 2 ), yielding per nucleotide per generation recombination rates of 1.9 3 10 À5 , 2.2 3 10 À5 and 1.6 3 10 À5 .\nInspection of Table 2 also shows that first-order interference coefficients were in general negative, indicating that a crossing over in one genomic segment increases the probability that a crossing over will occur in another genomic segment, while the second-order coefficient parameter had an average value close to zero with a large variance. The mechanism leading to these results will be discussed in the following section.\nOne major breakthrough in the work presented here lies in the space and time scales at which the experiments were performed. Indeed, the processes occurring within the course of a single infection of one multi-cellular host are of obvious biological relevance for any disease. Previous studies on viral recombination suffered from major drawbacks in this respect, basing their conclusions on experiments relying on complementation among non-infectious viruses or between viruses with undetermined relative fitness, on phylogenetically based analyses, or on experiments in cell cultures. For reasons detailed in the Introduction, the first two methods either do not provide information on the frequency of recombination, but only its occurrence, or address the question at a different temporal, and often spatial, scale. Results from cell cultures, on the other hand, impose cell coinfection by different viral variants, potentially overestimat- ing the frequency of recombination events. Our study circumvents these limitations by analyzing viral genotypes sampled from infected plants after the course of a single infection, and therefore the invasion and co-infection of cells in various organs and tissues is very close to natural. More than half of the genomes (53.8% 6 2.0%; see Table 1 ) present in a CaMV population after a single passage in its host plant were identified as recombinants, and these data allowed us to infer a per nucleotide per generation recombination rate on the order of 2 3 10 À5 to 4 3 10 À5 . The time length of one generation, i.e., the time required for a given genome to go from one replication to the next, is totally unknown in plant viruses. The only experimental data available on CaMV are based on the kinetics of gene expression in infected protoplasts, where the capsid protein is produced between 48 and 72 h [40] . The reverse transcription and the encapsidation of genomic DNA being two coupled phenomena [30] , we judged it reasonable to assume a generation time of 2 d and, thus, an average of ten generations during our experiments. In case this estimate is mistaken, we have verified a linear relationship between r and the number of generations, thereby allowing an immediate adjustment of r if the CaMV generation time is more precisely established. At this point, we must consider that all cloned genomes may not have been through all the successive replication events potentially allowed by the timing of our experiments. It was previously shown that about 95% of CaMV mature virus particles accumulate in compact inclusion bodies [41] , where they may be sequestered for a long time, as such inclusions are very frequent in all infected cells, including those in leaves that have been invaded by the virus population for several weeks. The viral population may thus present an age structure that could bias the estimation of the recombination rate. In order to minimize this bias, the clones we analyzed were collected in one young newly formed leaf, where the chances of finding genomes from ''unsequestered lines'' were assumed to be higher. In any case, our data analysis is conservative, since this age structure can only lead to an underestimation of the recombination rate.\nOur results show that interferences between pairs of loci are negative: a recombination event between two loci apparently increases the probability of recombination between another pair of loci. We believe that the most parsimonious explanation of these negative interferences is based on the way the infection builds up within plant hosts. Indeed, one can divide infected host cells into those infected by a single virus genotype and those infected by more than one viral genotype. In the former, analogous to clonal propagation, recombination is undetectable. In the latter, recombination is not only detectable but, as our results indicate, very frequent. Samples consisting of viruses resulting from a mixture of these two types of host cell infections will thus contain viruses with no recombination and viruses with several recombination events, thus yielding an impression of negative interference. These conceptual arguments are supported by mathematical models. It is indeed easy to show (detailed results not shown) that if a proportion F of the population reproduces clonally, analogous to single infections, while the remaining reproduces panmictically, negative interferences could be inferred even if they do not exist. For example, assuming a three-locus model with real recombination rates r 1 and r 2 and interference i 12 , the ''apparent'' recombination and interference parameters, would be r 1 = (1 À F)r 1 , r 2 = (1 À F)r 2 , and i 12 = À(F À i 12 )/(1 À F). Interestingly, this example also shows that our estimates of the recombination rate are conservative: that a fraction F of host cells are singly infected while others are multiply infected leads to an underestimation of the recombination rate. As judged by r 1 , r 2 , and r 3 , calculated between markers a-b, b-c, and c-d, respectively, we found evidence for recombination through the entire CaMV genome. The values for r 1, r 2 , and r 3 are remarkably similar, hence the recombination sites seem to be evenly distributed along the genome. We considered the template-switching model as the major way recombinants are created in CaMV. As already mentioned in the Introduction, hot spots of template switching have been predicted at the position of the 59 extremities of the 35S and 19S RNAs [21, 36, 42] . If other recombination mechanisms, such as that associated with second-strand DNA synthesis or with the host cell DNA repair machinery, act significantly, hot The various parameters are as follows: r1, recombination rate between markers a and b; r2, recombination rate between markers b and c; r3, recombination rate between markers c and d; i12, interference between crossovers in segments a-b and b-c; i23, interference between crossovers in segments b-c and c-d; i13, interference between crossovers in segments a-b and c-d; i123, second-order interference accounting for residual interference. The recombination rates are the maximum likelihood estimates (6 95% confidence intervals). The interference parameters were obtained numerically as explained in the Materials and Methods. DOI: 10.1371/journal.pbio.0030089.t002 spots would be expected at the positions of the sequence interruption D1, D2, and D3 [43] . Due to the design of our experiment and the position of the four markers, we have no information on putative hot spots at positions corresponding to the 59 end of the 35S RNA and to D1 (at nucleotide position 0). Nevertheless, the putative hot spots at the 59 end of the 19S RNA and at D2 and D3 (nucleotide positions 4,220 and 1,635, respectively) fall between marker pairs c-d, b-c, and a-b, respectively. Our results indicate that either these hot spots are quantitatively equivalent-though predicted by different recombination mechanisms-or, more likely, that they simply do not exist. Whatever the explanation, what we observe is that the CaMV can exchange any portion of its genome, and thus any gene thereof, with an astonishingly high frequency during the course of a single host infection. To our knowledge, the viral recombination rate has never previously been quantified experimentally for a plant virus [3] . In contrast, retroviruses and particularly HIV-1 have been extensively investigated in that sense. As we have already discussed for these latter cases, the quantification of the intrinsic recombination rate was carried out in artificially coinfected cell cultures. The estimated intrinsic per nucleotide per generation recombination rate in HIV-1 is on the order of 10 À4 [14, 15, 19] , less than one order of magnitude higher than our estimation for CaMV. Because for various reasons detailed above we probably underestimate the within-host CaMV recombination rate, we believe that the intrinsic recombination rate in CaMV is higher and perhaps on the order of that of HIV.\nOther pararetroviruses such as plant badnaviruses or vertebrate hepadnaviruses have a similar cycle within their host cells, including steps of nuclear minichromosome, genomic size RNA synthesis, and reverse transcription and encapsidation. Nevertheless, vertebrate hepadnaviruses (e.g., hepatitis B virus) infect hosts that are very different from plants in their biology and physiology, and this could lead to a totally different frequency of cell co-infection during the development of the virus populations. Thus, even though our results can be informative for other pararetroviruses because of the viruses' shared biological characteristics, they should not be extrapolated to vertebrate pararetroviruses without caution.\nViral isolates. We used the plasmid pCa37, which is the complete genome of the CaMV isolate Cabb-S, cloned into the pBR322 plasmid at the unique SalI restriction site [44] . To analyze recombination in different regions of the genome, we introduced four genetic markers: a, b, c, and d, at the positions 881, 3,539, 5,365, and 6,943, respectively, thus approximately at four cardinal points of the CaMV circular double-stranded DNA of 8,024 bp ( Figure 1 ). All markers, each corresponding to a single nucleotide change, were introduced by PCR-directed mutagenesis in pCa37, and resulted in the duplication of previously unique restriction sites BsiWI, PstI, MluI, and SacI in a plasmid designated pMark-S. Because, in this study, we targeted the possible exchange of genes between viral genomes, all markers a, b, c, and d were introduced within coding regions corresponding to open reading frames I, IV, V, and VI, respectively. Another important concern was to quantify recombination in the absence of selection, i.e., to create neutral markers. Consequently all markers consist of synonymous mutations (see below).\nProduction of viral particles and co-inoculation. To generate the parental virus particles, plasmids pCa37 and pMark-S were mechanically inoculated into individual plants as previously described [33] . All plants were turnips (B. rapa cv, ''Just Right'') grown under glasshouse conditions at 23 8C with a 16/8 (light/dark) photoperiod.\nThirty days post-inoculation, all symptomatic leaves were harvested and viral particles were purified as described earlier [45] .\nThe resulting preparations of parental viruses, designated Cabb-S and Mark-S, were quantified by spectrometry using the formula described by Hull et al. [46] . We fixed the initial frequency of markers to a value of 0.5, and a solution containing 0.1 mg/ml of virus particles of both Cabb-S and Mark-S at a 1:1 ratio was prepared. Plantlets were co-infected by mechanical inoculation of two to three leaves with 20 ll of this virus solution, using abrasive Celite AFA (Fluka, Ronkonkoma, New York, United States). The mixed CaMV population was allowed to grow during 21 d of systemic infection.\nEstimation of marker frequency within mixed virus populations. We designed an experimental protocol for quantifying marker frequency within a mixed Cabb-S/Mark-S virus population after a single passage in a host plant. Twenty-four individual plants, inoculated as above with equal amounts of Cabb-S and Mark-S, were harvested 21 d post-inoculation, when symptoms were fully developed. The viral DNA was purified from 200 mg of young newly formed infected leaves according to the protocol described previously [47] . After the precipitation step of this protocol, the viral DNA was resuspended and further purified with the Wizard DNA clean-up kit (Promega, Fitchburg, Wisconsin, United States) in TE 1X (100 mM Tris-HCl and 10 mM EDTA [pH 8]). Aliquots of viral DNA preparations were digested by restriction enzymes corresponding either to marker a, b, c, or d and submitted to a 1% agarose gel electrophoresis, colored by ethydium bromide and exposed to UV. Each individual restriction enzyme cut once in Cabb-S DNA and twice in Mark-S, thus generating DNA fragments of different sizes attributable to one or the other in the mixed population of CaMV genomes. After scanning the agarose gels, we estimated the relative frequency of the two genotypes in each viral DNA preparation and at each marker position, by densitometry using the NIH 1.62 Image program. The statistical analyses of the frequency of the four markers are described below.\nIsolation of individual CaMV genomes and identification of recombinants. To identify and quantify the recombinants within the CaMV mixed populations, aliquots from ten of the 24 viral DNA preparations described above were digested by the restriction enzyme SalI, and directly cloned into pUC19 at the corresponding site. In each of the ten viral populations analyzed, 50 full-genome-length clones were digested separately by BsiWI, PstI, MluI, and SacI, to test for the presence of marker a, b, c, and d, respectively. In this experiment, with the marker representing an additional restriction site, we could easily distinguish between the Cabb-S and the Mark-S genotype at all four marker positions, upon agarose gel (1%) electrophoresis of the digested clones. Clones with none or all four markers were parental genotypes, whereas clones harboring 1, 2, or 3 markers were clearly recombinants. Due to the very high number of recombinants detected, markers eventually appearing or disappearing due to spontaneous mutations were neglected.\nStatistical analysis. Here we present the different methods we used to quantify recombination in the CaMV genome. Because all these methods assume that the different markers are neutral, we first discuss assumption.\nWe used two datasets to test the neutrality of markers, both resulting from plants co-infected with a 1:1 ratio of Mark-S and Cabb-S. The first consisted of viral DNA densitometry data derived from 24 plants (described above), where for each plant we have an estimate of the frequency of each marker in the genome population. The second consisted of the restriction of 50 individual full-genome-length viral clones obtained from one co-infected plant (described above), yielding an estimate of the frequency of each marker, and this was repeated on ten different plants. The frequencies of the different markers were 0.508, 0.501, 0.516, and 0.507 for markers a, b, c, and d in the first dataset and 0.521, 0.518, 0.514, and 0.524 in the second dataset. We tested whether these frequencies were significantly different from the expected value under neutrality, 0.5, using either t-tests, for datasets where normality could not be rejected (seven out of eight cases), or Wilcoxon signed-rank non-parametric tests otherwise (marker c in the first dataset). In all cases p-values were larger than 0.05.\nThere are several cautionary remarks regarding these analyses. First, in all cases we found an excess of markers. Unfortunately, the two datasets cannot be regarded as independent because, even though the methods through which the frequency estimates were obtained were different, the plants used in the second dataset were a subset of the plants of the first. We thus have only four independent estimates in each case, and there is minimal power to detect significant deviations from neutrality with such a small sample size. It should be noted at this stage that deviations from the expected value could also be caused either by slight deviations from the 1:1 ratio in the infecting mixed solution, or by deviations from that ratio in the frequency of the viral particles that actually get into the plants. Second, because of the relatively small sample sizes and low statistical power, the tests presented above could have detected only large deviations.\nThe results clearly show, however, precisely that the markers do not have large effects, if any, and that therefore recombination estimates would be affected only very slightly by any hypothetical selective effects of the markers. Because of this, along with the fact that the introduced markers provoke silent substitutions in the CaMV genome, we assumed that markers were effectively neutral in the rest of the analysis.\nThe dataset used to estimate the recombination frequency consisted of the 500 full-genome-length viral clones (50 from each of ten co-infected plants) individually genotyped for each of the four markers. As discussed in detail in the Results, recombination was very frequent and concerned all four markers. Indeed, approximately half of the genotyped clones exhibited a recombinant genotype. It was therefore meaningful to try to obtain quantitative estimates of recombination from our data.\nOur aim was to analyze viral recombination in a live host. Consequently, we had to deal with the fact that more than one viral replication cycle occurred during the 21 d that infection lasted in our experiment (we had to wait that long for the disease to develop and to be able to recover sufficient amounts of viral DNA from each infection). Based on the kinetics of gene expression [40] , we postulate that each replication cycle lasts between 2 and 3 d, and that therefore seven to ten cycles occurred between infection and the sampling time. In case this assumption is incorrect, we did calculations assuming five, seven, ten, or 20 replication cycles during these 21 d. As shown, the results were not affected qualitatively, and only slightly quantitatively. It is important to note that we assumed that recombination occurred through a template-switching mechanism, and that therefore, from a recombination point of view, the CaMV genome is linear. The reverse transcription starts and finishes at the position 0 in Figure 1 , which is the point of circularization of the DNA genome. This implies that changes between contiguous markers a-b, b-c, and c-d can be considered as true recombination whereas those between a and d cannot, as they may simply stem from circularization of DNA, during the synthesis of which the polymerase has switched template once anywhere between a-b, b-c, or c-d.\nTo estimate the recombination rate between markers, we wrote recurrence equations describing the change in frequency of each genotype over one generation, assuming random mating and no selection (i.e., the standard Wright-Fisher population genetics model). We then expressed the frequency of all possible genotypes n generations later as a function of their initial frequency and of the recombination parameters. Subsequently we calculated the maximum likelihood estimates of the recombination parameters and their asymptotic variances given initial frequencies (we assumed that the two ''parental'' genotypes, Mark-S and Cabb-S, had equal initial frequencies of 0.5 and that all other genotypes had initial frequencies of zero) and frequencies after n generations (the observed frequencies; as stated above we used different values of n). All algebraic and numerical calculations were carried out with the software Mathematica.\nThe recombination parameters are the recombination rates between two adjacent loci, e.g., r 1 for the recombination rate between markers a and b, and the interference coefficients, e.g., i 12 for interference between recombination events in the segments between markers a and b and b and c. To define these parameters we followed Christiansen [48] , and in particular the recombination distributions for two, three, and four loci (respectively, Tables 2.7, 2.8, and 2.9 of [48] ). It is important to realize that given the definitions of these parameters, the estimator of the recombination rate between two loci is not affected by the number of loci considered. In other words, we obtain the same estimation of the recombination rate between markers a and b whether we consider genotypic frequencies at just these two loci, or the frequencies at these two loci plus a third locus, or the complete information to which we have access, the fourmarker genotypes. Information on additional loci only affects the estimates of the interference coefficients.\nIt proved impossible to carry out the calculations for four loci algebraically. Instead, we used a computer program to calculate the expected genotypic frequencies at all four loci after n generations, given the above stated initial frequencies and specified recombination parameters. For each combination of recombination parameters we calculated a Euclidean distance between the vector of the expected genotypic frequencies and the observed genotypic frequencies, and considered that the estimated recombination parameters were those yielding the minimal Euclidean distance. In all cases, the estimated recombination rates between pairs of loci were equal to the second decimal to those estimated algebraically from data for three or two loci." }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: The structure of an RNA molecule is widely recognized to play a role in many processes, including structurally organizing complex RNAs, the assembly of ribonucleoprotein complexes, and in translational recoding and regulation [reviewed in (1) ]. One common RNA folding motifs is a pseudoknot, the folding back of a single-stranded RNA onto itself to form two helical structures with single-stranded loops joining them (2) . Many such structures can be inferred from RNA sequences and frameshifting function has been demonstrated for some of these [reviewed in (3) (4) (5) ]. However, though much theoretical progress has been made in understanding how mRNA pseudoknots promote efficient À1 ribosomal frameshifting (6), a complete understanding of this mechanism remains untested.\nProgrammed À1 ribosomal frameshift signals are typically divided into three components. From 5 0 to 3 0 these are (i) a 'slippery site' in the form N NNW WWH, where N must be a stretch of any three identical nucleotides, where W is either three A or U residues, and H is A, C or U (spacing indicates the unshifted zero frame), (ii) a spacer region and (iii) an mRNA structural element, most often a pseudoknot. The general model posits that upon encountering the mRNA pseudoknot, an elongating ribosome is forced to pause such that the anticodons of its A-and P-site tRNAs are base-paired with the zero-frame codons of the slippery site. The nature of the tRNA-mRNA interactions is such that a relative slip of À1 nucleotide still allows base-pairing in the non-wobble positions. The slippage occurs during the ribosomal pause, and it has been shown that changes affecting ribosome pause times affect frameshift efficiencies [reviewed in (7) ]. An important observation is that even though mRNA pseudoknots and energetically equivalent stem-loop structures appear to promote ribosome pausing with equal effectiveness, mRNA pseudoknots are more efficient at promoting À1 PRF (8). Our '9 Å ' model (6) provided a refinement of the original 'simultaneous slippage' (9, 10) model of frameshifting by suggesting that rather than the entire ribosome having to slip one base in the 5 0 direction, slippage could be accomplished by moving the small section of mRNA in the downstream tunnel by one base in the 3 0 direction. We have proposed that this is accomplished by the bulky and difficult to unwind mRNA pseudoknot structures becoming wedged in the downstream entrance tunnel of the ribosome, preventing the downstream region of the mRNA from being pulled into the ribosome by the equivalent of one base during the accommodation step of elongation. This blockage would introduce tension into the spacer region, which could be resolved by unpairing the mRNA from the tRNAs, allowing the mRNA to slip 1 nt backwards, resulting in a net shift of reading frame by À1 base.\nThough the 9 Å model provides a partial explanation for why mRNA pseudoknots promote programmed À1 ribosomal frameshifting (À1 PRF) more efficiently than simple *To whom correspondence should be addressed. Tel: +1 301 405 0981; Fax: +1 301 314 9489; Email: [email protected] ª The Author 2005. Published by Oxford University Press. All rights reserved.\nThe online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact [email protected] stem-loop structures, it does not answer the question of how the mRNA pseudoknot directs the ribosome to pause at the correct position along the mRNA. A complementary 'torsional restraint' model addresses this issue (11) . When a stem-loop structure is unwound by an elongating ribosome, unwinding of the stem forces the loop to rotate. Since a simple stem-loop is not restrained, the loop can rotate freely and only the base pairs within the stem resist ribosomal movement, and thus the potential energy of unwinding should be distributed along the length of Stem 1 ( Figure 1A ). However, if the loop is anchored or restrained, as it is in a pseudoknot by Stem 2, since the intrinsic ribosomal helicase is processive (12) , Stem 1 cannot be fully unwound until Stem 2 is first denatured. Mechanically, as the ribosome begins to unwind the base of Stem 1, Stem 2 forces the supercoiling in the remainder of Stem 1, providing extra resistance to ribosome movement. At some specific point, the resistance to ribosome movement provided by the supercoiling counteracts the forward movement of the ribosome, increasing the likelihood that the ribosome will stop at a precise point along the mRNA. Energetically, since full unwinding of Stem 1 is dependent on complete denaturation of Stem 2, the potential energy of unwinding of the pseudoknot structure should similarly be directed toward one point. Viewed either mechanically or energetically, this point is where ribosomes will be directed to specifically pause on the mRNA. If it occurs with the tRNAs in ribosomal A-and P-sites positioned at the slippery site, then frameshifting is stimulated. This is summarized in Figure 1B . The efficiency of À1 PRF can thus be viewed as a function of (i) the fraction of ribosomes paused over the slippery site and (ii) the rate at which the structure can be denatured. There is increasing evidence from single molecule experiments that unfolding occurs in quick 'rips' at a particular force (13) , suggesting that in the case of unfolding pseudoknots, frameshifting efficiency is related to both the energy barriers to unfolding the pseudoknot structure and the resistance of the structure against the force of the ribosome. In the context of the torsional restraint model, this resistance is dependent on the ability of Stem 2 to remain intact while Stem 1 is being unwound.\nThere is experimental data that indirectly support this model: (i) disruption of the first 3 bp of Stem 1, which would displace the ribosome's pause site to a point 3 0 of the slippery site, has been shown to eliminate frameshifting (14) ; (ii) destabilizing Stem 2, which would allow it to be unwound more readily, has been shown to result in decreased frameshifting efficiency (15) (16) (17) ; (iii) replacing bulges in Stem 1 with base pairs would increase the energy required to unwind the first three bases, and a longer ribosomal pause over the slippery site would follow, yielding increased efficiencies in À1 frameshifting (15, 18) ; (iv) destabilizing the base of Stem 1 by replacing G:C base pairs with A:U pairs decreases À1 frameshifting efficiencies (19, 20) ; (v) the model eliminates the need for a 'pseudoknot recognizing factor', the evidence of which has not been forthcoming in either competition assays in in vitro translation systems (21) or by gel retardation assays (J. D. Dinman, unpublished data); and (vi) elimination of a potential torsion-restraining Stem 2, but not of a non-torsion-restraining Stem 2 in HIV-1, resulted in decreased À1 PRF efficiencies (22) .\nThough all of the cited studies support the torsional restraint model, none has directly addressed it. In the experiments presented in this study, a series of 'pseudo-pseudoknot' containing reporter constructs were used to test the torsional restraint hypothesis. In vitro frameshifting assays show that frameshifting can be significantly stimulated by limiting the rotational freedom of the loop region of a stem-loop structure, and that the degree of rotational freedom of Stem 1 is important in determining the extent of À1 PRF. Furthermore, mRNA toeprint analyses reveal a pseudo-pseudoknot-specific strong stop 16 nt 3 0 of the slippery site, consistent with this structure being able to direct ribosomes to pause with their A-and P-sites positioned at the slippery site.\nAll synthetic DNA oligonucleotides were purchased by IDT (Coralville, IA). The modified L-A viral À1 PRF signal containing the G GGU UUA slippery site followed by a simple stem-loop was amplified from pJD18 (23) using the primers luc5 0 b (5 0 -CCCCAAGCTTATGACTTCTAGGCAGGGTTT-AGG-3 0 ) and luc3 0 b (5 0 -CCCCCCATGGGACGTTGTAAA-AACGACGGGATC-3 0 ). These were digested with HindIII and NcoI (restriction sites are underlined) and cloned into the firefly luciferase reporter plasmid pT7-LUC minus 3 0untranslated region-A50 (24) . In the resulting reporter construct (pJD214-18), expression of firefly luciferase requires a À1 frameshift, and the 5 0 sequence of the Stem 2 is not able to base pair with the 3 0 sequence, so that only a stem-loop rather than a pseudoknot is able to form. The same primers were used to amplify DNA from pJDRC (23) to make pJD214-Ry. In this construct, complementary mutations (5 0 -GCUGGC-3 0 to 5 0 -CGACCG-3 0 ) in the 3 0 acceptor sequence of the pseudoknot-forming region of Stem 2 allow the formation of an mRNA pseudoknot that has previously been shown to promote frameshifting at the same frequency as the wild type (23) . The primer luc5 0 CON (5 0 -CCCCAAGCTTATGACTTC-TAGGCAAGGGTTTAGG-3 0 ) contains an additional A nucleotide upstream of the slippery site and was used to make pJD214-0, the zero-frame control. To eliminate the possibility of internal initiation occurring at the luciferase initiation codon downstream of the frameshift signals, the AUG codon was changed to AUA. The Stratagene Quik-Change kit was used to mutate pJD214-18 and pJD214-Ry into pJD336-18 and pJD366-Ry, respectively, using the oligonucleotides 5 0 -GGCGTTCTTCTATGGGACGTTGTA-AAAACGGATC-3 0 and 5 0 -GATCCGTCGTTTTTACAACG-TCCCATAGAAGACGCC-3 0 (the mutated codon is underlined). pJD366-18 was further mutated to make a zero-frame control by placing an A upstream of the slippery site using the oligonucleotides 5 0 -TGACTTCTAGGCAAGGGTTTAGGAG-TG and 5 0 -CACTCCTAAACCCTTGCCTAGAAGTCA (the inserted base is underlined). A series of synthetic DNA oligonucleotides were designed to join the loop acceptor region of mRNA transcribed from pJD366-18 to the downstream region that forms the pseudoknot in the wild-type L-A À1 PRF signal. In the J-oligos, the 3 0 sequences base pair with the loop of the mRNA transcribed from pJD366-18, and the 5 0 regions of these oligos base pair with the downstream sequence. This orientation is reversed for the R-oligos. These general orientations are shown in Figure 3B . The naming of the oligonucleotide names refers to the number of additional residues placed between the regions of complementarity. The bases complementary to the pJD366-18 sequence are underlined.\nPlasmid DNAs were prepared using the Qiagen mini-prep kits and were linearized with DraI in a total volume of 20 ml. Proteins were eliminated by the addition of 2 ml of 1 mg/ml proteinase K and SDS to a final concentration of 0.5% followed by digestion at 50 C for 30 min. Volumes were then increased to 100 ml, extracted twice with phenol/chloroform, and DNA was precipitated with 10 ml NH 4 Ac and 250 ml ethanol. The purified DNA was resuspended in DEPCtreated H 2 O. To prepare synthetic mRNAs, 2 ml of purified linear DNAs were used for in vitro transcription using the Ambion T7 mMachine mMessage kit. RNAs were precipitated using 30 ml DEPC H 2 O and 25 ml LiAc. The RNA was resuspended in 11 ml DEPC H 2 O (1 ml in 500 would give an OD 260 of 0.05-00.1; 1-2 mg/ml).\nTo anneal the oligonucleotides with the mRNA, J-oligos, R-oligos or the equivalent volumes of dilution buffer alone (20 mM Tris, pH 7.4, 2 mM MgCl 2 and 50 mM EDTA final concentration) were added to synthetic mRNA (0.5 mg), and the mixtures were first incubated in a 70 C heating block for 10 min; the block was then removed and allowed to cool to 37 C (30 min), after which they were briefly spun down and incubated on ice. In all experiments, the molar ratios of J-and R-oligos to synthetic mRNAs were 100:1. In experiments using the competing oligonucleotide (C-oligo), this was added to either 0.5:1 or 1:1 molar ratios with either J-or R-oligonucleotides. Reticulocyte lysates were thawed on ice, 15 ml of Àmet and 15 ml of Àleu master mixes plus 20 ml of H 2 O were added to 400 ml of lysate, and 19 ml of this was added to each annealed reaction to start the in vitro translation reactions. These were incubated at 30 C for 60 min (the reaction reached a plateau after 30-35 min where the greatest difference was seen between the zero-frame controls and the frameshifting plasmids) (data not shown), and the reactions were then placed on ice. An aliquot of 7.5 ml from each in vitro translation reaction was added to 50 ml of the prewarmed luciferase reagent, and luminescence readings were taken after a 3 s delay for 15 s in triplicate using a Turner 20/20 Luminometer.\nSynthetic transcripts generated from DraI-digested pJD366-18 ($1.7 kb) were 5 0 end labeled using [g-32 P]CTP. These RNAs (4 ml) were incubated with 1 ml of annealing buffer and either 1 ml of H 2 O or 1 ml of an oligo (0.25 ng) at 70 C. The heating block was allowed to cool at room temperature for 40 min before 8 ml of RNaseH buffer was added (20 mM HEPES, 50 mM KCl, 10 mM MgCl 2 and 1 mM DTT). An aliquot of 1 ml of enzyme was added (mung bean nuclease, RNaseH or RNaseT1) and the reactions incubated at 37 C for 1 h. The reactions were stopped by adding 4 ml of stop solution, the products separated through a 6% polyacrylamide-urea denaturing gel and visualized by autoradiography.\nmRNA toeprinting JD366-18 mRNA (1 mg in 8 ml) was annealed with 2 ml of 3 0 end-labeled toeprinting primer (5 0 -CGTACGTGATCTTCA-CC-3 0 , complementary to sequence 240 bp 3 0 of the slippery site) as described above. This was added to 15 ml of lysate (200 ml Ambion retic lysate, 7.5 ml of each master mix [Àleu and Àmet] and 70 ml of 250 mM KCl), except for 2 ml, which was added to 15 ml of RT buffer [50 mM Tris-HCl (25), 40 mM KCl, 6 mM MgCl 2 , 5 mM DTT and 575 mM dNTPs] to be used as a no-ribosome control. In vitro translation reactions were incubated at room temperature for 10 min, which was empirically determined to provide the optimum amount of time to allow ribosomes to initiate translation and pause at the frameshift signal. Subsequently, 15 ml of RT buffer containing RNasin inhibitor and cycloheximide (to a final concentration of 100 ng/ml) was added to stop translation. To this, 2 ml of Superscript II (Invitrogen) was added and the reaction incubated at room temperature for 10 min. Reactions were terminated by phenol:chloroform extraction and 15 ml of stop solution added. The toeprinting primer was also used in conjunction with pJD366-18 to produce sequencing ladders by standard dideoxynucleotide chain termination methods using Sequenase (USB). Products were separated though 6% polyacrylamide-urea denaturing gels and visualized using a Storm phosphorImager (Pharmacia).\nPseudo-pseudoknots stimulate frameshifting, and frameshifting efficiency changes with the degree of pseudo-pseudoknot rotational freedom\nWe previously showed in intact yeast cells that the pseudoknot containing mRNA produced from pJDRC was able to promote efficient À1 PRF, whereas one in which only a stem-loop can form, transcribed from pJD18, could not (23) . As a first step in this study, we tested the ability of synthetic mRNAs produced from pJD366-RC and from pJD366-18, two plasmids derived from these parental constructs, to promote À1 PRF. Total luciferase activities produced from these synthetic mRNAs were divided by the luciferase activity produced from the zero-frame control plasmid, pJD366-0, and multiplied by 100% to determine À1 PRF efficiencies. The results show that the trends observed in yeast were replicated in vitro, i.e. JD366-RC mRNA promoted $8% efficiency of À1 PRF as compared with $1.1% promoted by JD366-18 mRNA (Figure 2 ).\nThe 'torsional restraint' model predicts that conditions that would inhibit the rotational freedom of the loop region of the pJD366-18-derived mRNA should result in enhanced À1 PRF efficiency. The strategy used in this study was to anneal this mRNA with synthetic oligonucleotides complementary to both the loop region and to the sequence downstream that is normally involved in pseudoknot formation. These 'pseudopseudoknots' would be predicted to restore a pseudoknot-like structure to the mRNA. This is diagrammed in Figure 3A . Two different classes of oligonucleotides having different orientations relative to the mRNA were used to this end: 'joining' (J-) and 'reverse' (R-) oligos. The orientation of the J-oligos promotes the formation of a structure containing the equivalent of a Loop 2 region, while that of the R-oligos promotes a Loop 1 equivalent. The model also predicts that pseudo-pseudoknots having different degrees of rotational freedom should promote different frequencies of ribosome pausing over the slippery site, resulting in different efficiencies of À1 PRF. In order to control this parameter, increasing numbers of nucleotides were inserted between the mRNA hybridizing regions of the J-and R-oligos. The additional non-complementary bases in the J-oligos are 3 0 to the stem-loop residues involved in Stem 2, thus effectively increasing Loop 2. Similarly, the additional non-complementary bases in the R-oligos are 5 0 to the loop acceptor residues and correspond to an increased Loop 1. The structure of the stem-loop of pJD366-18 and its maximum base-paired interactions with representative J-and R-oligos are shown in Figure 3B . To demonstrate that an oligonucleotide-mRNA hybrid was capable of forming under the assay conditions, the J1-oligo was incubated with 5 0 [ 32 P]labeled JD366-18 mRNA and subjected to RNaseH digestion. Digestion of the RNA-DNA hybrid resulted in a labeled 110 nt fragment, demonstrating that the oligonucleotide bound to the mRNA at the position of the pseudoknot (Figure 4) .\nHaving demonstrated the utility of the in vitro frameshifting assay and that the J-and R-series of oligonucleotides were able to hybridize with synthetic mRNA produced from pJD366-18, the next step was to monitor frameshifting efficiencies promoted by these hybrid species. Significant increases in frameshifting were observed with the incubation of pJD366-18 mRNA with oligonucleotides J1 ($10%) and J2 ($35%), while only modest increases were seen with J3 and J4 ( Figure 5 ). These findings are consistent with the notion that changes in the degree of rotational freedom of the structure would affect the distribution of paused ribosomes in the vicinity of the slippery site. One potential complication with the J-oligos is the possibility that they could interact with the Loop 2-Stem 1 region. In the R-oligos, the additional bases are distal to any possible Loop 2-Stem 1 interactions and would be more analogous to increasing Loop 1. The R-oligos stimulated À1 PRF to an even higher extent than the J-oligos ( Figure 5 ). Importantly, increasing the length of the bridging regions in these oligonucleotides (R1 to R3), which is predicted to increase the rotational freedom of the stem-loop, resulted in decreased frameshifting activity as predicted by the torsional resistance model. However, addition of three residues between the two binding regions of the R-oligo (R4) resulted in an unexpected increase in frameshifting with a very large amount of variation.\nIn a series of control experiments, 8 nt oligos complementary to the 5 0 (Loop 1) and 3 0 Stem 2 forming regions of the pseudo-pseudoknot were hybridized to the SL mRNA and À1 PRF assays were performed. Neither of these were able to stimulate À1 PRF, even at concentrations in 100-fold molar excess to the mRNA template (data not shown). Though supportive of our central hypothesis, it is also possible that these results were due to the thermodynamic instability of the RNA:DNA duplexes through the course of the experimental protocol.\nTo determine whether the stimulation of frameshifting was specifically due to the bridging of the stem-loop with downstream sequence (the pseudo-pseudoknot), as opposed to the nonspecific presence of an RNA:DNA hybrid, the competing oligonucleotide (C-oligo) was designed to form a 15 bp duplex with JD366-18 mRNA, including the 3 0 Stem 2 forming region, which was expected to significantly out compete either the J-or R-oligos from binding to this site, thus disrupting formation of the pseudo-pseudoknot (see Figure 3A ). Additionally, in the presence of the C-oligo, the J-and R-oligos were still predicted to hybridize with the 5 0 Stem 2 forming region, enabling us to address the question of whether this interaction alone was able to stimulate frameshifting. The results demonstrate that the addition of the C-oligo severely inhibited the abilities of both the J-and R-oligos to promote efficient frameshifting ( Figure 6 ). These findings demonstrate that (i) frameshifting was specifically stimulated by bridging of the 5 0 and 3 0 Stem 2 forming regions by the J-and R-oligos, and (ii) that the presence of an RNA:DNA hybrid at the 5 0 Stem 2 forming region was not sufficient to stimulate frameshifting by itself.\nThe torsional restraint model predicts that pseudoknots should direct elongating ribosomes to pause at one specific location 1 2 3 4 5 6 7 8 . Efficient frameshifting is stimulated by pseudo-pseudoknots. In vitro translation assays were performed in retic lysates with mRNAs derived from pJD366-18 (SL) to which J-or R-oligos were annealed. Luciferase activities were divided by those obtained using mRNAs generated from pJD366-0, and the resulting ratios were multiplied by 100 to calculate percent frameshifting. The averages of three independent experiments performed in triplicate are shown. Error bars denote standard deviation. on the mRNA, rather than being distributed along Stem 1. We used mRNA toeprint assays to test this hypothesis. In mRNA toeprint reactions, the movement of reverse transcriptase is blocked by paused ribosomes, resulting in a strong stop positioned $16-18 nt 3 0 of the P-site of eukaryotic ribosomes (25) . Synthetic JD366-18 mRNAs were annealed with the sequencing oligonucleotide and either J1, R1 or no second oligo, and these were then used for in vitro translation reactions. After a period of time (10 min were empirically determined to be optimal), elongation reactions were stopped by the addition of cycloheximide, and reverse transcription reactions were initiated on the sequencing oligonucleotides. In parallel, control reverse transcription reactions were carried out using synthetic JD366-18 mRNA and oligonucleotides, but without in vitro translation. The results are consistent with the model, showing that the J1-and R1-oligos specifically promoted one strong reverse transcriptase stop 16 nt 3 0 of the P-site of the slippery site only the in the in vitro translation reactions ( Figure 7 ). As further predicted by the model, a broad distribution of stops of equal intensities was observed in this region with JD366-18 mRNA alone (Figure 7, lane 1) . Importantly, the +16 stop was not observed when toeprint reactions were carried in the absence of ribosomes. Additional strong stops were also of interest. One corresponding to the 3 0 end of the base of Stem 1 was observed in all samples, consistent with the presence of this structure. Both J-and R-oligo-specific pauses were also observed. The reason for the strong pause in the J-oligo is unknown. The R-oligo-specific pause is perhaps more revealing. It occurs at the 3 0 end of the RNA:DNA hybrid formed by this oligo and the mRNA, a structure that should also promote pausing of reverse transcriptase.\nThe results presented in this study provide strong support for the torsional restraint model of programmed À1 frameshifting. Specifically, we demonstrated that RNA:DNA hybrids that mimic mRNA pseudoknots can significantly stimulate frameshifting. As predicted by the model, changing the rotational freedom of the structure by altering the lengths in the J1-and R1-oligos between the 5 0 and 3 0 mRNA hybridizing regions resulted in changes in their abilities to stimulate À1 frameshifting. The demonstration that these 'pseudopseudoknot' structures cause elongating ribosomes to specifically pause with their A-and P-sites positioned at the slippery site provides independent evidence in support of the model. In the case of the J-oligo series, frameshifting was best stimulated by J2, suggesting the structure created and the rotational freedom allowed by it was optimal for À1 PRF. The experimental design is such that we assume a similar rate of unfolding for each oligo as the predicted maximum base pairing is the same for them all. However, we do note that the type of nucleotides separating the two, separately paired regions of the oligos, and their presentation, may play a role in À1 PRF efficiency. The recent NMR structural solution of the SRV-1 pseudoknot revealed a highly structured Loop 2-Stem 1 interface including base triples involving an A residue at the 3 0 end of Loop 2 (26) . The additional base in the J2oligonucleotide is also an A. Mutagenesis experiments in this region by other groups showed, for example, that replacing the 3 0 base in Loop 2 of IBV with an A residue promoted a significant increase in frameshifting efficiency (27) , and mutation or removal of the A residue at the 3 0 base in Loop 2 of the BWYV pseudoknot reduced frameshifting levels (17) . This part of the pseudoknot has been proposed to be important in a frameshifting model where differential transition state energy barriers (due to small differences in local structure, stability or dynamics) are the primary determinant of frameshifting efficiency (3). Indeed, a Loop 2-Stem 1 triplex interaction seen in smaller frameshifting pseudoknots from luteoviruses has been shown to be critical for À1 PRF, and that similar pseudoknots lacking the triplex are less efficient at frameshifting [(28) and references therein]. This extra structural feature would limit the rate of unfolding and provide extra anchoring of Stem 2 as the ribosome attempts to unwind Stem 1, i.e. it too would help to provide additional torsional restraint. It is also possible that although the J3-and J4oligonucleotides also help to form a pseudo-pseudoknot, the additional bases may interfere with the stabilization of Stem 1.\nWith the R-oligos, a general correlation was observed between minimization of rotational freedom and frameshifting efficiency, though this was not the case of the R4-oligo. Since the stability of the pseudo-pseudoknot generated with R4 should be similar to that of the other oligonucleotides based on the base-pairing, this result suggests that there are additional considerations to be uncovered with regard to the Frameshifting (% stimulated by R1) Figure 6 . Competition for J-or R-oligo binding sites inhibits its ability to promote efficient frameshifting. mRNA transcribed from pJD366-18 (SL) was annealed with either J-or R-oligos alone, or in combination with different concentrations of competing (C-) oligos (in ratios of 2:1 or equimolar as indicated). Sample marked SL is mRNA alone. Luciferase activities generated from in vitro translation reactions in rabbit reticulocyte lysates were divided by those obtained using mRNAs generated from pJD366-0, and the resulting ratios were multiplied by 100 to calculate percent frameshifting.\npseudoknot structure influencing frameshifting. Addition of residues in the R-oligos was analogous to lengthening Loop 1, which is typically short in À1 frameshifting pseudoknots. Limited and conflicting data are available on the importance of Loop 1 in À1 frameshifting pseudoknots. In one study, addition of three A bases to Loop 1 did not affect frameshifting efficiency (15) , while in another all the mutations made in this region were detrimental to frameshifting efficiency (17) . Given the complex interactions occurring between the helices and loops in this region, we cannot yet account for why the R4-oligo stimulated frameshifting so efficiently and with such variable results. Examination of the RNA toeprint data presented here reveals that both of the pseudo-pseudoknot structures formed by the J1-and R1-oligos promoted strong stops of the reverse transcriptase $16 nt 3 0 of the P-site codon of the slippery site, consistent with the hypothesis that the presence of Stem 2 forces ribosomes to pause with their A-and P-sites positioned over the slippery site. Previous studies mapping the lagging edge of paused ribosomes, i.e. mRNA heelprint studies, did not reveal any striking differences between the effects of pseudoknots versus stem-loops (8, 16) . Interestingly, using this method, the ribosomal pauses appeared distributed over a broader stretch of mRNA ($4 nt) than observed here. It is possible that some critical level of resolution is lost in the requirement for many additional manipulations of substrates using the mRNA heelprint as compared with the toeprint methods.\nA remaining question centers on whether the role of the RNA pseudoknot in À1 PRF is passive or active. In the '9 Å solution' (6), the frameshift mechanism is activated by movement of the A-site codon-anticodon complex by 1 base in the 5 0 direction upon accommodation. As currently described, the mRNA pseudoknot merely passively blocks entry of the downstream message into the ribosome, resulting in stretching of the segment of mRNA located between the codon-anticodon complex and the pseudoknot. By this model, all of the energetic input for the frameshift is derived from hydrolysis of GTP by eEF1A. However, it is possible that the pseudoknot may also actively contribute to the frameshift mechanism. Specifically, pulling the downstream message into the ribosome at accommodation could result in unwinding of Stem 1 of the pseudoknot by one additional base pair. The energetic cost of so doing would be to introduce an equivalent amount of torsional resistance into Stem 2. If Stem 2 were to release this resistance by 'pulling back', the base pair in Stem 1 would be re-formed, which in turn would contribute to the energy required to dissociate the A-and P-site codonanticodon complexes from the zero-frame. This would be followed by slippage of the mRNA by 1 base in the 3 0 direction relative to the ribosome, followed by the formation of À1 frame codon-anticodon complexes. As such, the proposed active role for the mRNA pseudoknot would further reduce the energetic barrier to À1 PRF. In sum, we suggest that the 'torsional restraint model' can be combined with the '9 Å solution' to mechanistically explain the original 'simultaneous \n3' mRNA + Ribos. mRNA Figure 7 . Pseudo-pseudoknots direct ribosomes to pause over the slippery site. mRNAs generated from pJD366-18 (SL) were annealed with the sequencing oligonucleotide and either J1-, R1-or no oligo (lanes 1, 2 and 3, respectively), and these were then used for in vitro translation reactions. Reactions were stopped after 10 min by the addition of cycloheximide, and reverse transcription reactions were initiated on the sequencing oligonucleotides. In parallel, control reverse transcription reactions were carried out using synthetic JD366-18 mRNA and oligonucleotides, but without in vitro translation (lanes 4-6). The positions of the slippery site, loops and stems of the pseudo-pseudoknots are indicated next to a sequencing reaction. Arrowheads indicate positions of reverse transcriptase strong stops and these are mapped to a representation of the stem-loop structure of pJD366- 18. slippage' model of À1 PRF (9, 10) . In other words, the 9 Å solution + torsional restraint = simultaneous slippage.\nTwo recent publications have also shown that oligonucleotide:mRNA duplexes can stimulate efficient À1 ribosomal frameshifting (29, 30) . These studies differed from the present one in a number of ways, particularly insofar as they examined the effects duplex structures immediately 3 0 of the slippery site rather than addressing mRNA pseudoknot related questions. The findings support the notion that the specific location of ribosome pausing on the mRNA plays a critical role in determining frameshifting, though they do come with caveats, e.g. neither study directly mapped ribosomal pausing, and the use of different slippery sites and downstream contexts likely contributed to disparate findings for the optimal distances between the 3 0 ends of slippery sites and 5 0 ends of frameshift-stimulating oligonucleotides. Although potentially useful therapeutically there are no known natural examples of frameshifting stimulated in this manner, and thus these results do not affect the hypothesis presented here. However, these studies are important in that they raise the possibility for a new role for micro-RNAs in regulating gene expression, and for therapeutic approaches to correcting inborn errors of metabolism due to the presence of frameshift mutations." }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: Methods for the automated chemical synthesis of oligonucleotides (1, 2) and their assembly into long double-stranded DNA (dsDNA) sequences by PCR (3, 4) and LCR (5) have enabled the chemical synthesis of genes and even entire viral genomes (6, 7) . These technological advances have helped spur the formation of the new field of synthetic biology (8) , which aims at defining the functional units of living organisms through the modular engineering of synthetic organisms. In addition, the demand for fully synthetic gene length DNA fragments of defined sequence has dramatically increased in recent years for use in applications such as codon optimization (9), construction of DNA vaccines (10) , de novo synthesis of novel biopolymers (11) , or simply to gain access to known DNA sequences when original templates are unavailable. The future demand for long synthetic DNA is likely to dramatically increase when it becomes cheaper/faster to synthesize a desired sequence than to obtain it by other means.\nThe assembly of DNA is currently limited by the presence of random sequence errors in synthetic oligonucleotides that arise from side reactions during synthesis (incomplete couplings, misincorporations, etc.) and resulting in 1-3 errors/kb (7, 12, 13) . The deleterious impact of these errors becomes more significant as the desired lengths of synthetic DNA increase. Indeed, in the remarkable assembly of the PhiX 174 bacteriophage genome (5386 bp) using gel-purified, synthetic oligonucleotides, the products contained an average of $2 lethal errors/kb resulting in 1 plaque-forming genomes per 20 000 clones (7) . A functional selection (plaque formation) was required in this study to identify a clone with the correct sequence. Thus, error reduction/correction is a requirement for the efficient production of long synthetic DNA of defined sequence. However, the process of sequencing multiple clones and manual correction of errors is both costly and time consuming.\nSeveral methods have been reported for the removal of error-containing sequences in populations of DNA. These methods rely upon the selective destruction (14, 15) or physical separation (16, 17) of mismatch-containing heteroduplexes. Smith and Modrich (14) reported the selective destruction of error-containing sequences in PCR products by generating dsDNA breaks upon overdigestion with the Escherichia coli mismatch-specific endonuclease MutHLS (18) . Gel purification and cloning of the remaining full-length DNA resulted in an apparent 10-fold reduction in the error rate for PCR products. However, the existing approaches are not well suited for error removal in long synthetic DNA sequences where virtually all members in the population contain multiple errors. The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact [email protected] Error correction with MutS is outlined in Figure 1 . The population of DNA molecules containing random errors is first re-hybridized to expose synthesis errors as mismatches ( Figure 1A ). Duplexes containing mismatches can then be removed from the population by affinity capture with immobilized MutS ( Figure 1B) , a process we term coincidence filtering, since both strands of the duplex must match to pass this filtering step. For long synthetic DNA sequences or for sequences with high error rates, coincidence filtering is ineffective, since the likelihood of both strands being perfectly matched after re-hybridization is very low. To generalize MutS error filtering for application on synthetic DNA, the synthetic DNA is cleaved into small overlapping fragments before MutS filtering. Fragments containing mismatches are selectively removed through absorption to an immobilized maltose-binding protein (MBP)-Thermus aquaticus (Taq) MutS-His 6 fusion protein (MBP-MutS-H6) (18) (19) (20) . The remaining mixture of fragments (enriched with fragments of the correct sequence) serves as a template for assembly PCR to produce the full-length product ( Figure 1C ). This process can be iterated until the consensus sequence emerges as the dominant species in the population. This approach is equivalent to DNA shuffling (21) with additional mismatch exposure and removal steps.\nIn this report, we assemble GFPuv from synthetic oligonucleotides and apply both coincidence filtering and consensus shuffling protocols to reduce errors in the resultant DNA populations. The error rates are characterized by gene function (fluorescence) and by DNA sequencing. We also provide a mathematical model describing the error reduction protocols to aid predictions about parameters influencing their effectiveness.\nChemicals were from Sigma. Restriction enzymes were from Promega and New England Biolabs. KOD Hot Start DNA Polymerase was from Novagen. Amylose resin was from NEB (catalog no. E8021S). Ni-NTA resin was from Novagen (catalog no. 70666). Ultrafiltration device from Millipore (catalog no. UFC900524). Slide-A-Lyzer dialysis membrane was from Pierce (catalog no. 66415).\nFull-length Taq MutS was amplified from template pETMutS (22) with primers 5 0 -AAA AAA CAT ATG GAA GGC ATG CTG AAG G-3 0 and 5 0 -AAA AAT AAG CTT CCC CTT CAT GGT ATC CAA GG-3 0 and cloned into the Nde1/HindIII sites of vector pIADL14 (23) to give plasmid pMBP-MutS-H6.\nE.coli strain BL21(DE3) transformed with pMBP-MutS-H6 was grown to OD 600 $1.0 and induced using 1 mM isopropylb-D-thiogalactopyranoside for 4 h at 37 C. Cells from 4 l of culture were pelleted and resuspended in 60 ml of buffer A (20 mM Tris-HCl, pH 7.4, 300 mM NaCl, 1 mM EDTA, 1 mM DTT and 1 mM phenylmethlysulfonyl fluoride). Cell suspension was sonicated on ice and insoluble material was removed by centrifugation at 50 000 g for 10 min at 4 C. Supernatant was applied to 5 ml amylose resin pre-equilibrated in buffer A. Bound MBP-MutS-H6 was washed three times using 20 ml buffer B (20 mM Tris-HCl, pH 7.4, 300 mM NaCl) and stored The re-hybridized gene synthesis products are fragmented, and error containing fragments are precipitated by MBP-MutS-H6 immobilized on amylose support. Error reduced fragments (orange, blue and red) are reassembled into the full-length gene followed by PCR amplification to generate error reduced products. Primers: black lines.\novernight at 4 C. MBP-MutS-H6 was eluted using 20 ml buffer B + 10 mM maltose. Eluate was applied to $4 ml of Ni-NTA resin pre-equilibrated in buffer B. Bound MBP-MutS-H6 was washed four times using 20 ml buffer B + 25 mM imidazole. Bound MBP-MutS-H6 was eluted using buffer B + 1 M imidazole. Eluate was concentrated via ultrafiltration using Amicon Ultra 5 kDa MWCO at 4 C. Concentrated sample was dialyzed extensively against 2· storage buffer (100 mM Tris-HCl, pH 7.5, 200 mM NaCl, 0.2 mM EDTA and 0.2 mM DTT) using a Slide-A-Lyzer 10 kDa MWCO cassette at 4 C. Protein concentration was determined using A 280 and a calculated extinction coefficient of 119 070 M À1 cm À1 . Dialyzed sample was diluted using an equal volume of glycerol and stored at À20 \nOligonucleotides were purchased from Qiagen with 'salt-free' purification. Sequence 261-1020 of pGFPuv (GenBank accession no. U62636 with T357C, T811A and C812G base substitutions) was assembled using 40mer (37) and 20mer (2) oligonucleotides with 20 bp overlap (Supplementary Table 1 ). Assembly reactions contained the following components: 64 nM each oligonucleotide, 200 mM dNTPs, 1 mM MgSO 4 , 1· buffer and 0.02 U/ml KOD Hot Start DNA Polymerase. Assembly was carried out using 25 cycles of 94 C for 30 s, 52 C for 30 s and 72 C for 2 min. PCR amplification of assembly products contained the following components: 10-fold dilution of assembly reaction, 25 mM of 20 bp outside primers, 200 mM dNTPs, 1 mM MgSO 4 , 1· buffer and 0.02 U/ml KOD Hot Start DNA Polymerase. PCR was carried out using 35 cycles of 94 C for 30 s, 55 C for 30 s and 72 C for 1 min followed by a final extension at 72 C for 10 min. PCR products were purified using the Qiagen QIAquick PCR purification kit with elution in dH 2 O followed by speed-vac concentration. Assuming an error rate of 1 · 10 À6 /bp/duplication for KOD DNA polymerase (24) , 35 cycles of PCR would be expected to introduce $0.053 mutations per assembled GFPuv molecule.\nAssembled GFPuv was diluted to 250 ng/ml in 10 mM Tris-HCl, pH 7.8, 50 mM NaCl and heated to 95 C for 5 min followed by cooling 0.1 C/s to 25 C. Heteroduplex for consensus filtering was split into three pools and digested to completion with NlaIII (NEB), TaqI (NEB) or NcoI plus XhoI (Promega) for 2 h following the manufacturer's protocols. Digests were purified using the Qiagen QIAquick PCR purification kit with elution in dH 2 O. Samples were pooled and the concentration was determined by measuring A 260 .\nMBP-MutS-H6 binding reactions contained $11.5 ng/ml DNA and $950 nM MBP-MutS-H6 dimers in 1· binding buffer (20 mM Tris-HCl, pH 7.8, 10 mM NaCl, 5 mM MgCl 2 , 1 mM DTT and 5% glycerol). Reactions were allowed to incubate at room temperature for 10 min before incubation for 30 min with an equal volume of amylose resin pre-equilibrated in 1· binding buffer. Protein-DNA complexes were removed by low-speed centrifugation and aliquots of supernatant were removed for subsequent processing.\nSupernatant (50 ml) from consensus filtering experiments was desalted using Centri-Sep spin columns (Princeton Separations) and concentrated. Purified and concentrated DNA fragments were reassembled as above with aliquots removed at varying cycles. Aliquots of assembly reactions were resolved on 2% agarose gels to monitor the reassembly process. Aliquots showing predominantly reassembled fulllength GFPuv were PCR amplified as above. Aliquots of supernatant from coincidence filtering experiments were diluted 10-fold and PCR amplified as above. PCR products were digested with BamHI/EcoRI and ligated into the 2595 bp BamHI-EcoRI fragment of pGFPuv. Ligations were transformed into E.coli DH5 and fluorescent colonies were scored using a handheld 365 nm ultraviolet (UV) lamp.\nPreparation of substrate for consensus shuffling from 10 non-fluorescent GFPuv clones Ten non-fluorescent GFPuv clones were pooled in equal amounts. The nature and location of the mutations in these clones is shown in Figure 4 . The GFP coding region was PCR amplified from the mixture and submitted to the consensus shuffling protocol with and without the application of the MBP-MutS-H6 error filter.\nTo create an error filter, we constructed a fusion protein between MBP (19) and the mismatch binding protein from T.aquaticus (22) with a C-terminal His 6 tag (MBP-MutS-H6). MBP-MutS-H6 was overexpressed and purified from E.coli to >95% purity (Supplementary Figure 1) . MBP-MutS-H6 immobilized on amylose resin was shown to selectively retain a 40mer heteroduplex containing a deletion mutation over wild-type homoduplex (Supplementary Figure 2) .\nTo demonstrate error correction, unpurified 40mer oligonucleotides were assembled by PCR (3) to produce a 760 bp gene encoding green fluorescent protein (25) (GFPuv). Two independent preparations of GFPuv containing typical gene synthesis errors (Figure 3 and Table 1 ) were re-hybridized and subjected to two iterations of coincidence filtering or consensus shuffling. For consensus shuffling, the GFPuv assembly product was split into three pools and digested into sets of overlapping fragments using distinct Type II restriction enzymes ( Figure 2 ). The digests were pooled and subjected to error filtering with or without added MBP-MutS-H6. The unbound fragments were reassembled into full-length products and PCR amplified. For coincidence filtering, unbound fulllength GFPuv was PCR amplified following treatment with the error filter. After cloning in E.coli, error rates were estimated by scoring colonies for fluorescence under a handheld UV lamp (Figure 3) . The actual error rates of the input and consensus shuffled populations were determined by sequencing plasmid DNA from randomly selected colonies (Figure 3) . The results show that two rounds of consensus shuffling increased the percentage of fluorescent colonies from $60 to >90% and Table 1 . Sequence errors in input and consensus shuffled DNA Table 1 .\nAlthough DNA shuffling has traditionally been used to create diversity through the combinatorial shuffling of mutations in a population, DNA shuffling also creates a sub-population of sequences with a reduction in diversity, as correct fragments can recombine to produce error-free sequences. Indeed, with consensus shuffling, it is possible to start with a population of DNA molecules wherein every individual in the population contains errors and create a new population where the dominant sequence is error free. To demonstrate this, 10 nonfluorescent GFPuv clones, each containing a deletion mutation (Figure 4) , were pooled and subjected to either DNA shuffling alone or two iterations of consensus shuffling. Products were cloned in E.coli, and the percentage of fluorescent colonies was monitored as an indication of progress toward the consensus sequence. DNA shuffling alone (no MBP-MutS-H6) increased the percentage of fluorescent colonies to 30% (387 colonies total) similar to a previous report (26) . Two rounds of consensus shuffling gave a new population that was 82% fluorescent (551 colonies total), indicating that the dominant species was likely the consensus sequence of the input population.\nBoth consensus shuffling and coincidence filtering protocols were effective in reducing errors in synthetic GFPuv populations ( Figure 3 ). In both cases, two iterations of either consensus shuffling or coincidence filtering increased fluorescent colonies from average values of $60 to >90%. Sequencing data from two independent experiments showed a 4.3-and 3.5-fold reduction in the error rate for the consensus shuffled populations compared with the input populations giving final error rates of 0.3 and 0.28 errors/kb, respectively. These results demonstrate the usefulness of the MBP-MutS-H6 error filter in both consensus shuffling and coincidence filtering protocols. Taq MutS has previously been shown to bind to deletion mutations with high affinity (27) , a mutation common in synthetic DNA. However, it is important to note that Taq MutS has lower affinity for specific point mutations and binds weakly to homoduplex DNA (27) . These factors may limit the stepwise efficiency of the error filter. Moreover, specific point mutations may be refractory to removal even after multiple rounds of consensus shuffling. Two rounds of consensus shuffling using the MBP-MutS-H6 error filter proved most effective in reducing deletions and G/C to A/T transitions, consistent with previous reports for the selectivity of Taq MutS (27) . However, it must be emphasized that each synthetic oligonucleotide point mutation would generate two heteroduplex DNA molecules containing unique mismatches after PCR amplification and re-hybridization ( Figure 1A and Table 1 ). For example, a G to A transition mutation in a synthetic oligonucleotide would generate heteroduplexes with G-T or A-C mismatches after PCR amplification and re-hybridization. For consensus shuffling, either of these mismatch containing heteroduplexes could evade precipitation by the MBP-MutS-H6 error filter and participate in the reassembly of full-length GFPuv. Therefore, Table 1 lists the pair of mismatches that could give rise to the observed transition or transversion mutation. These results show that the MBP-MutS-H6 error filter was most effective at removing insertion/deletion loops and G-T/A-C mismatches from the population.\nIt should be possible to generalize the consensus shuffling protocol to a large number of synthetic DNA constructs. GFPuv was chosen as the synthetic construct in this study for its advantages as a fluorescent reporter gene. This allowed easy optimization of our protocol without the need to sequence thousands of base pairs of DNA. We expect the results reported here for consensus shuffling to readily translate to synthetic DNA constructs of varied sequence, greater overall length and/ or higher initial errors/kb. Synthetic DNA constructs of varied sequence can be digested into a defined set of fragments using Type II restriction enzymes or fragmented into any desired size range using controlled DNase I digestion (26) . Digestion and reassembly of a large number of different genes is expected to be as robust as the protocol of DNA shuffling (28) , which has been broadly applied to a variety of gene sequences. Synthetic DNA constructs larger than GFPuv are expected to be amenable to error correction by consensus shuffling, as the error filtering is conducted on gene fragments before reassembly of the full-length gene. Thus, the errors/kb data presented in this study are expected to translate to larger genes with similar initial errors/kb (excepting mutations introduced by PCR amplification following the final application of the error filter). Synthetic DNA constructs of higher initial errors/kb are expected to be amenable for error correction by consensus shuffling. However, these constructs will require digestion into smaller sized gene fragments that may affect the efficiency of error correction. In contrast to consensus shuffling, an increase in the size of the synthetic DNA product or an increase in errors/kb would preclude the use of the coincidence filtering protocol, as every molecule in the population would contain one or more errors. As proof of the utility of the consensus shuffling protocol, 10 non-fluorescent GFPuv clones containing one or more errors (Figure 4 ) were converted into a population where 82% of the clones were fluorescent. It is important to note that DNA shuffling alone shows an improvement in percent fluorescent colonies in this example (from 0 to 30%). For synthetic DNA populations, DNA shuffling alone shows no improvement in percent fluorescent colonies (see Figure 3 'no MutS' treatments). DNA shuffling alone improves the overall number of correct sequences only for small DNA populations with low error rates. For example, when shuffling 10 clones with a unique mutation in each clone, one would expect the fraction of correct products to be (9/10) 10 = 35% (26), very close to the value of 30% that we observed. A mathematical model describing the error rates for shuffling and error filtering of synthetic DNA populations is presented below.\nTo estimate some parameters of consensus shuffling and coincidence filtering, a simple mathematical model (Equations 1-6) was constructed. An input population of dsDNA molecules of length N, containing E errors/base is re-hybridized, fragmented into shorter dsDNA fragments of average length S, error filtered and reassembled. P(F) is the probability a fragment of length S will have a correct sequence. We determine the probability that re-hybridized duplexes will have zero (C), one (H ) or both (I ) strands with errors.\nEquation 5 estimates the probability that a fragment will be correct after a cycle of MutS filtering, P(F 0 ), by applying a MutS selectivity factor (M ) to adjust the relative amounts of mismatch containing duplexes (I, H ) while accounting for the total fraction of correct strands in the re-hybridized duplexes. The probability of obtaining an error free assembly product, P(A), is then given by Equation 6 .\nFrom our consensus shuffling error rate data (Figure 3 ), we estimate the MutS selectivity factor M to be $2.2. Figure 5 shows some predictions that emerge from this model assuming typical length (2 kb), fragment sizes (200 bp) and error rates (1.8 errors/kb). Consensus shuffling is predicted to be most effective with smaller fragment sizes ( Figure 5A ). As mentioned above, smaller fragment sizes could be obtained by controlled digestion with DNase I (21) . In addition, multiple iterations of MutS filtering can have dramatic results on populations with few correct sequences ( Figure 5B ), although the model does not account for the differing specificity of MutS toward the various types of mismatches. The model also predicts that even modest improvements in the MutS selectivity factor through optimization of the MutS-DNA binding conditions and/or the use of a combination of MutS homologs with varying mismatch specificity (29) could dramatically improve the consensus shuffling protocol ( Figure 5C ). Coincidence filtering (N = S) is predicted to be effective for populations with low error rates per clone ( Figure 5D ) but becomes ineffective when the majority of re-hybridized duplexes contain mismatches.\nWe have demonstrated consensus shuffling and coincidence filtering as experimental methods to significantly reduce errors in synthetic DNA. Consensus shuffling should be generally applicable for error correction on synthetic genes of typical lengths and error rates. Two iterations of consensus shuffling ($6 h/iteration) generated a population with $1 error/3500 bp. This reduction in error rate will allow the identification of a correct clone after sequencing DNA from a reduced number of colonies. Coincidence filtering is a simple and effective procedure to reduce errors in synthetic DNA populations with low error rates per clone. These methods should significantly increase the speed and decrease the cost of production of synthetic genes.\nNote: While this manuscript was under review, Carr et al. (30) independently reported the application of Taq MutS in protocols for error reduction on synthetic DNA." }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: Purity and integrity of RNA are critical elements for the overall success of RNA-based analyses, including gene expression profiling methods to assess the expression levels of thousands of genes in a single assay. Starting with low quality RNA may strongly compromise the results of downstream applications which are often labor-intensive, time-consuming and highly expensive. However, in spite of the need for standardization of RNA sample quality control, presently there is no real consensus on the best classification criteria. Conventional methods are often not sensitive enough, not specific for single-stranded RNA, and susceptible to interferences from contaminants present in the sample. For instance, when using a spectrophotometer, a ratio of absorbances at 260 and 280 nm (A 260 :A 280 ) greater than 1.8 is usually considered an acceptable indicator of RNA purity (1, 2) . However, the A 260 measurement can be compromised by the presence of genomic DNA leading to over-estimation of the actual RNA concentration. On the other hand, the A 280 measurement will estimate the presence of protein but provide no hint on possible residual organic contaminants, considered at 230 nm (3) (4) (5) . Pure RNA will have A 260 :A 230 equal to A 260 :A 280 and >1.8 (1) . A second check involves electrophoresis analysis, routinely performed using agarose gel electrophoresis, with RNA either stained with ethidium bromide (EtBr) (6) (7) (8) (9) , or the more sensitive SYBR Green dye (10) . The proportion of the ribosomal bands (28S:18S) has conventionally been viewed as the primary indicator of RNA integrity, with a ratio of 2.0 considered to be typical of 'high quality' intact RNA (1) . However, these methods are highly sample-consuming, using 0.5-2 mg total RNA and often not sensitive enough to detect slight RNA degradation. Today, microfluidic capillary electrophoresis with the Agilent 2100 bioanalyzer (Agilent Technologies, USA) has become widely used, particularly in the gene expression profiling platforms (11, 12) . It requires only a very small amount of RNA sample (as low as 200 pg), the use of a size standard during electrophoresis allows the estimation of sizes of RNA bands and the measurement appears relatively unaffected by contaminants. Integrity of *To whom correspondence should be addressed. Tel: The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact [email protected] the RNA may be assessed by visualization of the 28S and 18S ribosomal RNA bands ( Figure 1A and B); an elevated threshold baseline and a decreased 28S:18S ratio, both are indicative of degradation. A broad band shows DNA contamination ( Figure 1C ). As it is apparent from a review of the literature, the standard of a 2.0 rRNA ratio is difficult to meet, especially for RNA derived from clinical samples, and it now appears that the relationship between the rRNA profile and mRNA integrity is somewhat unclear (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) . On the one hand, this may reflect unspecific damage to the RNA, including sample mishandling, postmortem degradation, massive apoptosis or necrosis, but it can reflect specific regulatory processes or external factors within the living cells. Altogether, it appears that total RNA with lower rRNA ratios is not necessarily of poor quality especially if no degradation products can be observed in the electrophoretic trace ( Figure 1D ).\nFor all these reasons, the development of a reliable, fully integrated and automated system appropriate for numeric evaluation of RNA integrity is highly desirable. Standardized RNA quality assessment would allow a more reliable comparison of experiments and facilitate exchange of biological information within the scientific community. With that prospect in mind, and with the aim of anticipating future standards by pre-normative research, we identified and tested two software packages recently developed to gauge the integrity of RNA samples with a user-independent strategy: one open source, the degradometer software for calculation of the degradation factor and 'true' 28S:18S ratio based on peak heights (24) and the freely available RIN algorithm of the Agilent 2100 expert software, based on computation of a 'RNA Integrity Number' (RIN) (25) . Both tools were developed separately to extract information about RNA integrity from microcapillary electrophoretic traces and produce a userindependent metrics. Using these tools, we assessed the purity and integrity of 414 RNA samples, derived from 14 different human adult tissues and cell lines, many of which representing tumors. Those results were compared with conventional RNA quality measurement approaches as well as with highly expert human interpretation. We evaluated the simplicity for users and examined the potential, accuracy and efficiency of each method to contribute to standardization of RNA integrity assessment upstream of biological assays. These procedures were further validated by real-time RT-PCR quantitation of the expression levels of three housekeeping genes, using the same RNA samples, at different levels of degradation.\nTotal RNA was prepared from human cell lines (especially from the ATCC bio-resource center, N = 50) and tissue samples (clinical samples, N = 285) from 13 different human adult tissue types, i.e. blood, brain, breast, colon, epithelium, kidney, lymphoma, lung, liver, muscle, prostate, rectum and thyroid. RNA purification was performed by cesium chloride ultracentrifugation according to Chomczynski and Sacchi (26) , by phenol-based extraction methods (TRIzol reagent, Invitrogen, USA), or silica gel-based purification methods (RNeasy Mini Kit, Qiagen, Germany; Strataprep kit, Stratagene, USA or SV RNA isolation kit, Promega, USA) according to the manufacturer's instructions with some modifications. Material was maintained at À80 C with minimal handling. RNA extraction was carried out in an RNase-free environment (see Supplementary Table 1 online) .\nThe commercially available RNA samples were the 'Universal Human Reference' (N = 75) distributed by Stratagene (USA), and human brain (N = 2) and muscle (N = 2) RNAs supplied by Clontech (USA).\nOnce extracted, RNA concentration and purity was first verified by UV measurement, using the Ultrospec3100 pro (Amersham Biosciences, USA) and 5 mm cuvettes. The absorbance (A) spectra were measured from 200 to 340 nm. A 230 , A 260 and A 280 were determined. A 260 :A 280 and A 260 :A 230 ratios were calculated. For microcapillary electrophoresis measurements, the Agilent 2100 bioanalyzer (Agilent Technologies, USA) was used in conjunction with the RNA 6000 Nano and the RNA 6000 Pico LabChip kits. In total, 39 assays were run in accordance with the manufacturer's instructions (see Supplementary Notes online). To evaluate the reliability of the classifier systems described in this study, replicate runs were done on a set of 56 RNA samples loaded on different chips, resulting in 2 (N = 41), 3 (N = 12), 7 (N = 2) and 50 (N = 1) data points per sample.\nHuman RNA integrity categorization RNA integrity checking was performed by expert operators who classified each total RNA sample within a predefined discrete category from 1 to 5, examining the integrity of the RNA from electropherograms (see Supplementary Table 2 online). A low number indicates high integrity. Reference criteria parameters include ribosomal peaks definition, baseline flatness, existence of additional or noise peaks between ribosomal peaks, low molecular weight species contamination and genomic DNA presence suspicion. A smearing of either 28S and 18S peaks, or a decrease in their intensity ratio indicate degradation of the RNA sample and results in the classification into the higher categories. To evaluate the robustness of this human interpretation, five highly experienced operators, trained in these cataloging steps, separately classified a subset of 33 samples from breast cancers. It included samples with varying levels of integrity: intact RNA (33%), low quality samples (20%) and a wide range of degradation (47%).\nBioanalyzer electrophoretic data were exported in the degradometer software folder (.cld format). For comparison of samples, the original data were re-scaled by the classifier system, first along the time-axis to compensate for differences in migration time, then along the fluorescence intensity-axis to compensate for variation in total RNA amount. As a result, fluorescence curves that have the same shape will have the same peak heights after re-scaling. Then, Degradation Factors (DegFact) and corrected 28S:18S ratios were calculated (see Supplementary Table 3 online) using the mathematical model developed by Auer et al. (24) , examining additional 'degradation peak signals' appearing in the lower molecular weight range and comparing them to ribosomal peak heights. Calculation of the DegFact is based on a numbering of continuous metrics, ranging from 1 to ¥; increasing DegFact values correspond to more degradation, and a new group of integrity is defined after 8 graduation steps. Once the classification of the RNA samples is completed, 4 groups of integrity are displayed, 3 showing an alert warning indicative of some measurable degradation (Yellow: 8-16, Orange: 16-24 and Red: >24), while all non-reliable data come together and form the fourth group (Black). We introduced a fifth class labeled White (<8), when no alert was produced by the software.\nSoftware and manual are freely available at http://www. dnaarrays.org/downloads.php. Degradometer version 1.4.1 (released in May 2004) of the software was used.\nBioanalyzer electrophoretic sizing files (.cld format) collected with biosizing software version A.02.12.SI292 (released in March 2003) were imported in the Agilent 2100 expert software (RIN beta release). The RIN algorithm allows calculation of RNA integrity using a trained artificial neural network based on the determination of the most informative features that can be extracted from the electrophoretic traces out of 100 features identified through signal analysis. The selected features which collectively catch the most information about the integrity levels include the total RNA ratio (ratio of area of ribosomal bands to total area of the electropherogram), the height of the 18S peak, the fast area ratio (ratio of the area in the fast region to the total area of the electropherogram) and the height of the lower marker.\nA total of 1300 electropherograms of RNA samples from various tissues of three mammalian species (human, mouse and rat), showing varying levels of degradation and an adaptive learning approach were used in order to assign a weight factor to the relevant features that describe the RNA integrity. A RIN number is computed for each RNA profile (see Supplementary Table 4 online) resulting in the classification of RNA samples in 10 numerically predefined categories of integrity. The output RIN is a decimal or integer number in the range of 1-10: a RIN of 1 is returned for a completely degraded RNA samples whereas a RIN of 10 is achieved for intact RNA sample.\nIn some cases, the measured electropherogram signals are of an unusual shape, showing for example peaks at unexpected migration times, spikes or abnormal fluctuation of the baseline. In such cases, a reliable RIN computation is not possible. Several separate neural networks were trained to recognize such anomalies and display a warning to the user or even suppress the display of a RIN number. Combining the results of the neural network for the RIN computation and the neural networks to detect anomalies, the RIN algorithm achieves a mean square error of 0.1 and a mean absolute error of 0.25 on an independent test set.\nThe beta release of the software and manual are freely available at http://www.agilent.com/chem/RIN. Agilent 2100 expert version B.01.03.SI144 (released in November 2003) of the software was used.\nExpression levels of three housekeeping genes (HKG)-GAPD, GUSB and TFRC-were measured by quantitative PCR using the TaqMan Gene Expression Assays according to the manufacturer's instructions (Applied Biosystems, USA). Sixteen aliquots of a unique batch of RNA sample (Universal Human Reference RNA, Stratagene, USA) of various levels of integrity (cf. Table 1 ) were used to test the influence of RNA quality on the relative expression of those three genes. In parallel, a 5 0 to 3 0 comparison was done using two separate GUSB and TFRC TaqMan probes.\nAn homogeneous quantity (0.8-1 mg) of the RNA samples was subjected to a reverse transcription step using the highcapacity cDNA archive kit (Applied Biosystems, USA) as described by the manufacturer. Single-stranded cDNA products were then analyzed by real-time PCR using the TaqMan Gene Expression Assays according to the manufacturer's instructions (Applied Biosystems, USA). Single-stranded cDNA products were analyzed using the ABI PRISM 7700 Sequence Detector (Applied Biosystems, USA). The efficiency and reproducibility of the reverse transcription were tested using 18S rRNA TaqMan probes. Five assays were used, GAPDH-5 0 (Hs99999905_m1), GUSB-5 0 (Hs00388632_gH), GUSB-3 0 (Hs99999908_m1), TFRC-5 0 (Hs00951086_m1) and TFRC-3 0 (Hs00951085_m1). In each case, duplicate threshold cycle (Ct) values were obtained and averaged; then expression levels were evaluated by a relative quantification method (27) .\nThe fold change in one tested HKG (target gene) was normalized to the 18S rRNA (reference gene) and compared to the highest quality sample (calibrator sample), using the following formula: Fold change = 2 ÀDDCt , where DDCt = (C t-target À C t-reference ) sample-n À (C t-target À C t-reference ) calibrator-sample . Sample-n corresponds to any sample for the target gene normalized to the reference gene and calibrator-sample represents the expression level (1·) of the target gene normalized to the reference gene considering the highest quality sample. Mean 2 ÀDDCt and SD were calculated, considering the samples either individually or grouped by quality metrics categories, based on RIN metrics or DegFact values, together with the lower and upper bound mean of 95% Intervals of Confidence (IC). Using this analysis, if the expression levels of the HKG are not affected by the RNA degradation, the values of the mean fold change at each condition should be very close to 1 (since 2 0 = 1) (27) .\nDescriptive statistics were executed using the XLSTAT software, version 7.1 (Addinsoft, USA), P = 0.05. Mean, SD and coefficient of variation (variation or CV) between and within groups of samples were calculated, together with a measure of the dispersion (range), inter-quartile range (1st and 3rd quartiles, Q1-Q3) and evaluation of the lower and upper bound mean of 95% Interval of Confidence (IC). Comparative statistical analyses between groups were completed, P = 0.05, using non-parametric statistical tests: two-independent Mann-Whitney U-test and k-independent Kruskal-Wallis test.\nWe analyzed 414 total RNA sample profiles from various human tissues (69%) and cell lines (31%) of either tumoral (85%) or normal (15%) origin, with varying levels of RNA integrity. Supplementary Table 1 online for details). Significant differences in A 260 :A 280 ratios were observed between specific groups of samples (i.e. tumoral versus normal or tissues versus cell lines). For instance, RNA extracted from normal samples displayed an improved ratio of 1.97, with 97% falling within the desired range ( Figure 2A ). In contrast, the distribution of A 260 :A 280 ratios was not found to correlate with either purification methods or tissues of origin.\nRNA integrity was further assessed by resolving the 28S and 18S ribosomal RNA bands using the Agilent 2100 bioanalyzer and the RNA 6000 protocol. The analysis was done on 399 RNA profiles; data from 15 samples was not obtained due to device problems during the runs. The system automatically provided 28S:18S ratios for 348 (87%) of the 399 profiles. Figure 2B shows the distribution of the 28S:18S computed values, with a median ratio around 1.7 and a variation of 54% from the mean (IC 1.9-2.1 and Q1-Q3 1.4-2.5). In addition, a significant degree of variability of the 28S:18S ratio (19-24%) was found for identical samples from replicate runs (2-50 times). Among those RNA samples, 28S:18S ratios of 2.0 or greater were rare, less than 44% of the values measured being within the theoretically desired range, except for the samples prepared from cultured cells ( Figure 2B ). The integration failed in the remaining 51 cases, displaying an atypical migration, with no clear 28S and 18S rRNA bands, and no 28S:18S ratio was computed (data not shown). \nExpert operators categorized the set of RNA samples by inspecting the electrophoretic traces of successful assays. Over the 399 RNA profiles checked, 379 (95%) were scored within predefined categories ( Figure 2C ), namely good [Human Categorization (HC)-level 1], regular (HC-level 2), moderate (HC-level 3), low (HC-level 4) and degraded (HC-level 5). The remaining 20 (5%) were flagged as displaying a temperature-sensitive profile: RNA samples initially found intact became highly degraded when heated, although no RNase contamination was observed (data not shown).\nEstimation of the robustness of this cataloging was done through comparison of qualifying criteria using a set of 33 breast cancer samples (see Materials and Methods). Integrity of the samples was evaluated independently by five expert operators, and categorization was found highly reliable with a coefficient variation (CV) $16%. This is low considering that individual interpretation is involved, but can be explained by the fact that very experienced operators accomplished the scoring based on a clearly defined set of instructions, thus limiting frequently observed subjective visual interpretation and inconsistency of human categorization. Predictably, a 28S:18S ratio of 2.0 denoted high quality for a majority of RNA samples, 91% being classified in HC-levels 1 to 3. However, 83% of total RNAs with 28S:18S > 1.0 but a low baseline between the 18S and 5S rRNA or front marker were also classified in HC-levels 1-3 (see Figure 1D ) and could be considered suitable for most downstream applications.\nRNA degradation was first assessed using the degradometer software (see Materials and Methods). Over the 399 RNA profiles checked, all were scored in one of the five predefined classes ( Figure 3A) . Altogether, 334 (84%) Degradation Factors (DegFact) values were computed, the remaining 65 RNA samples (16%) displaying profiles that could not be interpreted reliably; no DegFact values could be scored, and samples were flagged in the Black category ( Figure 3A ). Most of them (80%) correspond to samples previously classified by our operators as degraded (HC-level 5). The remaining cases had an average degradation factor of 7.5 (IC 6.7-8.3) with large variations over the entire set of samples (over 103% from the mean, range 1-52). A lower variability was persistently found when identical samples from replicate runs were considered, resulting in observed DegFact values with a 26-32% CV. In addition, statistically significant differences were found between DegFact values of samples sorted by types. The highest DegFact values were found characteristic of tissue samples, 41% of them displaying a DegFact > 8, as compared with 6% for the cell lines (data not shown).\nRemarkably, we found a significant linear relationship between the DegFact values distribution and the explicit human categorization. Most HC classes corresponded to an unambiguous DegFact distribution ( Figure 3B ), while HClevels 2 and 3 form a single class: HC-level 1, mean DegFact of 3.3, SD of 2.8 (IC 2.8-3.7); HC-level 2 and 3, mean Deg-Fact of 8.8, SD of 6.8 (IC 7.5-10.2); HC-level 4, mean DegFact of 15.9, SD of 7.8 (IC 12.7-19.1); HC-level 5, mean DegFact of 26.0, SD of 7.5 (IC 21.9-30.1). It is worth mentioning that the normalized heights of 18S and 28S peaks, and the interval between them after rescaling gradually decrease and then reverse with increasing degradation ( Figure 3B ).\nIntegrity of RNA samples was measured in parallel based on the RNA Integrity Number metrics using an artificial neural network trained to distinguish between different RNA integrity levels by examining the shape of the microcapillary electrophoretic traces (see Materials and Methods). Over the 399 RNA profiles checked, 363 (91%) were scored successfully ( Figure 4A) , with an average RIN of 7.7 (IC 7.4-8.0). The remaining 36 (9%) samples were associated with various unexpected signals, disturbing computation of the RIN using default anomaly detection parameters. In each case, a flag alert was added corresponding to critical anomalies including unexpected data in sample type, (or) ribosomal ratio, (or) baseline and signal in the 5S region (data not shown).\nRIN categorization was found regular, variability between replicate runs, compared to the other methods, being consistently very small (CV 8-12%). As expected, the highest RIN were characteristic of cell line samples, 72% of them displaying a RIN > 9, as compared with 47% for the tissue samples (data not shown).\nA first group, corresponding to 295 (82%) of the 363 RNA profiles, was analyzed using the default settings of the RIN system, but with a lower threshold of RNA quantity loaded (20 ng) for reliable detection of anomalies than that recommended by the manufacturer (50 ng). A significant linear relationship was found between the RIN number and both the explicit human classification provided by our operators, Figure 3 . RNA degradation characterization. Integrity of 399 RNA sample profiles was scored using the degradometer software. (A) A total of 334 RNA profiles were successfully categorized into 5 predefined alert classes using a mathematical model that quantifies RNA degradation and computes a degradation factor (DegFact). Four classes (White, Yellow, Orange and Red) are associated with different levels of degradation. A fifth class, Black alert corresponds to samples that the system was not able to qualify with accuracy (n.d.). The distribution is represented by the number of records in each class. (B) Comparative analysis was done using human evaluation (x-axis) based on electrophoresis analysis as a reference for RNA integrity classification; observations of rRNA peak heights and DegFact values were taken at each of the 5 HC levels. Histograms refer to the mean 28S and 18S rRNA peak heights and 95% confidence intervals (fluorescence intensities; left scale). Mean DegFact values and 95% confidence intervals (arbitrary unit, right scale) are plotted with the means joined. and the DegFact values calculated by the degradometer software ( Figure 4B ). Each distinct HC class corresponds to an explicit RIN number, with HC-levels 2 and 3 forming once again a single class: HC-level 1, mean RIN of 9.6, SD of 0.7 (IC 9.5-9.7); HC-level 2 and 3, mean RIN of 8.6, SD of 0.9 (IC 8.4-8.9); HC-level 4, mean RIN of 6.1, SD of 1.5 (IC 5.2-7.1); HC-level 5, mean RIN of 3.7, SD of 2.0 (IC 2.9-4.5).\nFor the remaining 68 samples (assay done with <20 ng of RNA), two separate groups were considered: 41 samples with a computed RIN below 5.0, and 27 above 7.0. All samples in the first group were derived from RNA 6000 Nano assays, with mean RNA quantities loaded below 10 ng (Q1-Q3, 5-12 ng), i.e. below the lower limit of quantitation indicated by the manufacturer. All but 8 of these samples were estimated by our operators to be of poor quality (HC-level 4; N = 3) or degraded (HC-level 5; N = 30), and all but 4 were flagged Black by the degradometer software and no DegFact values were scored. These RNA profiles could not be interpreted reliably, possibly due to either the low RNA concentration or the unusual migration behavior and shifted baseline values of degraded samples. Thus, the two automated systems were in disagreement for these samples; while human interpretation was in most cases in agreement with the RIN system, with less than 20% of inconsistency. In the second group of 27 samples, 20 of the profiles were derived from RNA 6000 Pico assays with RNA quantities loaded being on average below 4 ng (Q1-Q3, 0.5-0.8 ng), which is within the manufacturer specifications. All but 3 of them were estimated by our operators to range from high (HC-level 1; N = 12) to correct (HC-level 2 and 3; N = 12) quality levels. In addition, all RNA profiles except 1 were scored by the degradometer software, most of them displaying an alert flag (N = 20); some slight degradation was detected, associated to a low mean DegFact value of 9.7 (IC 8.1-11.3; Q1-Q3, 6.2-12.6). Thus, both automated systems and human interpretations agreed in most of these cases, with <11% of inconsistency.\nThe influence of RNA quality categorization obtained with both user-independent classifiers on gene expression profiling was explored using real-time RT-PCR. The expression levels of three housekeeping genes (HKG)-GAPDH, GUSB and TFRC-were measured in 16 aliquots of a unique RNA displaying various integrity metrics ( Table 1 ). The mean correlation coefficient (r) between the threshold cycle (Ct) among the 16 samples and both quality metrics was found high: r = À0.87 considering the RIN metrics and r = 0.85 considering the DegFact values. The values of the mean fold changes, calculated according to the 2 ÀDDCt quantification method (see Materials and Methods), were found lower than 1.0, corresponding to the expression level (1·) in the sample exhibiting the highest RNA quality (Table 2 and Figure 5 ). Considering that HKG expression was measured relative to the reference sample, an obvious decline of the relative expression levels was observed, up to 24, 70 and 82%, in samples categorized according to the RIN metrics ( Figure 5A) and DegFact values ( Figure 5B ). These results indicate that 2-to 7-fold differences may be expected in the relative expression levels of genes in samples that differ only by their quality (Table 2 ). These fold differences are much larger than those measured for RNA samples of comparable integrity, consistently lower than 1.6 (Table 2 and Figure 5 ). In addition, an unambiguous gap in the distribution may be defined ( Figure 5A and B) , distinguishing the RNA samples of the higher quality categories (RIN > 8 and DegFact values < 7) from those of the lower categories (RIN < 8 and DegFact values > 12).\nIt would be expected that measuring expression of an intact mRNA would yield approximately equal results regardless of the region being probed, and if mRNA fragmentation had occurred, then some sequences may be more abundant than others. We thus tested the effect of PCR probe location on the RNAs. The 5 0 and 3 0 GUSB probes, separated by 1209 nt, were associated with highly similar threshold cycle (Ct) measures (r = 0.98, b parameter = 0.88) ( Figure 5C ). Similar results were obtained for TFRC, with probes separated by 2066 nt (r = 0.84, b parameter = 0.92, data not shown). It seems therefore that the region being probed is not a source of variation in our results.\nIt is universally accepted that RNA purity and integrity are of foremost importance to ensure reliability and reproducibility of downstream applications. In the biomedical literature (PubMed, November 2004), from the 485 090 articles that relate to RNA, and the 287 515 or 40 395 including respectively the 'quality' or 'integrity' term, less than 100 were found to contain 'RNA quality' or 'RNA integrity' terms. Interestingly, half of them were published between 2001 and 2004; but none is proposing a standard operational procedure for RNA quality assessment to the scientific community. Except for two studies (24, 25) , those reports are based on 10 to 15 years old methods (1), indicating that they represent the established and currently mostly used methods. Our results strongly challenge the reliability and usefulness of those conventional methods, demonstrating their inconsistency to evaluate RNA quality.\nFirst, the A 260 :A 280 and A 260 :A 230 ratios are reflecting RNA purity, but are not informative regarding the integrity of the RNA. Available RNA extraction and purification methods yield highly pure RNA with very little DNA or other contaminations, resulting most often in both ratios )1.8, although 18% of the samples were found degraded and 7% more of poor quality. The high A 260 :A 280 ratios are indicative of limited protein contaminations, whereas high A 260 :A 230 ratios are indicative of an absence of residual contamination by organic compounds such as phenol, sugar or alcohol, which could be highly detrimental to downstream applications. Nonetheless, samples displaying low A 260 :A 230 ratios ((1.8) did not exhibit any inhibition during downstream applications, such as cDNA synthesis and labeling or in vitro transcription (data not shown). Second, due to a lack of reliability, the 28S:18S rRNA ratios may not be used as a gold standard for assessing RNA integrity. When ribosomal ratios were calculated from identical samples but through independent runs, a large degree of variability (CV 19-24%) was observed. Moreover, using the biosizing software, we found 28S:18S rRNA ratios evaluation compromised by the fact that their calculation is based on area measurements and therefore heavily dependent on definition of start and end points of peaks. In 13% of the cases, the system was unable to localize the ribosomal peaks, and therefore no 28S:18S ratios were computed. For the remaining samples, no clear correlation between 28S:18S ratios and RNA integrity was found although RNAs with 28S:18S >2.0 were usually of high quality. Most of the RNAs we studied (83%), displaying a 28S:18S > 1.0, could be considered of good quality. Interestingly, Auer et al. (24) in a study on 19 tissues from seven organisms, reported that an objective measurement of the RNA integrity may possibly be done through comparison of re-scaled 28S and 18S peak heights, but not of the corresponding areas. Actually, we observed a linear relationship between RNA integrity and differences in normalized 28S and 18S peak heights. Increased degradation resulted in a significant decrease in the scaled corrected heights of the ribosomal peaks, with inversion of the ratio at the highly degraded stages (cf. Figure 3B ). In comparison to the area computation, 28S:18S rRNA re-scaled peak height measurement produced more consistent values, with a CV reduced to 12-14%, and displayed clear concentration-independent values (see Supplementary Tables 1 and 3 online) . Human evaluation of the integrity of RNA through visual inspection of the electrophoresis profiles provided very consistent data. Variability between classifications produced by five independent expert operators (CV 16%) was lower than with automated management of more conventional control 28S:18S area values (CV 19-24%). It is, however, very time-consuming and strongly dependent on individual competence. Even with highly trained specialists, 5% of the set of RNA samples could not be allocated to any of the five predefined categories; their corresponding profiles were considered by our experts as atypical, displaying a temperature-sensitive shape (data not shown).\nThese strategies appear unsuitable for standardization and quality control of RNA integrity assessment, which require simple but consistent expert-independent classification, facilitating information exchanges between laboratories. The N-value corresponds to the number of samples by category. The mean quality metrics, i.e. RIN and DegFact and the mean fold change (2 ÀDDCt ) relative to the reference sample are indicated, together with the 95% confidence intervals. Observed technical variation (IC-rep, P = 0.05) is also specified, considering duplicate (two per gene per target sample) and replicate (six per gene per calibrator sample) measures. The reference sample exhibits a RIN of 9, a DegFact value of 4.9 and by default mean fold change set to 1. The observed decrease in the expression (relative expression, %) relative to the reference sample is calculated. The fold differences refer to the fold-ratios that are expected in the expression levels for a gene, across categories (between categories), given that the samples only differ by their quality, and within each category (within categories), considering RNA of comparable integrity. The fold-ratios (technical variation) that may be expected by chance in the gene expression levels, P = 0.05, from some technical reasons, are also considered.\nWe therefore investigated the performance of two recently developed user-independent software algorithms (24, 25) . The degradometer software provided a reliable evaluation of RNA integrity based on the identification of additional 'degradation peak signals' and their integration in a mathematical calculation together with the ribosomal peak heights. It allowed characterization of the integrity of 84% of the samples tested, one-third with an alert flag, which was first found to be fairly informative, as it strongly reduces the complexity of the metrics by introducing three distinct classes labeled Yellow, Orange and Red, and can be used as a first straightforward simple filtering step. However, degradation factors (DegFact) metrics yield precise measures with less than 32% CV and are much more valuable than flag alerts for the purpose of standardization. The same is true for the RNA Integrity Number 'RIN' software which allowed the characterization of the integrity of 91% of the RNA samples tested, with a RIN value for 363 RNA sample profiles with less than 12% CV. In general, there was a good agreement between the human classification, the degradation factor and the RIN (see Figure 4B ). This provided a cross-validation of the user-independent qualification systems tested. Both resulted in the refinement of human interpretations, validating four statistically relevant classes of samples, namely good (HC-level 1), regular/ moderate (HC-level 2 and 3), poor (HC-level 4) and degraded (HC-level 5). Moreover, the 5% RNA samples previously flagged by the operators as displaying an atypical temperature-sensitive shape were unambiguously assigned to one or the other category of samples [RIN = 7.3 (IC 6.8-7.8); DegFact = 11.9 (IC 9.5-14.2); data not shown]. Altogether, we found the degradometer and RIN algorithms to be highly reliable user-independent methods for automated assessment of RNA degradation and integrity. The RIN system is a slightly more informative tool, able to compute assessment metrics for 91% of the RNA profiles, compared to 84% with the degradometer software; the remaining being flagged respectively as N/A or Black alert. For samples available below a low limit of 20 ng (N = 80) the RIN system provided Figure 6 . Workflow of operational procedure for RNA quality assessment. Integrity of the RNA, once extracted and purified from cell lines, clinical or biological tissues samples, is controlled from the widely used bioanalyzer electrophoretic traces. As standard part of the Agilent analysis software (25), a RIN metrics is first calculated, scoring each RNA sample into 10 numerically predefined categories of integrity (RIN, from 1 to 10; N is a threshold value). As an independent control, a degradation factor metrics (DegFact, from 1 to ¥; N 0 is a threshold value) may optionally be allocated to each RNA sample using the bioanalyzer-independent degradometer software (24) . In a standard operating procedure, RIN and/or DegFact metrics will first be used as a standard exchange language to document RNA integrity and degradation, second to classify the RNA in homogeneous groups, and finally to select samples of comparable RNA integrity to improve the scheme of meaningful downstream experiments. The standard operating procedure will benefit from feedback information that will help users to define threshold integrity metrics values based on the results of RNA-based analyses. metric values for 85% of them, compared to only 46% with the degradometer software. Similarly, the RIN system was able to provide metric values for 81% of poor quality samples (including low quality and degraded samples; N = 96), whereas the degradometer software could classify only 44% of them. Another advantage with the RIN classifier is that, if there are critical anomalies detected (including genomic DNA contamination, wavy baseline, etc.), threshold settings may be changed and a reliable RIN value computed. This was the case for 25 of the 363 RNA sample profiles successfully classified by the system.\nWhile intact RNA obviously constitutes the best representation of the natural state of the transcriptome, there are situations in which gene expression analysis may be desirable even on partially degraded RNA. Some studies report collection of reasonable microarray data from RNA samples of impaired quality (28) , leading to meaningful results if used carefully. Moreover, Auer et al. (24) recently concluded that degradation does not preclude microarray analysis if comparison is done using samples of comparable RNA integrity. We confirmed the direct influence of the RNA quality on the distribution of gene expression levels, by detecting using Q-PCR a significant (up to 7-fold) difference in the relative expression of genes in samples of slightly decreased RNA integrity, which is much larger than the variation within comparable RNA quality categories (cf. Figure 5 and Table 2 ). This may correlate with ratio discrepancies in gene expression experiments, and therefore with false positive and false negative rates of differential gene expression when comparing two samples. Therefore, computing reliable metrics of RNA integrity, even if the RNA is found to be partially degraded, may be highly valuable. The straight and unambiguous relationships established between human interpretations and both RIN and DegFact distributions indicates that, using these metrics, it should be possible to distinguish specific samples that are too disparate to be included in comparative gene expression analyses without compromising the results. Although the information provided by these user-independent classifiers is not a guarantee for successful downstream experiments, it gives a more comprehensive picture of the samples and can be used as a safeguard against performing useless and costly experiments.\nThus, the RIN system may be used as simple metrics that can be easily integrated in any sample tracking information system for definition of standard operating procedures under quality assurance following a scheme such as the one described in Figure 6 . In this context, we suggest that the growing number of laboratories performing RNA Quality Control by microcapillary electrophoresis should be offered the option to report objective RNA quality metrics as part of the 'Minimum Information About a Microarray Experiment' MIAME standards (29) . Through registration of RNA profiles in a public electronic repository, such standardized information should enable and facilitate comparisons of RNA-based bioassays performed across laboratories with RNA samples of similar quality, in much the same way as sequencing traces are compared." }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: The principal mechanism of translation is the accurate decoding of the triplet codon sequences in one reading frame of mRNA. Specific signals built into the mRNA sequences can cause deviations from this rule. Viruses exploit several translational 'recoding' mechanisms, including translational hopping, stop codon readthrough and programmed ribosomal frameshifting (PRF) [reviewed in (1, 2) ], for regulating the amount of proteins produced from their polyproteins. For positive-stranded RNA viruses, À1 PRF is the prevailing recoding mechanism and an essential determinant of the stoichiometry of synthesized viral proteins. Most viral À1 PRF signals are regulating the production of replication-associated proteins. Depending on the virus, the efficiency of À1 PRF can vary between 1 and 40% (3) , and changes in the efficiency can inhibit virus assembly and replication (4) (5) (6) . Therefore, À1 PRF can be regarded as a potential target for antiviral agents (4, 7) . However, the development of efficient antiviral drugs is still hindered, since little is known about the trans-acting factors and the biophysical parameters affecting the À1 PRF efficiencies. Database searches have identified putative frameshift signals from a substantial number of chromosomally encoded eukaryotic mRNAs (8) . Thus, À1 PRF may also have an impact on the complexity of the proteome of several eukaryotic organisms.\nTwo cis-acting signals, a slippery heptamer X XXY YYZ (the incoming reading frame indicated) and a downstream secondary structure, direct the slippage and are therefore essential for this event (9) . À1 PRF takes place after the accommodation step in the slippery sequence by simultaneous slippage of both tRNAs into the overlapping À1 frame XXX YYY (9, 10) . The sequence of the heptamer allows postslippage base-pairing between the non-wobble bases of the tRNAs and the new À1 frame codons of the mRNA. Downstream RNA secondary structures [reviewed in (11) ] force the ribosomes to pause, and place the ribosomal A-and P-sites correctly over the slippery sequence (12) . However, the pausing of the ribosomes is not sufficient for À1 PRF to occur (13) ; in fact, the duration of the halt does not necessarily correlate with the level of the À1 PRF observed (12) . Crystallographic, molecular, biochemical and genetic studies suggest that a pseudoknot restricts the movement of the mRNA during the tRNA accommodation step of elongation by filling the entrance of the ribosomal mRNA tunnel (14) . This restriction can be eased either by unwinding the pseudoknot, which allows the mRNA to move forward, or by a slippage of the mRNA one nucleotide backwards. Chemical agents such as *To whom correspondence should be addressed. Tel: +358 9 19158342 ; Fax: +358 9 19158633; Email: [email protected] ª The Author 2005. Published by Oxford University Press. All rights reserved.\nThe online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact [email protected] antibiotics, certain mutations in the translation apparatus, and in translation elongation factors that change the translation fidelity and kinetics, have been shown to influence À1 PRF efficiency [(10,15) ; reviewed in (16) ].\nThe parameters known to contribute to the efficiency of À1 PRF are the sequence of the slippery heptamer, the downstream secondary structure, and the length and sequence of the spacer between the two cis-acting signals. Up-and downstream sequences such as termination codons in the vicinity of the À1 PRF signals, or even several kilobases away from them, can affect the À1 PRF efficiencies (3, (17) (18) (19) (20) (21) (22) . A specific sequence in the Barley yellow dwarf virus (BYDV) 3 0 untranslated region (UTR), 4 kb downstream from the slippage site, is vital for À1 PRF (6, 19) . A stimulating effect is achieved through the formation of a tertiary structure, where complementary nucleotides from the 3 0 UTR base pair with a single-stranded bulge in the cis-acting stem-loop (6). Human immunodeficiency virus (HIV) was also shown to require a more complex secondary structure instead of a simple stemloop for optimal À1 PRF in vivo (21, 22) . These investigations suggest that À1 PRF studies carried out with minimal frameshift signals may lead to inaccurate estimates of the stoichiometry of synthesized viral protein products during infection.\nCocksfoot mottle virus (CfMV; genus Sobemovirus) infects a few monocotyledonous plant species such as barley, oats and wheat. It has a monopartite, single-stranded, 4082 nt long, positive-sense RNA genome (23, 24) . The polyprotein of CfMV is translated from two overlapping open reading frames (ORFs) 2A and 2B by a À1 PRF mechanism (25) . In this study, we wanted to determine the in vivo À1 PRF efficiency guided by the CfMV U UUA AAC heptamer and the stem-loop structure. In addition to the minimal signal (18) , we decided to test the effect of flanking CfMV sequences for their ability to contribute to À1 PRF. We found that the surrounding viral sequences promoted more efficient À1 PRF than the minimal signal sequence in vivo when measured with the dual reporter vector system developed by Stahl et al. (26) . Therefore, we carried out an expression pattern and deletion analysis to understand the molecular basis of the observed upregulation. In addition, we critically analysed the suitability of the implemented experimental system for this type of a recoding study. An interesting possibility is that the viral proteins produced via À1 PRF could regulate À1 PRF. This hypothesis was tested by co-expressing the CfMV proteins P27 and replicase together with the dual reporter vectors.\nThree regions from the CfMV polyprotein ORFs ( Figure 1A) were cloned into the NheI and BclI sites between the lacZ and the luc ORFs in pAC74 (26) . This dual reporter vector was a generous gift from Dr J. Rousset of the Universite Paris-Sud, France. The inserted sequences 1602-1720 (A region), 1386-2137 (B region) and 1551-1900 (C region), were amplified by PCR using pAB-21 as a template (18) . Primers were used to introduce NheI and BglII sites to the flanking ends of the inserts. Since NheI digestion removed lacZ ORF, it was reintroduced into the plasmids as a final cloning step. The resulting plasmids were named pAC-A, pAC-B and pAC-C.\nCorresponding inframe controls, where one nucleotide was added in front of the slippery heptamer, were generated by PCR-based mutagenesis (Exsite, Stratagene) and named as pAC-Am, pAC-Bm and pAC-Cm, respectively. Deletion plasmids pAC-AB/ABm (1602-2137), pAC-AC/ACm (1602-1900), pAC-BA/BAm (1386-1720) and pAC-CA/ CAm (1551-1720) were also generated. The target sequences are shown in Figure 1B . The base numbering refers to the CfMV genome as in (23) . Transcription was driven from SV40 promoter. Plasmids encoded leucine (LEU2) and b-lactamase (ampicillin resistance) as selective markers. Plasmids were transformed into Saccharomyces cerevisiae H23 [MATa hsp150::URA3 ura3-1 his3-11 15leu2-3 112trp1-1 ade2-1 can100]. Dual reporter plasmid pAC1789 and the inframe control pAC1790 containing a 53 bp sequence from the HIV-1 frameshift region (26) were used as a positive control for monitoring the À1 PRF efficiency.\nTo analyse the proteins produced during À1 PRF, lacZ-A/ Am/B/Bm/C/Cm-Fluc fragments were cloned inframe with the N-terminal 6xhistidine-tag in pYES2/NT KpnI and XhoI sites (Invitrogen). Reporter fusions were amplified by PCR using pAC-A/Am, pAC-B/Bm or pAC-C/Cm as templates. The resulting plasmids were named pYES2/NT-A/ Am, B/Bm and C/Cm. Protein expression was regulated from GAL1 promoter. Two CfMV encoded proteins, P27 (C-terminal end of ORF2A) and replicase (ORF2B), were cloned into pYES2 (Invitrogen). Translation initiation codons were introduced within the oligonucleotides during PCR. The resulting plasmids were named pYES-P27 and pYES-Rep. Control plasmids, which lacked the translation initiation codons were prepared by PCR-based mutagenesis (pYES-P27DAUG and pYES-RepDAUG) and the resulting plasmids were verified by sequencing. Plasmids encoded auxotrophic marker for uracil (URA3).\nAll cloning steps were performed using standard protocols. Plasmids were amplified either in Escherichia coli DH5a or JM110, and purified with Qiagen columns. Inserts were verified by sequencing. Yeast transformations were done using the LiAc method (27) , and transformants were selected on a synthetic minimal defined medium (SC) lacking the corresponding auxotrophic marker(s) encoded by the used plasmid(s). Bacteria (E.coli DH5a) were grown in LB-medium containing ampicillin, whereas yeast cells were grown either in YPD, or in an SC medium. Protein expression from GAL1 promoter was repressed during growth at SC medium containing 2% glucose. Expression was induced by replacing glucose with 2% galactose and 1% raffinose.\nReporter fusions were expressed in S.cerevisiae INVSc1 (his3D1/his3D1, leu2/leu2 trp1-289/trp1-289 ura3-52/ura3-52) (Invitrogen) overnight. Protein fusions were purified in denaturing conditions using Ni-NTA agarose (Qiagen), and analysed in 6% SDS-PAGE gels. Proteins were visualized either by Coomassie staining, or by using antisera raised against the CfMV polyprotein region 1386-1724 encoding CfMV VPg (28) . Protein antibody complexes were visualized with horseradish peroxidase-conjugated anti-rabbit antibodies (Sigma) and ECL chemiluminescent reagents (Amersham).\nPlasmids pYES-P27, pYES-Rep, pYES-P27DAUG, pYES-RepDAUG, or empty pYES2 were co-expressed with pAC-A or with the corresponding pAC-Am inframe control in S.cerevisiae EGY48 strain (MATa, ura3, trp1, his3, 6lexAop-LEU2) (Invitrogen). Transformants were grown overnight in SC-Leu-Ura media in non-inducing conditions, and used to inoculate induction medium. Cells were harvested at late logarithmic phase. Expression of the CfMV proteins was confirmed by western blotting using polyclonal antisera against the CfMV ORF 2a and 2b proteins (28) . Determining the enzymatic activities as described below monitored the effect of CfMV P27 and replicase on À1 PRF.\nFor the in vitro analysis, the lacZ-gene of pAC-A/Am, -B/Bm and -C/Cm vectors was replaced with PCR-amplified Renilla luciferase (Rluc) gene from pRLnull vector (Promega). The resulting pACRF plasmids were used as templates for PCR in order to add T7 promoter upstream of the Rluc gene. These PCR products were used for RNA synthesis with RiboMax kit (Promega). Transcripts were treated with RQ1-DNase (Promega), purified with Qiagen RNeasy columns, and quantified spectrophotometrically. The integrity of the transcripts was checked in agarose gels. In vitro translations were carried CfMV À1 PRF test and control sequences were cloned between the b-galactosidase (LacZ) and firefly luciferase (Luc) genes into a dual reporter vector pAC74 (26) . Inframe control constructs had one extra nucleotide inserted in front of the slippery heptamer, which fused the reporters into the same reading frame. Thus, translation of the inframe control results in the production of a b-galactosidase-CfMV-firefly luciferase fusion. Translation of the test constructs in the incoming 0-frame yields a b-galactosidase-CfMV fusion, whereas À1 PRF produces a b-galactosidase-CfMV-firefly luciferase fusions identical to those produced from the inframe controls. À1 PRF efficiencies were calculated from the firefly luciferase activities after b-galactosidase normalization with the given formula. (B) CfMV polyprotein is encoded by two overlapping ORFs, 2A and 2B via À1 PRF. Sequence regions tested in the dual reporter vectors for their activity to promote À1 PRF are indicated. The numbering refers to the CfMV RNA sequence as published in (23) . out in wheat germ extract (WGE) according to the manufacturer's protocols (Promega). Reactions were incubated in room temperature for 60 min, and stopped on ice prior to enzymatic measurements.\nCell cultures were started from at least three independent clones and grown until the late exponential phase. Cells were collected by centrifugation, frozen in liquid nitrogen and stored at À70 C. Bacterial cells were lysed by sonication (3 · 15 s), and yeasts by vortexing with glass beads (0.5 vol) in +4 C for 30 min. Lysates were cleared by centrifugation, and enzymatic activities were determined immediately. Total protein concentrations were measured by using a Bradford protein assay reagent (Bio-Rad). b-Galactosidase (LacZ) and firefly or Renilla luciferase (LUC or RUC) activities were measured with commercial kits from Promega according to the manufacturer's instructions. LacZ activity was determined as the colour intensity at A414 nm. Luciferase activities were measured as relative light units (RLUs) with luminometer (Biohit or ThermoLabsystems). À1 PRF efficiencies were calculated from normalized firefly luciferase activities with the following formula: [(LUC activity from the test construct)/(LacZ or RUC activity from the test construct)]/[(LUC activity from the inframe control)/(LacZ or RUC activity from the inframe control)] · 100%.\nIn CfMV, the motif for À1 PRF is the slippery heptamer U UUA AAC and a stem-loop structure 7 nt downstream (25) . The efficiency of À1 PRF directed by CfMV cis-acting signals was assayed in vivo using a dual reporter vector system ( Figure 1A ). Since reporters are produced from one single mRNA, factors that affect the stability of the mRNA as well as the rate of translation initiation have a similar influence on the expression of both reporters, and these variations can be monitored as changes in the activity of the upstream reporter. We quantified À1 PRF by comparing the b-galactosidase normalized firefly luciferase activities derived from the test constructs via À1 PRF to those obtained from the inframe controls, in which identical b-galactosidase-CfMV-firefly luciferase fusions are produced without À1 PRF due to the added nucleotide in front of the slippery heptamer (see Figure 1A ). Similar vectors have been shown to detect even small changes in the recoding efficiencies resulting from alterations in the cis-or trans-acting factors (26, (29) (30) (31) (32) .\nThree inserts of varied lengths from the CfMV polyproteinencoding region (ORF2A/2B) were introduced between the two reporters ( Figure 1B) . The A-region, which at 119 bp was the shortest, represented approximately the minimal frameshift signal proven to be functional in vitro (18) . The longest region was the B-insert. At 752 bp, it started from the 5 0 -terminus of the 12 kDa viral genome-linked protein (VPg) gene and continued to the end of ORF2A. This region encodes CfMV protein P27 with an unknown function (28) . Since the minimal requirements for the functional frameshift signal in vivo were not known, an intermediate 349 bp C-sequence was also selected for the analysis. A well-characterized 53 bp frameshift cassette derived from HIV-1 RNA was used as a positive control. Our results regarding the HIV À1 PRF efficiency, 0.7 -0.1% in bacteria, and 4.5 -1.1% in yeast (Figure 2) , are corroborated by those published earlier (26, 33, 34) indicating that our dual reporter system was fully functional.\nb-Galactosidase has been shown to retain its specific activity well, irrespective of the C-terminal fusions (35) . This is important, since the first reporter serves to control the variations among the abundance and translation rates of the studied mRNAs (26, 30) . In addition to changes in specific activities, heterologous fusions can cause alterations in the solubility and conformation, which can expose cryptic protease target sites and reduce the stability of the proteins (36) . Therefore, for a reliable quantification of À1 PRF, it was important to test that equimolar amounts of fusions produced from the corresponding test and control constructs had similar enzymatic activities. Most inframe controls and the analogous test constructs had equal absolute b-galactosidase activities ( Table 1) . Comparable results were obtained, if activities were normalized with total protein concentration (data not shown). These results indicated that the length of the fusion as such did not affect the specific activities. The b-galactosidase activity from pAC-Am inframe control was also comparable to activity obtained from an empty pAC74, where this enzyme has no fusion (data not shown). This further supported the view that the few observed variations in the b-galactosidase activities more likely resulted from the changes in translatability or stability of the transcripts. In addition to pAC-Cm, two inframe controls pAC-Am and pAC-ACm showed $25% lower b-galactosidase activities when compared to the equivalent test constructs ( Table 1 ), indicating that the productivity from these constructs was reduced. Taken together, b-galactosidase seemed to fit well to be used as the first reporter and thus normalization factor in the in vivo experiments of this study.\nCfMV frameshift signals generated significant À1 PRF in yeast. À1 PRF level measured from pAC-A was 3-fold higher than from HIV RNA (Figure 2A and B) . The extent of À1 PRF directed by the minimal region A in yeast, 14.4 -1.9%, was at the same level as that reported for the CfMV minimal frameshift signal in vitro (12.7%) (18) . In contrast to our earlier in vitro observations (18) , the longer CfMV sequences upregulated À1 PRF in vivo. In yeast, the level of upregulation was 2-fold for pAC-B, the À1 PRF frequency being 26.3%, and almost 5-fold for pAC-C resulting in efficiency close to 70% (Figure 2A) , which is an extremely high value, if compared to the other values published earlier (3). CfMV frameshift signals directed À1 PRF at a lower level in bacteria than in yeast ( Figure 2B ). The extent of À1 PRF directed by region A in bacteria was 2.4 -0.7%. As in yeast, the longest B region stimulated À1 PRF 2-fold in bacteria when compared with pAC-A. However, region C did not further improve À1 PRF, but programmed À1 PRF to similar levels as pAC-B, the percentages being 4.7 -1.6% for pAC-C and 5.5 -1.5% for pAC-B.\nTo identify the sequence(s) responsible for the enhancement of À1 PRF in vivo, a deletion analysis was carried out. The 5 0 -or the 3 0 -sequences flanking the A-region were deleted from pAC-B/Bm or pAC-C/Cm as indicated in Figure 1B , which generated vectors pAC-AB/ABm, pAC-BA/BAm, pAC-AC/ ACm and pAC-CA/CAm. À1 PRF frequencies were determined in yeast ( Figure 2C ). Increased À1 PRF was observed in all deletion constructs in comparison to the À1 PRF directed by the A region. The BA and AB regions promoted À1 PRF as efficiently as the B region, whereas regions CA and AC were better than region A, but not as good as region B. In other words, the presence of nucleotides 1386-1720, or downstream nucleotides 1602-2137, was sufficient to increase À1 PRF to the level directed by the region B. Thus, the deletion analysis did not identify single specific sequence region as being responsible for the increased À1 PRF frequencies.\nThe expression pattern of the test and control constructs was analysed to understand the basis for the observed upregulation in yeast. Cassettes containing the reporters and the studied intercistronic sequences were expressed and purified as N-terminal histidine fusions. This allowed us to capture all the N-terminally intact products. The affinity-purified proteins were separated in SDS-PAGE gels, and visualized either by Coomassie staining (data not shown), or by western blotting with the CfMV-specific anti-VPg antibodies. The expected b-galactosidase-CfMV fusions terminating at the end of the 0-frame in the test constructs were detected. Also, the longer transframe b-galactosidase-CfMV-firefly luciferase fusion proteins were present in both the test and the inframe constructs ( Figure 3) . Comparison of the Coomassie-stained gels with the western blots revealed that the antisera recognized the products terminating at the CfMV-encoding regions better than the transframe products. Furthermore, the small size of the CfMV-specific region in the pYES2/NT-Am decreased the binding of the antibodies to these inframe control fusions. Thus, this data were not suitable for quantitative analysis of À1 PRF. Interestingly, an additional protein, which reacted with CfMV-specific antisera, was co-purified from the cells expressing pYES2/NT-Bm and pYES2/NT-Cm inframe controls ( Figure 3 ). The size of these fusions suggested that translation had terminated approximately at the site for À1 PRF signals. If such putative termination products were also present in cells expressing the test constructs, the correctly terminated 0-frame products in the western blots masked these products.\nA closer look at the absolute b-galactosidase and firefly luciferase activities revealed that firefly luciferase expression from pAC-Cm was clearly reduced (data not shown). In fact, expression from the inframe control was comparable to the corresponding pAC-C test construct. This was also obvious when the firefly luciferase activities were normalized with the total protein amount. After setting the activity from pAC-Am to a relative value of one, the corresponding values from pAC-Bm and pAC-Cm were 0.80 and 0.28. Although the b-galactosidase measurements (Table 1) suggested that the overall translatability of the pAC-Cm mRNA was also reduced to some extent, it explained the decrease in firefly luciferase expression only partially. In the light of these findings, the extremely high À1 PRF frequency estimate calculated for the C-region could be explained with more frequent translation termination at the frameshift signals of the pAC-Cm mRNA, which reduced firefly luciferase activity in relation to b-galactosidase.\nÀ1 PRF was also assayed in vitro in WGE. Although LacZ-encoding gene is suitable for the in vivo studies, it is an unsuitable first reporter for the in vitro determination of À1 PRF efficiencies due to its big size (30) . In good agreement with this, we observed several unexpected products in the in vitro translations programmed with LacZ-CfMV-luc mRNAs (data not shown). Renilla luciferase has been shown to retain its specific activity irrespective of the C-terminal fusions (30) . Therefore, we decided to use Rluc-CfMV-luc transcripts to determine the À1 PRF efficiencies in the cellfree system. First, we verified the suitability of Renilla luciferase for the intended in vitro experiments as described in (30) . Transcripts encoding monocistronic Renilla luciferase and Renilla luciferase fused to firefly luciferase (Rluc-Am/ Cm-luc) were mixed in different ratios and used to program the in vitro translations. Increasing concentrations of transcripts encoding the Rluc-Am-luc fusion resulted in linearly growing firefly luciferase activities. At the same time Renilla luciferase activities remained constant, which showed that its enzymatic activity was not sensitive to the C-terminal fusions ( Figure 4A ). Similar results were obtained with Rluc-Cm-luc mRNA (data not shown). À1 PRF efficiencies were then determined with transcripts that contained CfMV regions A, B and C, and their corresponding inframe controls. In all cases, slightly higher À1 PRF frequencies were obtained than in vivo. In nice correlation with the in vivo results, enhanced À1 PRF was observed with the region B, although the effect was weaker than in vivo. In this context, region C did not differ from the minimal region A in its capacity to program À1 PRF ( Figure 4B) .\nThe ratio between the CfMV P27 and replicase is regulated by À1 PRF during CfMV infection (28) . We studied whether these proteins could regulate the À1 PRF process. P27, replicase, or an empty expression vector was co-expressed in yeast together with the dual reporter vectors containing the minimal À1 PRF test and inframe control regions as intergenic sequences (pAC-A and -Am). P27 and replicase expression was verified by a western blot analysis ( Figure 5) . A faint band having nearly the same mobility as the replicase was detected in cells grown under repressing conditions. However, due to the small size difference, this protein was not regarded as replicase.\nEnzymatic activities were measured from yeast lysates prepared from induced cultures. Measurements showed comparable levels of b-galactosidase in all the samples, indicating that P27 or replicase expression did not affect the stability of the dual reporter mRNA or the translatability of the first reporter ( Table 2 ). The effect of P27 or replicase expression was monitored by comparing the reporter activity ratios to those measured from cells harbouring the empty expression plasmids (Table 2) . Co-expression of CfMV replicase did not affect the normalized firefly luciferase expression (LUC/LacZ) from the inframe control, whereas slightly increased luciferase expression from the test construct was observed. In contrast, P27 expression reduced firefly luciferase expression both from the test and the inframe constructs. The effect was stronger in the presence of inframe control as normalized firefly luciferase levels reached only 54% of expression measured from the empty vector control.\nTo verify that the observed differences in firefly luciferase production depended on the studied CfMV proteins, we co-expressed the dual reporter vectors with plasmids having the first translation initiation codons of P27 and replicase deleted (pYES-P27DAUG and pYES-RepDAUG). Western blot analysis with antisera against ORF2A or 2B did not detect any proteins produced from these vectors (data not shown). The obtained LUC/LacZ ratios were compared to those measured from cells expressing the CfMV proteins (pYES-P27 or pYES-Rep). LUC/LacZ ratios measured from cells expressing replicase were slightly lower than the ratios calculated from cells harbouring pYES-RepDAUG plasmids, being $90% when co-expressed with pAC-A and $84% when co-expressed with pAC-Am. In the presence of P27, LUC/ LacZ ratio of pAC-A reached $81% of expression measured from cells transformed with pYES-P27DAUG. Again the effect of P27 expression was more evident with pAC-Am inframe control as P27 expression reduced LUC/LacZ ratio to half ($48%) when compared to the corresponding value measured from the cells harbouring pYES-P27DAUG. This verified that CfMV P27 was able to reduce the downstream reporter expression from dual reporter mRNAs. Since CfMV P27 had a proportionally stronger effect to firefly luciferase production from the inframe control mRNAs in comparison to the test mRNAs (Table 2) , the calculated À1 PRF efficiency increased from 14.7 to 22.4%.\nSince À1 PRF studies are affected by a huge number of different parameters, it is not an easy task to determine the real ratio between the proteins produced via this mechanism in vivo. However, in viral systems, the efficiency of À1 PRF is an essential determinant of the stoichiometry of synthesized viral protein products, which must be rigidly maintained for efficient propagation of the virus. For example, frameshifting in retroviruses determines the ratio of structural (Gag) to enzymatic (Gag-Pol) proteins, and plays a critical role in viral particle assembly (5) . In this study, the capacity of CfMV frameshift signals to direct efficient À1 PRF was analysed in vivo by using dual reporter vectors. The length of the CfMV sequence clearly affected the actual efficiency percent in vivo. The PRF efficiency was elevated when longer viral sequences were directing the À1 PRF, but the deletion analysis did not identify any specific region as being solely responsible for the enhancement. Up-and downstream sequences nearby or far away from the cis-acting signals have been reported to enhance À1 PRF in other viruses, such as HIV, human T-cell leukaemia virus and BYDV (6, 19, 20) . Also out-of-frame stop codons have been shown to influence À1 PRF frequency in vitro in retroviruses (17) and in CfMV (18) . A study on the spacer sequences located between the cis-acting signals showed that high slippage frequencies were obtained when the first three nucleotides were G/U, G/A and G/A, the first two being the most important (37) . In CfMV, the spacer starts with UAC, which partially explains the capacity of the CfMV sequence to promote high slippage levels. In this study, the observed enhancement of À1 PRF was, however, caused by sequences that were not in the immediate vicinity of the Figure 5 . Co-expression of CfMV P27 or replicase simultaneously with the minimal frameshift signal construct pAC-A or the corresponding inframe control pAC-Am in yeast. Yeast total protein samples were separated in 12% SDS-PAGE gels, transferred onto PVDF membranes, and immunocomplexes detected by ECL chemiluminescent system. CfMV P27 expression was verified by western blotting with antisera raised against ORF2A (A), and CfMV replicase expression was detected with antisera raised against ORF2B (B). Abbreviations: À, repressed; +, induced; C1, pMAL-VPg $53 kDa; and C2, baculovirus expressed CfMV replicase. slippery sequence thus indicating that CfMV sequences further away also have an influence on the level of frameshifting in vivo. We conclude that the most reliable estimates for À1 PRF and consequently for the amount of replicase versus the 0-frame translation product P27 can be obtained only by using the full-length viral sequences. In reality, such a study would however be hampered by the non-quantitative nature of the western blot analysis, the presence of different polyprotein processing intermediates, and the differences in the stabilities of the end products in the infected cells.\nThe overall competence of CfMV signals to direct À1 PRF was high, when compared to related plant viruses, such as Potato leaf roll virus and BYDV. À1 PRF values of $1% have been reported for these viruses when measured with reporter-based assays (6, 38) . We can hypothesize that one reason for the high efficiency is the slippery tRNA Asn encoding the AAC triplet of the CfMV heptamer. Equal U UUA AAC slippery heptamer has been measured to induce 20-40% of À1 PRF in a diversity of animal viruses [(39); reviewed in (3)]. The low fitness of CfMV À1 PRF signals in bacteria is in agreement with the poor functioning of the eukaryotic slippery heptamers of the order X XXA AAC in prokaryotes (40) (41) (42) . IBV RNA, having an identical shifty heptamer, has been shown to direct À1 PRF at similar 2-3% level in bacteria (41) . A recent study reported that XXXAAAC heptamers dictate À1 PRF to occur via the slippage of two adjacent tRNAs placed over the heptamer, irrespective of whether the host is an eukaryote or a prokaryote (42) . Therefore, the inability of prokaryotic translation systems to direct efficient À1 PRF from this heptamer is not an inherited property of prokaryotic tRNA Asn , but results from differences in the ribosomes (42) .\nPaused ribosomes can pass the À1 PRF site by À1 frameshifting, resumption of 0-frame translation, or termination (43) . Transient polypeptide intermediates that result from the pausing of ribosomes in the slippery sequences have been observed during IBV and S.cerevisiae L-A virus polyprotein synthesis (12, 13, 43, 44) . A pseudoknot structure formed by IBV mRNA causes a translational pause at fixed position upstream the secondary structure regardless of whether the slippery heptamer is present or absent (12) . Based on the findings of this study, we propose that also here a certain percent of ribosomes stalled at the secondary structure of the frameshift site in our inframe control and test mRNAs in yeast, and this led to the prematurely terminated products observed with the inframe control constructs pAC-Bm and -Cm. Although not unambiguously proven by this study, high frequency of termination of translation especially at the frameshift site of the pAC-Cm mRNA would nicely explain the extremely high calculated À1 PRF efficiency.\nFactors that change the translation fidelity and kinetics have been shown to influence À1 PRF efficiency [ (10, 15) ; reviewed in (16) ]. Autoregulation of +1 frameshifting by mammalian ornithine decarboxylase antizyme has been reported (45) . This mechanism allows modulation of frameshifting frequency according to the cellular concentration of polyamines. One could speculate that such a regulation mechanism could also be useful to adjust the amounts of the replicationassociated proteins to match the requirements of different phases in viral replication cycle. This hypothesis was studied by expressing CfMV proteins P27 and replicase together with pAC-A and pAC-Am in yeast cells. Since b-galactosidase production remained constant regardless of the presence or absence of CfMV proteins, they did not interfere with translation initiation from pAC-A/Am mRNAs per se. However, P27 expression caused a reduction in the firefly luciferase production especially from the inframe control, whereas replicase production only slightly increased the firefly luciferase production from pAC-A, but not from pAC-Am. Since replicase expression had only a faint effect on the normalized firefly luciferase production via À1 PRF, our conclusion is that CfMV replicase had no pronounced effect on translation at the frameshift site. Co-expression of the non-translatable form of P27 with the dual reporter vectors verified that P27 truly affected firefly luciferase expression on the protein level. Therefore, we propose that CfMV protein P27 may influence translation at the frameshift site. If CfMV P27 indeed interferes with viral protein synthesis during CfMV infection, the mechanism, its specificity and the possible biological role needs to be elucidated in the future." }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: Public health is a small component of the health system, both in terms of budgetary allocation at either state or national level and in terms of the number of practitioners. It incorporates a myriad of activities; legislation and regulation for health protection, preventive services directed at specific diseases and populations, and health promotion programs geared towards particular risk factors and vulnerable groups in the community. As such, it looks like a disparate collection of programs and investments.\nIn Australia, there is also confusion about the very terminology of 'public health'. Despite its extensive history and global understanding, in Australia the term is used variously; to refer to publicly funded health services, and interventions (regardless of the funding source) which are aimed at primary prevention and the promotion and protection of the public health ('rats and drains'). This has led to an increasing number of jurisdictions adopting the label 'population health'.\nRenovation of the public health system has been on the international agenda for some years. In the US, the Institute of Medicine released reports during 2003 about the public health workforce required for 21 st century challenges [1], as well as re-visited and updated its landmark report, The Future of Public Health in the 21st Century [2] . In the UK, following the path-breaking review of the NHS by Derek Wanless [3] the Treasury commissioned him, in 2003, to undertake a review of whole-of-government effort in public health. Arising in part from the challenges that confronted Canada during the outbreak of sudden acute respiratory syndrome (SARS) in 2003, a new public health agency, at arms length from government, is being created.\nPublic health in Australia, meanwhile, remained fragmented -by programs, across jurisdictions (particularly the states and territories) -and without a systematic approach to funding, organisation, or conceptualisation. In 2003/04, the gap between rhetoric and funding continued to be noticeable, along with the tension between framing priorities for popular appeal versus the technical language of the evidence base.\nThis article will examine some of the indicative developments of public health in Australia in 2003/04. The key developments are identified, and a number of them are selected for in-depth analysis. In this article, we use the traditional meaning of the term 'public health' and focus on activities which are usually designed to promote and protect the health of the population. The drivers for these developments, their short term implications and some signposts for the future are suggested.\nWhile early global anxiety over SARS occupied headlines between February and May, the more persistent popular headline in 2003 focused on obesity. Summits were held in NSW and Victoria, while the National Obesity Taskforce was convened under the auspice of the Australian Health Ministers Council (AHMC).\nWhen Kay Patterson was the Federal Health Minister, she declared that prevention was the fourth pillar of Medicare and she wanted to be 'Minister for Prevention'. Indeed, the 2003/04 federal budget, although limited, contained a bundle of initiatives entitled \"Prevention on the Health Agenda\". In particular, a number of immunisation and health promotion programs were included.\nSignificant amongst the funding initiatives for public health announced in 2003/04 was government support for the meningococcal vaccine. Although this was the culmination of many months of careful planning, a perception existed that this only occurred after considerable public interest in and anxiety about deaths from outbreaks of this disease.\nFurther changes to the recommended schedule in 2003 were made by the Australian Technical Advisory Group on Immunisation (ATAGI), in particular the inclusion of pneumococcal and varicella vaccines; however, these did not result in similar prescribed vaccine programs or in similar funding. These three developments are reviewed in greater detail in the next section.\nThe National Public Health Partnership (NPHP) and the AHMC adopted the influenza pandemic plan in October 2003, and with the advent of the newly-identified disease SARS, as well as outbreaks of meningococcal disease, management and prevention of communicable diseases was prominent. Following on from the significant funding boost for bioterrorism preparedness in 2002/03, public health preparedness became a more generic theme.\nThe arrival of SARS occupied the national popular and political imagination as well as tested the infrastructure capacity of public health. Australia fared well during the outbreak. Apart from escaping with only six Australian cases, it provided an opportunity to establish a coordinated approach between the Commonwealth and the states/territories and also contributed to the global epidemiological investigation and prevention effort. SARS also prompted amendments to the Quarantine Act [4] .\nWhile the recall following the Pan Pharmaceutical crisis put the Therapeutic Goods Administration (TGA) under the spotlight, it also managed to conclude negotiations that had been in train for several years on a Trans-Tasman regulatory regime and authority. Also on the regulatory front, the Australian New Zealand Food Regulation Ministerial Council endorsed a nutrition, health and related claims policy guidelines and established a review of genetically modified (GM) labeling of foods [5] . All these developments pointed to the global nature of public health, and the intersection between public health activities and the economy.\nPolicy development in public health has never been confined to a set of health programs, and in 2003/04, the lead was often taken from outside the health sector. Most significant was the adoption of the National Agenda for Early Childhood [6] , pushed by public health advocates for child health since the mid 1990s. The National Public Health Partnership responded by coordinating a scoping of child health strategies across Australia. Elsewhere in Government, \"Promoting and Maintaining Good Health\" was adopted as one of the National Research Priorities [7] . Healthy ageing also emerged as a policy theme in Ageing Research.\nPublic health workforce development was pursued outside the mainstream education and training arrangements for public health in universities. The Community Services and Health Training Board commissioned a consultative process to develop population health competencies for the Vocational Education and Training (VET) sector [8] . New population health qualifications and competencies were proposed for incorporation into the Health Training Package -including certificates in population health and in environmental health, and diplomas in population health and in indigenous environmental health.\nThe release in 2003 of the report \"Returns on Investments in Public Health: an epidemiological and economic analysis\" [9] (often referred to as the Abelson report), may have a significant impact in subsequent years. Commissioned several years earlier by the Population Health Division of the Department of Health and Ageing (DoHA), the report experienced a relatively low profile until Derek Wanless visited from the UK. Having chaired a review that contributed to a significant budgetary increase for the NHS, Wanless had been commissioned by the British Treasury to examine prevention across government. In September 2003, at a meeting in Canberra with senior officials across key agencies, Wanless marveled at the value of the Abelson report, described in more detail below.\nAlthough 2004 was an election year, public health policy was neither visible during the campaign or in policy development more generally. The Federal Government's initiative to wind up the National Occupational Health and Safety Commission received little publicity and comment, even though it indicated the Commonwealth's increasing tendency to pursue its own pathway, separate from states and territories, and to bring the functions of statutory bodies into departments.\nJurisdictional and annual reports show that across the states and territories, there were multiple plans, draft guidelines, meetings, episodic training and programs across a broad range of areas. Some health issues are being taken up across jurisdictions -particularly tobacco control, sexually transmitted infections, Aboriginal health, and vaccination. Innovative activities were reported in some jurisdictions, such as a new Health Impact Assessment Branch and a new public health training program in Western Australia. There was, however, no apparent consistency in health priorities across the nation, and an apparent divergence in the interests of the states/territories and the federal government.\nWhile the \"prevention and management of overweight and obesity\" agenda may have appeared to many observers as a new issue in 2003, its arrival was preceded by several years of intensive work. The NHMRC had released Acting on Australia's Weight: Strategic plan for the prevention of overweight and obesity in 1997 [10] , the same year the ABS published the findings from the 1995 National Nutrition Survey, revealing that 45% of men and 29% of women in Australia were overweight, with an additional 18% of men and women classified as obese [11] . Furthermore, overweight and obesity were more common in lower socio-economic groups, in rural populations, in some immigrant groups, and in Aboriginal and Torres Strait Islander (ATSI) peoples.\nDespite longstanding national cooperation on nutrition (since the days of the National Better Health Program in the late 1980s), and even more recent national cooperation on physical activity, public and political imagination was not captured until the same issues were recast as 'obesity', with a focus in particular on childhood obesity. Following from the NSW Childhood Obesity Summit in late 2002, the Australian Health Ministers agreed that a national approach was required and established a National Obesity Taskforce [12] .\nIn 2003, NSW Health released it's response to the Summit recommendations and supported the vast majority of the 145 resolutions [13] . The Victorian Department of Human Services also held a summit [14] , while Healthy Weight 2008 -Australia's Future was released by the Commonwealth [15] . The NHMRC joined in with release in late 2003 of clinical practice guidelines for general practitioners and other health professionals [16] .\nWhile the specifics vary, the major themes and strategies are captured in Healthy Weight 2008. These are summarised in the Table 1. The Commonwealth strategy is, however, relatively weak on intersectoral policy and regulatory measures. As an illustrative example of the contrast at the state level, implementation in NSW now ranges from school physical activity and nutrition survey, to a school canteen strategy, to negotiating with Commercial Television Australia about their code of practice on advertising in peak children's viewing hours. The Commonwealth apparently chose not to consider how it might exercise its relevant taxation or legislative powers, despite the history of health promotion pointing to the importance of public policy measures beyond the health system.\nAn examination of the manner in which the obesity issue was framed, and the details contained in the national strategy, raises a number of issues and questions:\n-Why was framing the issues as 'obesity' more successful than the focus on 'nutrition' and 'physical activity'? Why did 'obesity' gain traction while the other terms did not?\n-Why did the Commonwealth opt for the softer programmatic approach, rather than tackle obesity with stronger public policy measures (such as taxation and regulation), and demonstrate its national leadership capacity? -Was the absence of stronger public policy measures because 'obesity' is regarded as largely a health issue, rather than a whole-of-government issue? Or was the Government waiting to see if the US opposed the WHO Global Strategy on account of the strength of the industry lobby?\n-After a number of years of public concern about eating disorders and whether they arise in part because of promotion of certain types of body image, was the 'obesity' label a backward step for mental health and a return to traditional images of beauty?\n-Is there a risk that people, including children, who are labeled as 'overweight and obese' will be stigmatised? To what extent have the voices of affected communities been incorporated into the development of national strategies, if at all? -Given the correlation between obesity and socioeconomic disadvantage, how would the proposed strategy not exacerbate those inequalities? -Were children targeted because they are a \"captive audience\" and therefore easy targets or did the evidence suggest the best return on investment (in terms of health gain and managing demand on the health care system) would come from a focus on children?\n-Was the move to appeal to a populist agenda, while simultaneously progressing the longer-term agenda of tackling health inequalities through multi-sectoral partnerships, a triumph for public health advocates?\nThese complex threads are interwoven. For the moment, the publicly enunciated agenda represents a confluence of a number of rationales.\nDuring 2003-4 three new vaccines were added to the schedule of recommended vaccines for Australians (an additional change to the schedule, recommending that polio immunisation be changed from oral to injected (IPD) vaccine, will not be discussed here). These vaccines protect against serogroup C meningococcal disease, some strains of Pneumococcal disease, and chicken pox [17] . For the first time, not all of these recommended vaccines will be funded by Government.\nPrior to the introduction of these vaccines, the quality of information about the epidemiology and burden of disease caused by these three infections was extremely variable. Meningococcal disease has been notifiable for many years, and in Australia almost all is caused by serogroups B and C. Whilst serogroup B predominantly occurs in young children, a new strain of serogroup C [18] was causing increasing anxiety amongst public health professionals, microbiologists, staff of accident and emergency departments, intensive care units and of course the public and media.\nThe cause of anxiety amongst health professionals was based on the fact that this new strain carried a high fatality rate with severe after-effects in a high proportion of survivors. The attack rate, although still small, was increasing exponentially each year and reaching an important trigger point, and the majority of cases were now healthy teenagers and young adults. Although an initial accelerated catch-up programme was introduced for teenagers (the major risk group), the new conjugated vaccine was also introduced to the childhood schedule at age one, as from that age, only one dose (at a cost of $30-$60) was considered necessary for full protection from serogroup C disease.\nPneumococcal disease became notifiable in 2001, however, with such a short surveillance history, not much is certain locally, epidemiologically speaking, about risk groups and effects (although there is no reason to suppose that it has a different epidemiological pattern from other developed countries). Pneumococcal disease is thought to occur at least four times as often as meningococcal disease, is known to carry major sequelae and has a high case fatality rate. For some time it has been known to be even more common amongst the indigenous Australian population with attack rates of up to 1 in 500 each year, knowledge which underpinned the 1999 decision to target Aboriginal people for free vaccination as soon as the new vaccines became available. Unfortunately at about $120 per dose, conjugate pneumococcal vaccine is very expensive and, for the protection of the very young children who bear the brunt of this disease, it is licensed only to be given as a three dose course, making provision of this vaccine to all Australian children prohibitively expensive.\nVaricella, predominantly a childhood disease, is caused by a Herpes virus known as herpes virus 3 or varicella-zoster virus or VZV. It is not notifiable in Australia; therefore no epidemiological population data are available. A reliable varicella vaccine has been available since the mid 1990s in the USA and is part of American routine immunisation schedule. This vaccine became available in Australia in 2000, at a cost of about $75-$90 per dose, with two doses being required for full protection.\nIn 2003 the Commonwealth provided its periodic update on the Australian Standard Vaccination Schedule, the list of vaccines it provides as appropriate at no cost to all Australians [19] . For the first time it differed from the National Immunisation Program recommendations in that besides meningococcal serogroup C conjugate vaccines, pneumococcal vaccine, varicella vaccine and also inactivated polio (injected) vaccine were also recommended: however, funding was only secured for meningococcal conjugate vaccines, with a continuation of the provision of pneumococcal vaccines for indigenous children. As a result, although recommended, pneumococcal and varicella vaccines were not funded and parents would have to decide whether or not to pay for them.\nThese funding decisions had important implications. Vaccines protect most of their recipients from unpleasant and sometimes life-threatening disease. One view, subscribed to in the UK, is that ethically, children should not be denied access because of their parents' inability to pay. These vaccines have been the subject of several cost-benefit studies, with generally favourable to extremely favourable pro-vaccination results. Table 2 summarises the various models for framing policy.\nThe policy of funding meningococcal serogroup C vaccine was built on a sustained program of epidemiological evidence, ethical decision-making and public support (and was arguably honed by public pressure). Pneumococcal disease and varicella vaccination programs however, were neither supported by good local epidemiological evidence nor respectable levels of public awareness about these diseases. There had not been a similar program of sustained policy building to support or drive a decision to fund these vaccines. As a funding policy, this was noteworthy in that it marked a departure from previous policies where all recommended vaccines were fully funded by governments. National vaccination policy is designed to advise vaccination policy makers and practitioners of the most up-to-date thinking about optimal vaccination schedules for Australian children, and is not therefore proscriptive, unlike the United Kingdom (UK). Changing or adding vaccines to the recommended schedule is therefore an advisory matter, and the question of funding the vaccination program is decided separately.\nCost benefit studies indicate pneumococcal polysaccharide and conjugate vaccines can be cost-effective although vaccine costs clearly affect ratios of cost to benefit greatly [20, 21] . Varicella vaccine is more contentious, because this disease is more severe in older cases, and it is possible that one result of a vaccination program could be an increase in older cases (and therefore severe disease). Whilst the vaccine undoubtedly works, there is no consensus about precisely who should be vaccinated for maximum population health as well as cost benefit, and again potential financial savings are highly dependant upon vaccine costs [22, 23] .\nThe costs of preventive vaccine programs and curative medicine are funded from different sources. Vaccines are currently funded by the Commonwealth and subsidised through the states according to local vaccination policies, whilst the costs of curing cases of these diseases is broadly funded through the Medicare and private health insurance systems. Savings to Medicare and health insurance funds, as a result of successful vaccination programs, are not automatically transferred to the Commonwealth to fund the vaccine programs. Savings -or costs -in one area are of little interest or importance to other program areas.\nIn 2004 the Government revised this funding policy, providing funding for conjugate pneumococcal vaccines population immunisation program for all children under seven years of age (as well as specific people in other risk groups) to commence in January 2005. The Australian Technical Advisory Group on Immunisation (ATAGI) completed Ministerial reports on both varicella and polio (injected as well as oral) vaccination late in 2004, and it is possible that programs for these vaccines will also be funded in the future.\nThe 2002/2003 Federal Budget papers stated that \"the Government is committed to making disease prevention and health promotion a fundamental pillar of the health system\": however, this was not evident in the subsequent 2003/2004 budget. The Government's Focus on Prevention Package in 2002/03 aimed to incorporate disease prevention into the core business of the primary health care system and was reflective of how the public health agenda was evolving at the national level [24] . The package was comprised largely of a range of measures directed at specific diseases, plus a bundle of initiatives for general practitioners, also referred to as the \"primary health care system\".\nAmongst health conditions affecting Australians, breast cancer received the most attention, with the National Breast Cancer Centre being funded to develop a partnership approach to the review and dissemination of new information, along with information, support and management initiatives for rural women diagnosed with breast cancer. Hepatitis C also received some attention, with funding for national education and prevention projects. Financial support was offered for the SARS efforts that had been undertaken by states and territories, in particular for providing medical personnel at international airports. A clear process for assessing priorities under the broad banded National Public Health Program was also flagged.\nFor purposes of the budget, primary health care was defined as general practitioners, and the measures funded included:\n• \"Lifestyle prescriptions\" to help GPs \"raise community awareness and understanding of benefits of preventive health\";\n• Collaborative approach to learning, training education and support systems;\n• Coordinated care plans for people with chronic or terminal conditions; and\n• Involvement in multidisciplinary case conferencing.\nThe budget did not adopt a comprehensive approach to the primary health care system, perhaps because many community health services, which represent the other important arm for delivery of public health services, are the responsibility of states. The timetable for renewing Public Health Outcome Funding Agreements (PHOFAs) between the Commonwealth and states and territories in 2004 raised in the minds of some stakeholders, the possibility that the Commonwealth might adopt a more comprehensive and strategic approach, linking public health and primary health care funding streams.\nJudging by the actual quantum of funds made available in the 2003/2004 budget, it would seem that most elements from the package did not actually receive additional funding, as shown in Table 3 . Indeed, many of the GP initiatives, previously cast as improving primary health care, were subsequently packaged as 'prevention'.\nThe combination of these measures reflected a tight fiscal climate, with little growth in the overall health budget, as well as that of other portfolios. It was also a package that demonstrated relatively limited imagination, with support for established issues (such as breast cancer) and repackaging general practice measures that were already in train. With Medicare spending \"uncapped\" (and targeted public health programs \"capped\"), attaining more prevention dollars through the GP sector may appear to be one of the few ways to 'grow' dollars for prevention. Although this could be considered to be consistent with the Ottawa Charter of \"reorienting health services\", many GPs are not trained in a population-based approach to practice, and simply providing new for payments to all represents an undifferentiated, uncoordinated and untargeted approach to prevention. If there is limited support to GPs, and little monitoring, then these measures are unlikely to translate into improved health outcomes. Funding for the Tough on Drugs strategy was announced outside the Focus on Prevention package; perhaps due to Source: [28] the fact that the Tough on Drugs was the responsibility of the Parliamentary Secretary therefore requiring a separate communications strategy, or because the Prime Minister has a strong personal interest in the illicit drug strategy. The range of measures funded (which included introduction of retractable needle and syringe technology, addressing problems related to increased availability and use of psycho stimulants, establishing a research fund, supporting alcohol and drug workforce development needs, promoting access to drug treatment in rural areas, and tackling problems faced by drug users with concurrent mental health problems) certainly suggested more serious government interest and commitment to illicit drugs. \nDuring the course of the Howard Government, there has been a gradual process of re-casting the \"landscape\" of interest groups and policy constituencies. Strong support for breast cancer and zero-tolerance on illicit drugs contrasts sharply with the delays experienced in renewal of the National HIV/Hepatitis C Strategy. The new prominence given to meningococcal vaccine, child health and obesity creates space for other interest groups: even if the re-framing was shaped by nutrition and physical activity lobbies, other clinical interests have been brought into the picture. These developments illustrate how 'political' considerations are important in determining 'public health policy'.\nIt was interesting however, to observe the interest in prevention from outside the health portfolio, particularly from Treasury. This was motivated in part by the Intergenerational Report and concerns about both the sustainability of Medicare as well as the social and economic cost burden arising from an ageing society. This helped to ensure interest in the Abelson Report [9] .\nFew countries have conducted research on return of investment from prevention efforts. Australia was praised by Derek Wanless at a high-level consultation for completing such an analysis, during his visit to Canberra while conducting a review for the UK Treasury, \"Securing Good Health for the Whole Population\" [26] . His final report pointed to Australia and Netherlands as two countries that were increasingly using economic evaluation in public health programs. It will be interesting to see if public health policy analysts and Treasury officials draw on this report in future years. In the future it will be interesting to see if the focus on high-visibility programs can demonstrate short-term economic returns.\nGiven 2004 was an election year, the \"political economy\" of prevention programs could arguably have become a focus of future public health policy, with the 2003/4 agenda providing the Government with the opportunity to gauge public reaction to this new positioning and design their election campaign appropriately. This was, however, not the case. The American emphasis on 'preparedness' appears not to resonate with the Australian public in the same way.\nFrom the perspective of public health policy advocates, some lessons that can be drawn from 2003/04 are:\n• Government's response to public health proposals are shaped by its understanding of the popular interest and desire to communicate directly with the general public;\n• Longer term public health issues which have struggled to gain support can be progressed if they are cleverly shaped to fit the Government's \"formula\";\n• Develop and nurture new advocates, particularly in seeking to engage with the broader health system; and\n• Work with the media as partners rather than adversaries\nThese lessons need to be learned well and quickly, to assist with moving the forum for public health policy debate more into the public domain; beyond an essentially \"in house\" discourse between politicians, researchers and public health advocates. If a more engaged and informed community takes up a public health issue, government will be more likely to respond. " }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: As of 2005, the world is confronted by 338 generic infectious diseases, scattered in a complex fashion across over 220 countries and regions. Each new day confronts health care workers with unexpected outbreaks, epidemics and heretofore unknown pathogens. Over 2,000 named bacteria, viruses, fungi and parasites are known to cause human disease; and are confronted by 328 anti-infective agents and vaccines. Experts working in Health Geographics share an obvious and immediate need for comprehensive and timely data on the status of infection around the globe. A recent outline of GIDEON addressed uses for the Infectious Diseases clinician [1] . This review will focus on the Global Health aspect of the program.\nIn 1990, we initiated a project to design computer systems to follow all diseases, outbreaks, pathogens and drugs. The initial DOS-based program was written in Paradox for floppy disks, later evolving through a compact disk-based program in Windows. A commercial web-based program was eventually released under the name, GIDEON (Global Infectious Diseases and Epidemiology ON-line, Gideon Informatics, Inc, Los Angeles, California) at http:/ /www.GideonOnline.com. The current version is available on CD (updated every three months) or web subscription (updated every week).\nThe program consists of four modules: Diagnosis, Epidemiology, Therapy and Microbiology. Program modules of peripheral interest in Health Geographics (Therapy and Microbiology) will be discussed only briefly.\nThe Diagnosis module is designed to generate a ranked differential diagnosis based on signs, symptoms, laboratory tests, incubation period, nature of exposure and country of disease origin. Figure 1 depicts the data entry screen for a patient suffering from fever and joint pain following a trip to Indonesia. The lower 'Personal notes' box is used to record additional case data, and can be written in the user's own language. The differential diagnosis list for this case (figure 2) indicates that this patient may be suffering from Chikungunya. The appearance of many diseases on the list indicates that the user failed to enter all positive, and negative findings. For example, the fact that cough was absent would have reduced the likelihood of the second disease listed (Mycoplasma infection) and increased the statistical probability of Chikungunya.\nAt this point, the user can generate a hard copy or e-mail report, access a table comparing the clinical features of the diseases listed, or examine the ranking or omission of specific diseases. If the user clicks on a specific disease name, clinical and epidemiological data on the disease in question are depicted (figure 3). The differential diagnosis list is generated by a Bayesian formula which compares the product of disease-incidence and symptom incidence, for all compatible infectious diseases. In the above example, a number of diseases known to occur in Indonesia were capable of producing fever, and joint pain. The statistical likelihood of Chikungunya in this case can be computed by a simple Bayesian formula, as follows: Two spreadsheets in the GIDEON database respectively follow the incidence of all symptoms for every disease, and the incidence of all diseases for every country. When a clinical case is \"entered\" into GIDEON, the program identifies all compatible diseases and ranks their relative likelihoods as determined by the above formula, ie: P-(C/ S) vs. P-(D2/S) vs. P-(D3/S) ... vs. P-(Dn/S).\nA blinded study of 500 cases conducted by this author found that the correct diagnosis was listed in the differential list in 94.7% of cases, and was ranked first in 75% [2] . A second study of hospitalized patients in Boston found that the correct diagnosis was listed in only 69%, and was ranked first in 60% [3] . It is likely that inclusion in the differential diagnosis list may be more important than disease ranking in such systems [4] .\nA \"Bioterrorism\" option generates the differential diagnosis for diseases associated with suspected bioterror scenarios. In Figure 4 , \"<bioterrorism simulator>\" has been substituted for Indonesia, given the above constellation of fever, joint pain, etc. The resulting differential diagnosis lists Ebola (42.9% probability), followed by Crimean-Data entry screen for a bioterrorism scenario Figure 4 Data entry screen for a bioterrorism scenario.\nCongo hemorrhagic fever (12.6% probability). A similar \"Worldwide\" option can be used to explore all of the worlds diseases consistent with given clinical features, and access text on the global status for individual diseases.\nIn theory, data entry by users can be monitored at the server level for purposes of surveillance. For example, if one or more users in China were to enter cases of fatal pneumonia, a \"red-flag\" at any monitoring agency (i.e., the World Health Organization) could indicate the possible appearance of SARS -long before submission of specimens or reporting of the case to local authorities. Similarly, the appearance of multiple cases of \"dysentery\" by users in a given community could indicate a possible outbreak of shigellosis.\nThe Epidemiology module presents detailed country-specific information on the status of each disease, both globally and within each relevant country. The current version contains over two million words in 12,000 notes. All data are derived from Health Ministry publications, peer-review journals, standard textbooks, WHO and CDC websites and data presented at conferences. The user may also access over 30,000 graphs which follow disease incidence, rates and other numerical data. The main Epidemiology screen is shown in Figure 5 . Note that the user can append custom \"personal notes\" -in any national language or font-regarding the status of every disease in their own institution. Such notes would be accessible by all colleagues using GIDEON on the local network.\nMaps which depict the global distribution of each disease can be accessed through the 'Distribution' tab ( Figure 6 ).\nEpidemiology module, main screen Figure 5 Epidemiology module, main screen. The 'images' tab has been pressed, to access thumbnail images of Plague. These can be maximized and copied to PowerPoint, etc. Note addition of 'Personal notes' by the user, at lower right.\nText outlining country-specific data for the disease ( Figure 7) is available through either a list of countries displayed in this module, or by clicking the relevant 'red dot' on the map. These text boxes also include data sets which automatically generate incidence / rate graphs (Figure 8) , a chronological account of all disease outbreaks, and numbered reference links to relevant journal publications and reports of ongoing outbreaks from ProMed http:// www.promedmail.org. A separate 'Graphs' option allows the user to generate custom-made graphs comparing multiple disease rates, or rates in multiple countries. (Figure 9 ).\nAdditional tabs access the descriptive epidemiology and clinical background of each disease. Synonym tabs generate lists of alternative terms for diseases and countries in Spanish, German, Norwegian, etc. Historical data record the incidence of individual diseases and significant outbreaks spanning decades. An additional \"Fingerprint\" option generates a list of diseases compatible with any set of epidemiological parameters. For example, in Figure 10 we see that ten parasitic diseases are transmitted by fish in Japan.\nThe Therapy module follows the pharmacology and application of all drugs and vaccines used in Infectious Diseases. The current version contains 264 generic drugs and 64 vaccines. Various sub-modules present the mechanism of action; pharmacology, dosages, drug-drug interactions, contraindications, spectrum, and susceptibility testing standards. An international synonym lists contains over 9,500 trade names. As in other modules, users may add Epidemiology module Figure 6 Epidemiology module. Map depicting the global distribution of plague. Specific map areas can be expanded, and all elements can be copied for reproduction as necessary. Country-specific notes regarding plague appear when corresponding red dots are clicked.\ncustom notes in their own language for each drug or vaccine: prices, resistance patterns, local trade names, etc.\nThe Microbiology option is similar to the Diagnosis module. Users may enter any combination of phenotypic tests, and obtain a ranked probability list of compatible bacteria. The current version incorporates more than 1,300 taxa. The Microbiology module is also designed to list the phenotype, prior names, ecology and disease association for any organism, or compare the phenotypes of any combination of organisms selected by the user.\nSince the graphic and mapping functions of GIDEON treat individual countries as whole units, data presentations lack a certain degree of \"granularity.\" Thus, the dif-ferential diagnosis of fever in Venezuela will include malaria, even if the patient is living outside of the endemic, southern region. This problem is corrected to a large extent by text in the associated country-specific notes and the general knowledge base of the treating physician. In theory, the manufacturer could follow the incidence of each disease for every state, district, province and oblast; but variability would still exist according to occupation, rural vs. urban setting, season, etc.\nAn additional problem relates to the availability and quality of valid epidemiological data. Disease reporting varies widely from country to country. For example, AIDS reporting statistics from sub-Saharan Africa are generally inadequate. Where necessary, the spreadsheets used by GIDEON record published true estimates rather than questionable reports. In other instances, Health Ministry Figure 7 Plague in Tanzania. Clicking on relevant data sets will generate incidence and rates graphs. Note several numbered links to journal publications.\nPlague -Worldwide incidence and rates per 100,000 Figure 8 Plague -Worldwide incidence and rates per 100,000.\ndata conflict with reports of the World Health Organisation, a fact which is recorded in relevant GIDEON country notes. Occasionally, major diseases are not reported at all. For example, several recent cases of cholera in Japan originated from Thailand; but Thailand has not officially reported a single case in many years. Where possible, the GIDEON data base relies on published best estimates, and at times 'educated guesses' when data are entirely lacking.\nThus, there are few published data for disease incidence in Togo, and the program is forced to rely on publications for neighboring Ghana.\nThe reader is referred to the GIDEON website http:// www.GideonOnline.com for an extensive listing of data sources, published reviews, technical background and pricing information.\nGraph contrasting AIDS rates among user-selected countries Figure 9 Graph contrasting AIDS rates among user-selected countries.\nPublish with Bio Med Central and every scientist can read your work free of charge " }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: Secondly: Understanding globalization as a subject matter itself needs certain benchmarks and barometers of its successes and failings. Health is one such barometer. It is a marker of social infrastructure and social welfare and as such can be used to either sound an alarm or give a victory cheer as our interconnectedness hurts and heals the populations we serve.\nAnd lastly: In as much as globalization can have an effect on health, it is also true that health and disease has an effect on globalization as exemplified by the existence of quarantine laws and the devastating economic effects of the AIDS pandemic.\nA balanced view would propose that the effects of globalization on health (and health systems) are neither universally good nor bad, but rather context specific. If the dialogue pertaining to globalization is to be directed or biased in any direction, then it must be this: that we consider the poor first.\nI am pleased to introduce 'Globalization and Health', a peer reviewed, open access (free to the end user) journal. In this, the début editorial, I will briefly outline the purpose and scope of this journal highlighting our intention to publish a balanced mixture of opinion on the subject.\nThat the journal be 'Open Access' is entirely appropriate. Knowledge, at its best utility, is a 'public good' i.e. nonrival, non-excludable. While this journal will deal with the subject matter of creating 'global public goods for health', it will also by virtue of its very existence, contribute toward that process. Globalization and Health's 'Open Access' policy changes the way in which articles are pub-lished. First, all articles become freely and universally accessible online, and so an author's work can be read by anyone at no cost. Second, the authors hold copyright for their work and grant anyone the right to reproduce and disseminate the article, provided that it is correctly cited and no errors are introduced [1] . Third, a copy of the full text of each Open Access article is permanently archived in an online repository separate from the journal. Globalization and Health's articles are archived in PubMed Central [2], the US National Library of Medicine's full-text repository of life science literature, and also in repositories at the University of Potsdam [3] in Germany, at INIST [4] in France and in e-Depot [5], the National Library of the Netherlands' digital archive of all electronic publications. Importantly, the results of publicly funded research will be accessible to all taxpayers and not just those with access to a library with a subscription. As such, Open Access could help to increase public interest in, and support of, research. Note that this public accessibility may become a legal requirement in the USA if the proposed Public Access to Science Act is made law [6]. Added to this, a country's economy will not influence its scientists' ability to access articles because resource-poor countries (and institutions) will be able to read the same material as wealthier ones (although creating access to the internet is another matter [7] ).\nThe rationale behind starting a journal dedicated to globalization and health is three fold:\nFirstly: Globalization is reshaping the social geography within which we might strive to create health or prevent disease. The determinants of health -be they a SARS virus or a predilection for fatty foods -have joined us in our global mobility. Driven by economic liberalization and changing technologies, the phenomenon of 'access' is likely to dominate to an increasing extent the unfolding experience of human disease and wellbeing.\nSecondly: Understanding globalization as a subject matter itself needs certain benchmarks and barometers of its successes and failings. Health is one such barometer. It is a marker of social infrastructure and social welfare and as such can be used to either sound an alarm or give a victory cheer as our interconnectedness hurts and heals the populations we serve.\nAnd lastly: In as much as globalization can have an effect on health, it is also true that health and disease has an effect on globalization as exemplified by the existence of quarantine laws and the devastating economic effects of the AIDS pandemic.\nA balanced view would propose that the effects of globalization on health (and health systems) are neither univer-sally good nor bad, but rather context specific. The extent to which individual states are able to engage the process of globalization on their own terms differs widely from one country to the next. Child mortality, for example, changes quickly in response to subtle changes in purchasing power in impoverished communities. In affluent communities however, a small change in income has little effect on utility in either direction. As we consider the effects of globalization on wellbeing it becomes apparent that we need to consider both the long term scenarios for populations as a whole, and the immediate effects for the more vulnerable within those populations who are dependent on fragile local economies.\nIf the dialogue pertaining to globalization is to be directed or biased in any direction, then it must be this: that we consider the poor first." }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: (termed tRNA Ser CAG), and revealed its decoding mechan-Bioscience and Biotechnology, Tokyo Institute of Technology, ism by means of an in vitro translational assay system Nagatsuta, Midori-ku, Yokohama 227, Japan (Yokogawa et al., 1992; Suzuki et al., 1994) . Furthermore, 2 Corresponding authors when we investigated the distribution of this non-universal genetic code in fungi, as well as C.cylindracea, eight other In some Candida species, the universal CUG leucine\nCandida species-C.albicans, C.zeylanoides, C.lusitaniae, codon is translated as serine. However, in most cases, C.tropicalis, C.melbiosica, C.parapsilosis, C.guilliermonthe serine tRNAs responsible for this non-universal dii and C.rugosa-were found to utilize the codon CUG decoding (tRNA Ser CAG) accept in vitro not only serine, for serine instead of leucine, all having tRNA Ser CAG as but also, to some extent, leucine. Nucleotide replacethe mediator in the unusual decoding (Ohama et al., 1993 ; ment experiments indicated that m 1 G37 is critical for Ueda et al., 1994) . Several other investigators have also leucylation activity. This finding was supported by the shown that the codon CUG is actually translated as serine fact that the tRNA Ser CAGs possessing the leucylation in vivo in C.albicans and C.maltosa (Santos and Tuite, activity always have m 1 G37, whereas that of Candida 1995a; Sugiyama et al., 1995; Zimmer and Schunck, 1995) .\nOne of the most remarkable structural features observed A37. Quantification of defined aminoacetylated tRNAs in most of these tRNA Ser CAGs is that the nucleotide 5Јin cells demonstrated that 3% of the tRNA Ser CAGs adjacent to the anticodon (position 33) is occupied not by possessing m 1 G37 were, in fact, charged with leucine the conserved U residue (U33) but by a G residue (G33).\nIt has been speculated that U33 is necessary for forming of C.maltosa possessing this type of tRNA Ser CAG also the U-turn structure of the anticodon loop in all tRNAs suggested that the URA3 gene inactivated due to the reported so far (Quigley and Rich, 1976 ; Sprinzl et al., translation of CUG as serine was rescued by a slight 1996) . Moreover, the nucleotide at position 37, 3Ј-adjacent incorporation of leucine into the polypeptide, which to the anticodon CAG, is 1-methyl guanosine (m 1 G) in demonstrated that the tRNA charged with multiple almost all tRNA Ser CAGs except for that of C.cylindracea amino acids could participate in the translation. These (A37), while all the serine tRNAs in fungi corresponding findings provide the first evidence that two distinct Introduction Normanly and Abelson, 1989; Shimizu et al., 1992; McClain, 1993; Schimmel et al., 1993) . This line of study The universality of the genetic code was once considered began with the artificial conversion of leucine tRNA of to be one of the essential characteristics of life, which led Escherichia coli to serine tRNA by Abelson's group 10 to the conception of the 'frozen accident theory'. This years ago (Normanly et al., 1986) . Recently, tRNA identity theory proposes that all extant living organisms use the elements of Saccharomyces cerevisiae leucine tRNA were universal genetic code, which was born by accident and elucidated using unmodified variants synthesized by T7 'frozen', and that they originate from a single, closely RNA polymerase (Soma et al., 1996) , indicating that in interbreeding population (Crick, 1968) . However, in recent addition to the discriminator base, A73, the second letter years a number of non-universal genetic codes have been of the anticodon, A35, and the nucleotide 3Ј-adjacent to reported in various non-plant mitochondrial systems, as the anticodon, m 1 G37, are important for recognition by well as in several nuclear systems (reviewed in Osawa leucyl-tRNA synthetase (LeuRS). The majority of Candida et al., 1992; Osawa, 1995) , which contradict the frozen tRNA Ser CAGs have A35 and m 1 G37, while the discriminaccident theory.\nAmong these deviations from the universal codes, ator is occupied by a nucleoside other than adenosine (mostly G73). In this respect, tRNA Ser CAG seems to be a potentially chimeric tRNA molecule capable of being recognized not only by seryl-but also by leucyl-tRNA synthetases.\nPreviously, we showed that these tRNA Ser CAGs would have originated from the serine tRNA corresponding to codon UCG . This suggests an evolutionary pathway in which conversion from A to m 1 G would have taken place at position 37 just after the emergence of tRNA Ser CAG had brought about a change in the universal code. Since such a mutation at position 37 might potentially result in the leucylation of tRNA Ser CAG, we attempted to elucidate the charging properties of these tRNA Ser CAGs both in vitro and in vivo. Based on the results of in vitro aminoacylation reactions using tRNA variants constructed by the microsurgery method, the direct analysis of aminoacylated tRNAs in cells and a genetic approach, we demonstrate here that these serine tRNAs are actually leucylated both in vitro and in vivo. Furthermore, m 1 G at position 37 was found to be indispensable for the leucylation of tRNA Ser CAGs. In fact, the tRNA Ser CAG of C.cylindracea, which has A at position 37, exhibits no leucylation activity. C.cylindracea has a high GϩC content (63%) and utilizes CUG as a major serine codon. However, the other Candida species have no such high GϩC content and utilize the CUG as a minor serine codon (Kawaguchi et al., 1989; Lloyd and Sharp, 1992; our unpublished observation) . Considering the relationship between the usage of the codon CUG as serine and the leucylation properties of tRNA Ser CAG, it seems that only Candida species with a genome in which the incidence of the CUG serine codon is very low possess serine tRNA Ser CAG that can be leucylated. Furthermore, such tRNA Ser CAGs charged with heterogeneous amino acids should be utilized equally in the translation process. This is the first demonstration that a single tRNA species is assigned to two different amino acids in the cell. We propose designating this type of codon having multiple amino acid assignment as a 'polysemous codon'. The correlation between the dual-assignment state and the pathway of genetic code diversification is also discussed. \nand C.cylindracea (Yokogawa et al., 1992; Ohama et al., 1993 ). The numbering system and abbreviations for modified nucleotides conform Candida zeylanoides tRNA Ser CAG is leucylated to Sprinzl et al. (1996) and Crain and McCloskey (1996), respectively. in vitro (B) Time-dependent aminoacylation with SerRS or LeuRS from First the leucylation of tRNA Ser CAGs from C.zeylanoides C.zeylanoides cells. Aminoacylation reactions were carried out with and C.cylindracea was examined using LeuRS partially 0.7 µM tRNAs and with same amounts of enzyme activities calculated using cognate tRNAs. Serylation and leucylation are shown by dotted purified from C.zeylanoides, since it is known that leucine and solid lines, respectively. The right-hand frame shows the solid tRNAs of yeast have one of their identity determinants at curves from left-hand frame plotted with an enlarged ordinate. The position 37 (Soma et al., 1996) and tRNA Ser CAGs of aminoacylation of C.zeylanoides tRNA Ser CAG (s) and of C.zeylanoides and C.cylindracea have different nucleo-C.cylindracea tRNA Ser CAG (u) are compared; C.cylindracea tRNA Ser GCU (j), having no leucylation activity, is shown as a tides at this position (m 1 G and A, respectively) (Figure control. (C) TLC analysis of acetylleucyl-tRNA fragments derived 1A). Both tRNAs showed almost full serylation activity from leucylated tRNA Ser CAGs. After leucylation with [ 14 C]leucine, (~1200-1500 pmol/A 260 unit), as shown in Figure 1B . The leucyl-tRNAs were acetylated with acetic anhydride. Acetyl-tRNA Ser CAG of C.zeylanoides was evidently leucylated leucylated at all, as was the case when another species tRNA Ser CAG by gel-electrophoresis under acidic con- observed with LeuRSs from both C.cylindracea and S.cerevisiae (data not shown). of serine tRNA specific for codon AGY (Y: U or C) m 1 G37 is responsible for recognition by (tRNA Ser GCU) was employed as a control substrate leucyl-tRNA synthetase ( Figure 1B , right-hand graph). The K m value of C.zeylan-Among the tRNA Ser CAGs of several Candida species, oides LeuRS towards tRNA Ser CAG (5.0 µM) is only one that of C.cylindracea is unique because it alone possesses order of magnitude larger than that of the serylation of no leucylation capacity. A sequence comparison of these this tRNA (0.22 µM) as well as that of leucylation toward tRNAs ( Figure 1A ) prompts us to speculate that the the cognate leucine tRNAs of S.cerevisae (0.34 µM; Soma nucleotide at position 37 is strongly associated with et al., 1996) .\nleucylation, because all tRNA Ser CAGs possessing leucyl-In order to verify that the leucylation activity observed ation activity have m 1 G in common, while only the for the tRNA Ser CAG of C.zeylanoides actually came from tRNA Ser CAG of C.cylindracea, which possesses no leucylthe tRNA Ser CAG itself, and not from a trace amount ation activity, has A at this position. of leucine tRNA contaminating the tRNA sample, the To examine the validity of this speculation, a series of leucylated 3Ј-terminal RNA fragment derived from leucyl-tRNA Ser CAG variants was constructed by the in vitro tRNA Ser CAG was analyzed in the following manner. 14 Ctranscription method using T7 RNA polymerase, as well leucylated tRNA Ser CAG from C.zeylanoides was first as by the microsurgery method, and the leucylation activity acetylated with acetic anhydride to prevent deacylation, of each variant was measured. When the tRNA Ser CAG of and then digested with RNase T1. The resulting 3Ј-C.zeylanoides synthesized by in vitro transcription was terminal fragment with 14 C-labeled acetylleucine was employed as a substrate, no leucylation activity was analyzed by cellulose TLC. The results are shown in detected, not even for the tRNA transcript having G37 Figure 1C . If leucylated tRNA Ser CAG were digested ( Figure 3A ). On the other hand, as shown in Figure 3A , with RNase T1, 14 C-labeled acetylleucyl-CCA should be serylation activity exceeded 1000 pmol/A 260 unit. These released as a labeled fragment ( Figure 1C , lane 3), because results strongly suggested that some nucleoside modifica-G is located at position 73 of the tRNA Ser CAG (Figure tion is necessary in tRNA Ser CAG for recognition by 1A, left-hand structure). Any contaminated leucine tRNAs, LeuRS. We thus attempted to replace the m 1 G37 of if they exist, will give some 14 C-labeled fragments larger C.zeylanoides tRNA Ser CAG with G (the variant is symbolthan the tetramer ( Figure 1C , lane 4), because all the ized as m 1 G37G) or A (m 1 G37A), by the microsurgery leucine tRNAs of yeasts so far analyzed (Sprinzl et al., method (Figure 2A and B; for details, see Materials 1996) including those of C.zeylanoides (T. Suzuki, unpuband methods) to examine the contribution of m 1 G37 to lished result) are known to have A73 at their 3Ј-ends, leucylation and the contribution of A37 of C.cylindracea which are resistant to RNase T1. The mobility of the tRNA Ser CAG to the prevention of leucylation. acetylleucyl-oligonucleotide derived from tRNA Ser CAG When aminoacylation of m 1 G37A and m 1 G37G was from C.zeylanoides ( Figure 1C , lane 1) was identical to examined ( Figure 3A ), the results indicated that both that of acelylleucyl-CCA prepared from the RNase U2 substitutions lead to complete loss of leucylation (Figure digests of leucyl-tRNA Leu s from C.zeylanoides (lane 3).\n3A, right-hand graph), although no apparent influence was This observation clearly demonstrates that leucine is observed on serylation ( Figure 3A , left-hand graph). These definitely attached to the tRNA possessing G73; the tRNA findings strongly indicate that the methyl group of m 1 G37 therefore must be tRNA Ser CAG and not tRNA Leu . Thus, plays a crucial role in enhancing the leucylation activity it is concluded that the tRNA which incorporated leucine of tRNA Ser CAG. in vitro is in fact tRNA Ser CAG. This deduction is supported\nThe slight reduction in leucylation activity observed in by the results of an additional experiment: incorporation the control variant z-G33G ( Figure 2A ) compared with of [ 14 C]leucine into the tRNA Ser CAG sample with LeuRS native tRNA ( Figure 3A , right-hand graph) was found to was reduced by the addition of SerRS and non-labeled have resulted from the partial deacetylation of 4-acetyl serine to the reaction mixture (data not shown), which cytidine (ac 4 C) due to acid treatment of the 5Ј-half clearly indicates that the same tRNA molecule is comfragment of tRNA Ser CAG (see Materials and methods). petitively aminoacylated by these two enzymes. This is considered further in the Discussion. To conclude that tRNA Ser CAG is aminoacylated with leucine, we carried out a further experiment. The G33 acts as a modulator of leucylation tRNA Ser CAG was charged with serine and serylated In addition to m 1 G37, another unique feature of the serine tRNA Ser CAGs in these Candida species is the presence tRNA Ser CAG was separated from non-aminoacylated The effect of mutation at position 33 in these two each variant was confirmed to have been replaced as expected (shown tRNAs was found to be quite different. In the case of the by arrows).\nC.cylindracea tRNA, none of the mutations at position 33 caused leucylation of the tRNA, as was observed with the native tRNA Ser CAG, and there was no reduction in of G at position 33, where a pyrimidine (mostly U) is completely conserved in usual tRNAs (Sprinzl et al., serylation activity ( Figure 3C ). In contrast, the replacement of G33 by pyrimidines in C.zeylanoides tRNA Ser CAG 1996). Since we considered it is possible that this notable feature may be in some way related to the unusual considerably enhanced the leucylation activity ( Figure 3B , right-hand graph), while no significant difference was aminoacylation characteristics described above and/or to the translation of non-universal genetic code, we examined observed in the serylation activity ( Figure 3B , left-hand graph). The kinetic parameters of leucylation for the the effect of residue 33 on the aminoacylation and transla- show the spots corresponding to acetylleucine and acetylserine as markers, respectively. (D) Analysis of acetylamino acids attached to tRNA fragments on a TLC plate. Lane 2 shows the spot corresponding to the acetylamino acids derived from the RNase T1 fragment of C.zeylanoides tRNA Ser CAG. Lanes 1 and 3 indicate the spots corresponding to acetylleucine and acetylserine, respectively. Ten micrograms of [ 14 C]acetylaminoacyl-tRNA Ser CAG from C.zeylanoides was digested with RNase T1 and developed on cellulose TLC plates under the same conditions as (C). CCA fragments with [ 14 C]acetylamino acids were scraped from the plate from which the fragments were eluted with H20 and desalted by Sep-pak C18 under the conditions described in the literature (Wang et al., 1990) .\n[ 14 C]acetylamino acids discharged from the fragments were developed on TLC and visualized by an imaging analyzer (BAS-1000, Fuji Photo Systems). variants of C.zeylanoides tRNA are shown in Table I . It\nEvidence for leucylation of C.zeylanoides tRNA Ser CAG in vivo is notable that the K m values of the two pyrimidine At this point, we had established that the tRNA Ser CAG mutants, z-G33U (1.4 µM) and z-G33C (1.3 µM), are of C.zeylanoides is actually able to accept leucine in vitro. clearly lower than those of the two purine mutants, z-G33A However, considering the facts that SerRS and LeuRS (6.7 µM) and z-G33G (5.6 µM). The V max value of z-G33U coexist in cells and, judging from their K m values, that (1.2 pmol/min) is 39% of that of z-G33C (3.1 pmol/min), the affinity of tRNA Ser CAG toward SerRS is one order of which could explain why z-G33U shows lower leucylation magnitude higher than that toward LeuRS, we needed to activity than z-G33C despite having nearly the same K m ascertain whether the tRNA Ser CAG of C.zeylanoides is in value ( Figure 3B , right-hand graph). Judging from the fact leucylated in vivo. For this purpose, we adopted a sequence analysis (data not shown), the slight reduction newly developed method for quantifying an individual in the leucylation of z-G33G (5.6 µM) compared with aminoacyl-tRNA in cells (Suzuki et al., 1996) . that of the native tRNA Ser CAG (5.0 µM) is probably due Aminoacyl-tRNAs separately prepared from cells of to the partial deacetylation of ac 4 C at position 12, as C.zeylanoides and C.cylindracea were immediately submentioned above. This was confirmed by the observation jected to acetylation using [1-14 C]acetic anhydride to label of a slight reduction in leucylation activity also in acidthe amino acids as well as to stabilize the aminoacylated treated native tRNA Ser CAG (data not shown). It is thus tRNAs. From each of the acetylated aminoacyl-tRNA concluded that replacement of a pyrimidine by a purine mixtures, tRNA Ser CAGs from C.zeylanoides and C.cylindat position 33 has a repressive effect on leucylation of the racea were fished out by a solid-phase-attached DNA tRNA Ser CAG of C.zeylanoides.\nprobe as described previously (Tsurui et al., 1994 ; Wakita The translation efficiencies of the variants with a muta et al., 1994) . A single band for each of the aminoacyltion at position 33 were also examined in a cell-free tRNAs was detected by staining ( Figure 4A ) with which translation system of C.cylindracea (Yokogawa et al., the radioactivity coincided in each case ( Figure 4B ). 1992; Suzuki et al., 1994) , to evaluate the effect of G33.\nAcetylated amino acids attached to these tRNAs were A change from G to U at position 33 apparently enhanced deacylated by alkaline treatment and analyzed by TLC. the translation activity 2.5-fold, although their decoding As shown in Figure 4C , acetylserine was observed as a properties did not change at all (data not shown). We thus major amino acid derivative in both tRNA Ser CAGs, but consider that G33 serves as a modulator of leucylation of acetylleucine was detected only in the C.zeylanoides tRNA Ser CAG, despite a slight disadvantage in transla-tRNA Ser CAG; the acetylserine and acetylleucine spots were identified as described previously (Suzuki et al., tion activity. 1996) . The radioactivities remaining on the origins probably came from the direct acetylation of some nucleotides in the tRNAs, as discussed previously (Suzuki et al., 1996) . From comparison with the radioactivity of acetylserine, it was calculated that~3% of the tRNA Ser CAG was attached with acetylleucine. These results were reproducible.\nDigestion of purified acetyl-aminoacyl tRNA Ser CAG with RNase T1 also gave only a 14 C-labeled CCA fragment, as shown in Figure 1C . When the acetylated amino acid released from the fragment purified from the corresponding spot on TLC was analyzed by TLC, the ratio of acetylleucine to acetylserine was also found to be 3% ( Figure 4D ), indicating that acetylleucine is covalently attached to the tRNA Ser CAG fragment with G73. It thus became clear that the tRNA Ser CAG of C.zeylanoides was in fact charged with leucine by 3% of the amount of serylation of the same tRNA Ser CAG in C.zeylanoides cells.\nAminoacylation has generally been considered to be the final stage determining translational accuracy (reviewed in Parker, 1989; Kurland, 1992; Farabaugh, 1993) . However, in the case of tRNA Gln charged with glutamate in the chloroplast, Glu-tRNA Gln is rejected by an elongation factor so that the chloroplast translation machinery does not employ the mischarged aminoacyl-tRNA (Stanzel et al., 1994) . It is likely that this is an exceptional case due to the lack of glutamyl-tRNA synthetase in the chloroplast.\nIn order to prove that leucylated tRNA Ser CAGs actually participate in the translation process in Candida cells without such a rejection mechanism, we utilized a URA3 gene expression system derived from S.cerevisiae in C.maltosa, which was developed by Sugiyama et al. (1995) . Candida maltosa utilizes the codon CUG as serine and possesses the relevant tRNA Ser CAG gene (Sugiyama et al., 1995; Zimmer and Schunck, 1995) . Since the et al., 1995) . In the present study, this URA3 gene, with the CTG codon replaced by various leucine or serine codons, was utilized as a marker gene ( Figure 5A ). First, ADE1/ura3::C-ADE1) (Ohkuma et al., 1993) , the growth of which was monitored on minimal medium SD plates a plasmid in which the S.cerevisiae URA3 gene was inserted downstream of a C.maltosa-specific promoter in the presence and absence of uracil. When uracil was supplied to the SD plate for the (C-p) was constructed and designated as pCSU-CTG (Sugiyama, 1995) . As controls, mutant plasmids of pCSU-positive control experiments, all the transformants grew normally ( Figure 5B , middle row). However, in the absence CTG, in which the codon CTG was replaced by either the serine codon TCT or the leucine codon CTC, were of uracil, cells harboring pCCU and pCSU-CTC showed normal growth, whereas no growth was observed in those constructed and named pCSU-TCT and pCSU-CTC, respectively. In addition, a plasmid (pCCU) consisting of harboring pCSU-TCT and pUTH18 that contained no URA3 gene insertion. Cells harboring pCSU-CTG showed the URA3 gene of C.maltosa having a CTT leucine codon at the corresponding site, combined with the C.maltosa-weak but significant growth ( Figure 5B, uppermost row) . These results demonstrate that if the codon at position 45 specific promoter, was also used as a positive control. These variant plasmids were introduced into a URA3-is translated as leucine, active ODCase will be produced and the cells will be able to grow, but translation of the defective C.maltosa strain CHU1 (his5, ade1, ura3::C-codon with serine will produce inactive ODCase and the We believe that tRNA Ser CAG is the only molecule responsible for the leucine insertion corresponding to cells will be unable to grow. The result with cells harboring pCSU-CTG clearly demonstrates that the URA3 mutation codon CUG in C.maltosa cells, based on the following obervations. We have purified and sequenced a number on the C.maltosa chromosome was in some way complemented by the introduced pCSU-CTG plasmid, suggesting of leucine and serine tRNAs from Candida species, in which codon CUG is translated as serine, and failed in that the CTG codon was read at least partially as leucine in C.maltosa cells possessing tRNA Ser CAG.\nfinding tRNA with the anticodon sequence potentially complementary to codon CUG other than tRNA Ser CAG In order to quantify the growth rate of the cells harboring pCSU-CTG, the viability of the cells was examined in (Yokogawa et al., 1992; Ohama et al., 1993; Suzuki et al., 1994; Ueda et al., 1994; our unpublished observation) . liquid medium without uracil. As shown in Figure 5C , whereas translation of the CTG codon as serine completely Futhermore, tRNA genes for serine and leucine from these Candida species were sequenced following the blocked cell growth in the case of pCSU-TCT, and full complementation was observed in the case of pCSU-CTC amplification by cloning and/or PCR methods, and we found that only tRNA Ser CAG is able to translate codon in which the CTC codon was read as leucine, intermediate cell growth was observed in the case of pCSU-CTG, CUG (Yokogawa et al., 1992; Ohama et al., 1993; Suzuki et al., 1994; Ueda et al., 1994 ; our unpublished observ-indicating that ODCase was expressed in an active form, albeit at a low level, when there was a slight incorporation ation). Thus, it could be concluded that only the tRNA Ser CAG species inserts leucine into polypeptide of leucine at the CTG codon. The slow growth of the cells harboring pCSU-CTG was not due to the spontaneous corresponding to codon CUG. reversion of the CTG codon to another leucine codon or due to any other mutation, because the cells harvested Discussion from the colony on the SD-plate show the same growth phenotype. These results are unlikely to reflect the different The observations presented here clearly demonstrate that, in certain living organisms, a single codon can be simul-expression levels of the URA3 gene variants because the URA3 mRNA level is not altered by mutations at position taneously assigned to two distinct amino acids. Most codons in the genetic code degenerate, but our findings 45 (Ohkuma, 1993) . Furthermore, the possibility that the URA3 gene with CTG at position 45 is translated more show that some amino acids are also able to degenerate with respect to a particular codon. Such codon ambiguity efficiently than the gene with TCT at the same site due to codon preference (Ikemura, 1982) is excluded by the is governed by a tRNA acceptable to two amino acids simultaneously, as described above. We propose to desig-fact that the TCT codon is the most preferred of all the serine codons, including the CUG codon, in C.maltosa nate a codon corresponding to multiple amino acids a 'polysemous codon'. (Sugiyama et al., 1995) .\nODCase activity resulting from the translation of the A high degree of accuracy in tRNA aminoacylation has been considered crucial for preserving fidelity in protein URA3 gene was examined in the presence of a pyrimidine analog, 5-fluoroorotic acid (5FOA), an inhibitor in synthesis. It has been established that aminoacyl-tRNA synthetase is able to discriminate precisely its cognate pyrimidine biosynthesis. Incorporation of 5FOA with ODCase results in the formation of 5-fluorouridylate, amino acid from other structurally related amino acids at the adenylation reaction step, and its cognate tRNAs from which is harmful to cell propagation (Boeke et al., 1984) . Thus, URA3-defective strains grow normally on a medium non-cognate ones (reviewed in Parker, 1989; Kurland, 1992) . The misacylation error in this process has been containing 5FOA, whereas cells possessing the active URA3 gene are unable to grow on this medium. Cells estimated to range between 10 -4 and 10 -5 (Lin et al., 1984; Okamoto et al., 1984) . Discrimination of cognate harboring the respective plasmids were cultivated in the presence of 5FOA in addition to uracil. tRNA from non-cognate tRNAs is mediated by positive and negative identity determinants localized on the tRNA As shown in the bottom row of Figure 5B , cells harboring pCSU-CTG exhibited similar growth on the molecule (Yarus, 1988; Normanly and Abelson, 1989) . The only exception reported so far is that tRNA Gln is agar plate to those with pCSU-TCT and pUTH18, although the transformants with pCSU-CTC and pCCU were unable aminoacylated with glutamate in Gram-positive bacteria and in some organelles (Lapointe et al., 1986 ; Schön to grow. These results indicate that the CTG codon at position 45 was mainly translated as serine in C. maltosa, et al., 1988) . However, this differs from misaminoacylation in that this process is indispensable to compensate for the so as to produce the inactive ODCase. However, when the liquid medium was supplied with 5FOA, a slight lack of glutamyl-tRNA synthetase in these organisms. In general, high fidelity in the aminoacylation process is reduction in the growth rate was observed in the case of pCSU-CTG, compared with pCSU-TCT ( Figure 5C ), considered to be indispensable for translating genes into functionally active proteins with a high degree of accuracy. while very slow growth was observed in the case of pCSU-CTC used as a control. In order to detect a low\nThe discovery of a polysemous codon in a Candida species contradicts the established notion of aminoacyl-level of ODCase activity arising from a slight incorporation of leucine at the CUG codon in the 45th position, we ation with high fidelity. We have shown that a single tRNA is acceptable to two different amino acids, and adjusted the ratio of 5FOA and uracil as shown in Materials and methods. This growth rate reduction clearly suggests that it can therefore transfer two different amino acids corresponding to a particular codon. The expression that the slow growth observed in the SD medium was due to low expression of active ODCase. Thus it is concluded experiment using the ODCase-encoding URA3 gene containing codon CUG at the site essential for its activity that the CUG codon is partially translated as leucine in C.maltosa cells.\n(see also Sugiyama et al., 1995) suggested that leucine could be incorporated into the gene product corresponding on experiments using an artificial mutation, and it does not reflect experimental observation in an extant living to codon CUG in C.maltosa, as judged from the complementation tests with the URA3 mutation. Although the organism.\nOn the basis of peptide sequences, several research amount of leucine incorporated per CUG codon was not quantitatively determined, it is clear that the incorporation groups have reported that codon CUG corresponds only to serine in C.maltosa (Sugiyama et al., 1995) and was mediated by the leucyl-tRNA Ser CAG. We thus concluded that codon CUG was simultaneously assigned to C.albicans (Santos and Tuite, 1995a; White et al., 1995) . No leucine-inserted peptide was detected in these studies. serine and leucine in the normal translation process in C.maltosa. A quantitative analysis of the amino acids However, we consider that any peptide with a leucine which was inserted for the codon CUG might have been attached to the tRNA indicated that 3% of tRNA Ser CAG is leucylated in C.zeylanoides cells. Such a high level of missed during purification or was undetectable in the peptide sequencing, because the amount of leucine-inserted leucylation is far beyond conventional misacylation, whose rate is estimated to be less than 10 -4 . Unless a proofreading peptide (~3%) would have been too low to be positively identified in sequencing experiments. mechanism exists on the ribosome, incorporation of leucine at CUG codon sites may reflect the relative ratio of\nWe have shown that tRNA Ser CAG in Candida species is a chimera of tRNA Ser CAG and tRNALeuCAG in so far tRNA Ser CAG leucylation, which is two orders of magnitude higher than that of conventional mistranslation.\nas it is the substrate for both SerRS and LeuRS. The K m value for LeuRS is 5.0 µM, which is only one order of To date, artificial manipulations of molecules participating in the translation process, such as the overproduction magnitude larger than that for SerRS (0.22 µM). In an in vitro aminoacylation experiment Ͼ30% of tRNA Ser CAG of aminoacyl-tRNA synthetase (Swanson et al., 1988) , mutations of tRNAs etc. and/or control of growth condi-subjected to the reaction could be converted to leucyl-tRNA Ser CAG using an increased amount of LeuRS and a tions, such as deprivation of amino acids in the medium (Edelmann and Gallant, 1977; O'Farrell, 1978; Parker and longer incubation time (data not shown). We observed that while the presence of SerRS and non-radioactive Precup, 1986), have been found to increase the error rate in translation (reviewed in Parker, 1989) . However, our serine reduced leucylation, complete loss of leucylation could not be achieved (data not shown), indicating that the observation is based on experiments using wild-type cells grown in a rich medium suitable for high viability. In affinity of LeuRS toward tRNA Ser CAG is relatively high. In proliferating cells of C.zeylanoides, the leucyl-these respects, the polysemous codon is a phenomenon completely different from these artificial translational tRNA Ser CAG in the cells was estimated to be 3% of the seryl-tRNA Ser CAG, which is much lower than that errors. It is known that many examples exist for alternative decoding of universal codons-initiation codons other obtained in the in vitro experiments. We consider that such a reduction in leucylation is due to the competition than AUG (Gold, 1988; Kozak, 1983) , leaky stop codons caused by nonsense suppresser or native tRNAs (Murgola, for the tRNA Ser CAG between SerRS and LeuRS in the cells. Despite this competition, the distinct detection of 1985), the UGA codon used for incorporation of selenocysteine (Leinfelder et al., 1988) and so on. However, leucylated tRNA Ser CAG in vivo supports the existence of an ambiguous aminoacylation reaction toward the single because of strong dependence on the context effects or possible secondary structures of mRNAs, these recoding tRNA Ser CAG species. The polysemous codon results from the coexistence of events are those which are programed in the mRNAs (Gesteland et al., 1992) . We have sequenced several genes tRNA identity determinants for serine and leucine in a single tRNA molecule. Construction of tRNA Ser CAG in Candida genomes, but we could not find any secondary structure around the codon CUG in these genes. Con-variants by the microsurgery method led to the finding that a single methyl moiety of m 1 G at position 37 sidering that the polysemous codon is mediated by a single tRNA, it is unlikely that a polysemous codon occurs under is involved in the leucylation process. In contrast, the tRNA Ser CAG of C.cylindracea, which has A at the same the influences of the neighboring regions in mRNAs. Alternative decoding of a polysemous codon CUG is position, is deprived of such leucine-accepting activity. Himeno and his co-workers noted that three nucleotides possible, assuming that LeuRS is overexpressed under a certain physiological condition. Depending on the of leucine tRNAs were strongly recognized by S.cerevisiae LeuRS using unmodified variants transcribed by T7 RNA increased amount of the LeuRS in cells, incorporation of leucine corresponding to codon CUG may occur fre-polymerase (Soma et al., 1996) . Although the discriminator base, A73, is the strongest recognition site among them, quently, which causes the production of polypeptides with new functions. This possibility should be examined in A35 and G37 in the anticodon loop also play roles as determinants in tRNA. They were able to compare the further experiments.\nThe idea of a polysemous codon also differs from the activities of variants mutated at position 37 with A or G using the variants with A at the discriminator position 'near-cognate' concept proposed by Schultz and Yarus (1994) . They claimed that ambiguous decoding may occur which effectively elevates leucylation activity. In our work, we utilized serine tRNA with a modified nucleoside as a consequence of an irregular codon-anticodon interaction induced by the 27-43 base pair at the anticodon and with G at the discriminator position as a substrate for LeuRS, because the T7 transcript of tRNA Ser CAG showed stem of the tRNA, resulting in a genetic code change transition state. The polysemous codon found in our study no activity for leucylation. Our experiments using microsurgery methods indicated that m 1 G is of great importance is caused by the tRNA aminoacylation process of tRNA with codon-anticodon interaction proceeding precisely in in leucylation, despite the fact that the presence of G at the discriminator position is unsuitable for the recognition the conventional manner . Furthermore, since the hypothesis of Schultz and Yarus is based of LeuRS. Some modified nucleotides in tRNA are known to be involved in recoginition of some synthetases (Muramatsu et al., 1988) . Pütz et al. (1994) showed that m 1 G at position 37 of yeast tRNA Asp is one of the negative determinants for arginyl-tRNA synthetase.\nWe have also demonstrated that the nucleotide at position 33, where only tRNA Ser CAG uniquely possesses G, modulates the leucine-accepting activity. G33 may prevent tRNA Ser CAG from excessive leucylation, which S.cerevisiae cells, but that the viability of the cells decreased substantially. This finding suggests the polysemous state may be tolerated only when the ambiguous recognizes its cognate leucine tRNA from the 3Ј-side of the anticodon loop, which is afforded by the uridine-turn translation is under a strict constraint. We consider that G33 functions as a negative modulator in the leucylation structure due to U33 ( Figure 6A ). The methyl moiety of m 1 G37 is directly recognized by LeuRS. In the case of tRNA Ser CAG, thereby controlling the relative seryl-to leucyl-tRNA Ser CAG ratio.\nof C.zeylanoides, the anticodon loop distorted by G33 decreases the affinity toward LeuRS, judging from the Several lines of experiment have suggested that U33 is involved in the tRNA function on ribosomes, such as in observation that G33 increased the K m value for leucylation approximately 4-to 5-fold in comparison with that with rigid codon-anticodon interaction, proper GTP hydrolysis of the ternary complex and the efficient translation of prymidine bases at the position ( Figure 6B ). In C.cylindracea, m 1 G is replaced by A, which means that the tRNA termination codons (Bare et al., 1983; Dix et al., 1986) . Indeed, the replacement of G33 by U in C.cylindracea has lost the two major determinants for LeuRS, m 1 G and the discriminator base ( Figure 6C ). Consequently, LeuRS tRNA Ser CAG increased the efficiency of in vitro translation by 2-to 3-fold (data not shown). The negative effect of is unable to recognize tRNA Ser CAG at all, and G33 concomitantly loses its function as a modulator. LeuRS G33 on translation may indicate involvement in some mechanism for decoding the polysemous codon. This is, of course, unable to recognize other serine isoacceptor tRNAs corresponding to universal codons, because they possibility needs to be clarified by further study. Nevertheless, we have shown here that one of the roles of G33 is have modified A at position 37. How did this interaction between LeuRS and the suppression of leucylation, and we consider that the nucleotide at position 33 is not directly involved in tRNA Ser CAG evolve? Candida species utilizing CUG as serine can be classified into two distinct groups: group 1 recognition by LeuRS. On the basis of our observation that no leucylation was detectable in the C.cylindracea contains the species that have tRNA Ser CAG with leucylation activity, and includes C.zeylanoides, C.maltosa and tRNA Ser CAG variants in which G33 was replaced by a pyrimidine base (c-G33U and c-G33C), we speculate that others (see Figure 6B ); group 2, which is represented solely by C.cylindracea, contains species that have tRNA Ser CAG G33 influences the location and/or conformation of m 1 G37, accompanied by the alteration of the anticodon loop without leucylation activity ( Figure 6C ). A plausible evolutionary process is that group 1 would have arisen structure, decreasing the affinity of LeuRS toward tRNA Ser CAG.\nprior to group 2 after the genetic code change, which is speculated on the basis of the following observations. It has been generally considered that reconstructed tRNA does not lose its activity during the several reaction First, the homology between tRNA Ser CAGs in group 1 and its isoacceptor tRNAs for codon UCG is higher than steps needed in the microsurgery method, such as cleavage of the tRNA strand and ligation of tRNA fragments that between the tRNA Ser CAG from C.cylindracea and its isoacceptor . Second, C.cylindracea (Ohyama et al., 1985) . However, a slight reduction of leucylation activity was observed in the control variant, (group 2) possesses high copy numbers of the tRNA Ser CAG genes (~20 copies) on the diploid genome (Suzuki z-G33G, compared with that of the native tRNA ( Figure 3A , right-hand graph), which turned out to result from et al., 1994) , while low copy numbers (two or four copies) are observed for group 1 tRNA Ser CAG genes (Santos the partial deacetylation of 4-acetyl cytidine (ac4C) due to acid treatment of the 5Ј-half fragment (see Materials et al., 1993; Sugiyama et al., 1995; T.Suzuki, personal observations) . Third, the codon CUG is utilized as a major and methods). Nevertheless, it is reasonable to deduce the effect of base replacement on the aminoacylation activity serine codon on several genes in C.cylindracea, such as lipase (Kawaguchi et al., 1989) and chitin synthase by comparing the activities of these reconstructed tRNAs, because the same 5Ј-half fragments were used for all the (unpublished results) , while CUG appears infrequently on the genomes of other species belonging to group 1 (Lloyd manipulated tRNA molecules of C.zeylanoides.\nA plausible mechanism by which LeuRS could recog-and Sharp, 1992; Sugiyama et al., 1995; T.Suzuki, personal observations) . During the course of the change in the nize cognate leucine and serine tRNAs specific for codon CUG is illustrated in Figure 6 . LeuRS contacts and genetic code, the genome should pass through a state group 2 (Ohama et al., 1993) . Fourth, the phylogenetic dextrose) and minimal medium SD [0.67% yeast nitrogen base without tree of these species and relatives constructed by using amino acids (Difco) and 2% dextrose] supplied with 24 mg/ml uracil were used for the cultivation of yeast cells.\nseveral genes also supports this evolutionary pathway SD-plates with or without uracil were prepared by adding agar at a (manuscript in preparation (Boeke et al., 1984) .\nfor codon UCG Pesole et al., 1995) . Thus, the nucleotide at position 37 seems likely to have\nIn order to introduce mutation at the 45th codon in the reading frame mutated in the direction modified A→m 1 G- Alternative splicing generates a multiple protein pUTH18 containing an autonomously replicating sequence of C.maltosa (Takagi et al., 1986) and C-HIS5 (Hikiji et al., 1989) were used as sequence from a single gene at the mRNA level. In when the codon appears infrequently, as observed in group was mutated from CTG to CTC, and pCCU (Sugiyama et al., 1995) synthesis caused by a polysemous codon. We speculate instruction manual. The electrified cells were spread on a SD-plate that such ambiguity could have given rise to proteins containing uracil and incubated at 30°C.\nwith multiple amino acid sequences in non-house-keeping genes, which may have conferred multifunctionality on\nIn vitro aminoacylation assay Seryl-or leucyl-tRNA synthetases were partially purified from C.zeylan-the proteins. Since the C.cylindracea strain was developed oides cells as described previously , both of the industrially for the production of lipase, such multifunc- Large-scale purification of tRNA Ser CAGs from C.zeylanoides and C.cylindracea mmol) and leucine (11.5 MBq/mmol) were from Amersham. 5-fluoroorotic acid monohydrate (5FOA) was from PCR inc. 3Ј-Biotinylated Candida cylindracea cells (3.1 kg) were treated with phenol, from which 150 000 A 260 units of unfractionated tRNA were extracted. Eighty DNA probes were synthesized by Sci. Media, Japan. Synthetic RNA oligomers and a chimeric oligonucleotide composed of DNA and 2Ј-O-thousand A 260 units of tRNA mixture were obtained by DEAE-cellulose chromatography with stepwise elution, which was then applied onto a methyl RNA were synthesized by Genset Co. Ltd. Most of the enzymes used for the microsurgery were from Takara Shuzo (Tokyo, Japan).\nDEAE-Sephadex A-50 column (6ϫ100 cm). Elution was performed with a linear gradient of NaCl from 0.375 to 0.525 M in a buffer Other chemicals were obtained from Wako Chemical Industries.\nconsisting of 20 mM Tris-HCl (pH 7.5) and 8 mM MgCl 2 . The fraction as CZE-37 (5ЈGmCmCmCmAmAmUmGmGmAmAmdCdCdTdG-CmAmUmCmCmAmUm3Ј), possessing a cleavage site between posi-rich in tRNA Ser was applied onto a RPC-5 column (1ϫ80 cm) and eluted with a linear gradient of NaCl from 0.4 to 1 M NaCl in a buffer tions 37 and 38 of C.zeylanoides tRNA Ser CAG. Two hundred micrograms of purified tRNA Ser CAG from C.zeylanoides was incubated at 65°C for consisting of 10 mM Tris-HCl (pH 7.5) and 10 mM Mg(OAc) 2 . As a result of these chromatographies, 300 A 260 units of purified tRNA Ser CAG 10 min with 14.4 nmol CZE-37 in a buffer consisting of 40 mM Tris-HCl (pH 7.7), 0.5 mM NaCl, 0.1 mM DTT, 0.0003% BSA and 0.4% were finally obtained.\nOne hundred and fifty thousand A 260 units of tRNA from C.zeylanoides glycerol (500 µl), and then annealed at room temperature. Magnesium chloride was added to the mixture up to a final concentration of 4 mM cells (3.7 kg) were fractionated on DEAE-Sepharose fast-flow column (3.5ϫ130 cm) with a linear gradient of NaCl from 0.25 to 0.4 M in a and the reaction was carried out at 30°C for 2 h by the addition of 600 units of RNase H (Takara Shuzo). About 60 µg of the cleaved 3Ј-half buffer consisting of 20 mM Tris-HCl (pH 7.5) and 8 mM MgCl 2 . About 300 A 260 units of C.zeylanoides tRNA Ser CAG were finally obtained by fragment was obtained by purification using 10% PAGE containing 7 M urea. Either of two synthetic oligo-RNAs, pCAGAp or pCAGGp, was further column chromatography with Sepharose 4B in a reverse gradient of ammonium sulfate from 1.7 to 0 M with a buffer consisting of ligated with the same 5Ј-half fragment digested by RNase T1 as the variants mutated at position 33 under the conditions described above. 10 mM NaOAc (pH 4.5), 10 mM MgCl 2 , 6 mM β-mercaptoethanol and 1 mM EDTA.\nThe ligated and dephosphorylated 5Ј-half fragments were annealed and ligated with the 3Ј-half fragment digested by RNase H. About 50 µg of each of the two variants from C.zeylanoides mutated at position 37-Construction of tRNA variants with mutation at position 33 The microsurgery procedures were basically carried out according to the m 1 G37A and m 1 G37G-was obtained by the phosphorylation of the 5Јend and purification by 12% PAGE containing 7 M urea. literature (Ohyama et al., 1985 (Ohyama et al., , 1986 . Limited digestion of 4 mg purified tRNA Ser CAG from C.zeylanoides with RNase T1 was performed at 0°C for 30 min in a reaction mixture containing 50 mM Tris-HCl (pH 7.5), Identification of amino acids attached to tRNA Ser CAGs in 100 mM MgCl 2 , 0.5 mg/ml of the tRNA and 25 000 units/ml RNase the cells T1 (Sigma). After phenol extraction, the resulting fragments were treated Identification of aminoacyl-tRNA Ser CAG from Candida cells was carried with 0.1 N HCl at 0°C for 12 h in order to cleave the 2Ј, 3Ј cyclic out by a new method developed recently by us (Suzuki et al., 1996) . phosphate of the 3Ј-end of the fragments formed in the limited digestion,\nThe experimental conditions were the same as those reported. To and then the 5Ј-and 3Ј-half fragments were separated by 10% PAGE fish out the aminoacyl-tRNAs, we designed two 3Ј-biotinylated DNA containing 7 M urea (10ϫ10 cm). Four hundred and thirty micrograms probes: 5ЈAGCAAGCTCAATGGATTCTGCGTCC3Ј for C.cylindracea of the 5Ј-half and 520 µg of the 3Ј-half fragments were recovered from tRNA Ser CAG and 5ЈGAAGCCCAATGGAACCTGCATCC3Ј for the gel. The purified 5Ј-half fragment was dephosphorylated with C.zeylanoides tRNA Ser CAG. These probes were immobilized with strepbacterial alkaline phosphatase (Takara Shuzo), and G33 at the 3Ј-end of tavidin agarose (Gibco BRL) as reported previously (Wakita et al., 1994) . the 5Ј-half fragment was removed by oxidation with sodium periodate as described in the literature (Keith and Gilham, 1974) . After dephos-" }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: RNA recombination is a very common phenomenon. It has been observed in all types of viruses using RNA as a carrier of genetic information: in positive-sense, single-stranded RNA viruses (1) (2) (3) (4) , in negative-sense, single-stranded RNA viruses (5, 6) , in double-stranded RNA viruses (7, 8) and in retroviruses (9) (10) (11) . Moreover, it has been shown that RNA recombination enables the exchange of genetic material not only between the same or similar viruses but also between distinctly different viruses (12) . Sometimes it also permits crossovers between viral and host RNA (13) (14) (15) (16) (17) . Taking into account the structure of viral genomic molecules and the location of crossover sites, three basic types of RNA recombination were distinguished: homologous, aberrant homologous and non-homologous (3, 4, 18) . The former two occur between two identical or similar RNAs (or between molecules displaying local homology), while the latter involves two different molecules. Most of the collected data suggest that RNA recombinants are formed according to a copy choice model (4, 18) . A viral replication complex starts nascent RNA strand synthesis on one template, called RNA donor and then switches to another template, called RNA acceptor. Accordingly, two main factors are thought to affect RNA recombination: the structure of recombining molecules and the ability of the viral replicase to switch templates.\nTo gain more knowledge of the mechanism of RNA recombination, several model experimental systems have been created. They provided us with some specific data describing homologous and/or non-homologous recombination in particular viruses, e.g. in poliovirus, (19) mouse hepatitis virus (20, 21) , brome mosaic virus (BMV) (4, 22) , turnip crinkle virus (23, 24) or tomato bushy stunt virus (25) . As a result, the involvement of viral replicase proteins in recombination has been demonstrated (26, 27) and a wide spectrum of RNA motifs supporting recombination have been identified (4, 23, (28) (29) (30) . In general, the collected data suggest that there exist two major types of RNA structural elements that induce recombination events: (i) universal ones mediating template switching by different viral replicases, e.g. regions *To whom correspondence should be addressed. Tel: +48 61 8528503; Fax: +48 61 8520532; Email: [email protected] The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact [email protected] of local homology (28, 31) or complementarity (32) (33) (34) (35) and (ii) virus-specific ones, e.g. promoter-like structures (36, 37) . Unfortunately, up till now there has been no in vivo recombination system that could be used to test the recombination activity of any given RNA sequence and consequently to verify the above hypothesis and find some general laws governing the studied process.\nIn our studies on genetic RNA recombination we have used the well-characterized in vivo system developed in BMV (30, 33) . BMV is a model (+)RNA virus of plants (38) . Its genome is composed of three segments called RNA1, RNA2 and RNA3. RNA1 and RNA2 encode BMV replicase proteins 1a and 2a, respectively. RNA3 encodes movement (3a) and coat proteins (CP) (38) . All three BMV RNAs possess an almost identical 3 0 -untranslated region (3 0 -UTR). The first BMV-based recombination system was created by Nagy and Bujarski (33) . They constructed a recombinationally active BMV mutant whose genome is composed of wtRNA1, wtRNA2 and modified RNA3 (PN0-RNA3 called the recombination vector, for details see Figure 1 ). Only 3 0 -UTR was modified in PN0-RNA3, while its 5 0 -UTR, intergenic and coding regions were unchanged. Despite the introduced changes, the recombination vector is stable and replicates when used together with wtRNA1 and wtRNA2 to infect plants. It starts to recombine if a recombinationally active sequence (RAS) is introduced just between the CP coding sequence and the modified 3 0 region (into the RAS-cloning site). Non-homologous recombination was observed when a 140-60 nt sequence complementary to RNA1 between positions 2856 and 2992 was inserted into PN0-RNA3 (a sequence from the 3 0 -portion of RNA1 was introduced in antisense orientation) (30, 33) . Interestingly, the same RNA1 fragment inserted in sense Figure 1 . The BMV-based recombination system. White, black and gray boxes represent coding, noncoding and recombinationally active sequences, respectively. The location of the primers (A and B) used for specific RT-PCR amplification of the 3 0 -portion of BMV RNA3 (parental or recombinant) is indicated by arrows. (A) BMV genome. The BMV genome consists of three RNA segments: RNA1, RNA2 and RNA3. All three BMV RNAs share an almost identical 3 0 -noncoding region with a tRNA-like structure at the very end. (B) Recombination vector. The PN0-RNA3 vector is a wtRNA3 derivative with a modified 3 0 -noncoding end [(for details see ref. (33) ]. The latter includes (i) the RAS cloning site, (ii) a 197 nt sequence derived from the 3 0 -noncoding region of cowpea chlorotic mottle virus RNA3 (marked as CCMV), (iii) the sequence of wtRNA3 between nt 7 and 200 (counting from the 3 0 end-marked as the region B) and (iv) the last 236 nt from the 3 0 end of BMV wtRNA1 (marked as A). (C) Non-homologous recombination. Non-homologous recombination was observed if an 140 nt sequence, called RAS1as (shown in A), complementary to wtRNA1 between positions 2856 and 2992 was inserted into the PN0-RNA3 vector. The presence of the RAS1as sequence in RNA3 derivative (called Mag1-RNA3) allows local RNA1-RNA3 hybridization that mediates frequent non-homologous crossovers. It is thought that polymerase starts nascent strand synthesis on the 3 0 end of RNA1 and then switches to RNA3 within a local double-stranded region. (D) Non-homologous recombinant. Non-homologous recombination repairs Mag1-RNA3 by replacing its modified 3 0 end with the 3 0 -noncoding fragment coming from RNA1. orientation did not support homologous crossovers (33) . Non-homologous recombination repaired the RNA3 vector by replacing its highly modified 3 0 end with 3 0 -UTR derived from RNA1. The resultant recombinants replicated and accumulated better than the parental RNA3 molecule, and so the latter was out competed from the infected cells. The above system is extremely efficient, since it employs selection pressure to support the accumulation of RNA3 recombinants. Because RAS is placed in two different segments of the BMV genome, we have proposed to name this system heteromolecular.\nUnfortunately, Nagy and Bujarski's BMV-based recombination system has one serious limitation. It was designed in such a manner that viable RNA3 recombinants can easily form only if a sequence derived from the 3 0 -portion of RNA1 or RNA2 is used as a RAS. Consequently, the heteromolecular system could not be applied for testing the recombination capacity of various RNA motifs. Olsthoorn et al. (39) attempted to solve that problem by inserting examined sequences into the 3 0 -noncoding region of BMV RNA2 and RNA3. This system was not further developed, since any changes in RNA2, which encodes BMV polymerase, could strongly affect the studied process.\nHere, we describe a new BMV-based recombination system. It has been constructed in such a way that both tested RASes are placed in the same segment of the BMV genome (in the modified RNA3 molecule); therefore, we have called this system homomolecular. To prove the usefulness of the homomolecular system, we have employed it to examine the recombination activity of sequences derived from the hepatitis C virus (HCV) genome. The examined sequences have been inserted into RNA3 as direct or inverted repeats. This demonstrated that the 101 nt hypervariable region of HCV efficiently supports both homologous and non-homologous crossovers, while the most conservative 98 nt portion of HCV's 3 0 -UTR induces only non-homologous recombination events. Moreover, a direct comparison of the hetero-and homomolecular systems revealed crucial differences between the mechanisms of homologous and non-homologous recombination. The former involves preferentially two different segments of the BMV genome and the latter occurs more easily between the same genomic RNAs.\nPlasmids pB1TP3, pB2TP5 and pPN0-RNA3 containing fulllength cDNA of BMV RNA1, RNA2 and modified RNA3 (recombination vector), respectively, were the generous gift from J. J. Bujarski (Northern Illinois University, DeKalb, IL).\nRestriction enzymes (EcoRI, SpeI and XbaI) T7 RNA polymerase, RNasine, RQ DNase RNase free, MMLV-reverse transcriptase, Taq polymerase and pUC19 cloning vector were from Promega.\nThe following primers were used for the construction of pMatNH-pMatH-, pMatNH-HVR-, pMatH-HVR-, pMatNH-X-, pMatH-X-RNA3: \nPlasmids pMag1-and pMagH-RNA3 contain full-length cDNA of the RNA3 vector carrying the recombinationally active sequence RAS1 inserted in antisense or sense orientation, respectively. Both plasmids were constructed in the same way: pPN0-RNA3 was linearized with SpeI endonuclease and ligated with SpeI cut RAS1 cDNA. Then plasmids carrying RAS1 in antisense (pMag1-RNA3) and sense (pMagH-RNA3) orientation were identified (30) .\nTo prepare pMatNH-RNA3 and pMatH-RNA3 plasmids (containing full-length cDNA of MatNH-RNA3 and MatH-RNA3), pMag1-RNA3 and pMagH-RNA3 were digested with KpnI and EcoRI endonucleases. Then, the deleted fragment was replaced with a KpnI-EcoRI cut 379 nt cDNA fragment corresponding to the BMV RNA1 3 0 end (containing the entire 3 0 -UTR and RAS1). The latter were obtained by PCR involving primers 1, 2 and pB1TP3 as a template.\nTo construct pMat0-RNA3, i.e. a plasmid containing cDNA of the universal recombination vector Mat0-RNA3, the following modifications were introduced into pMatNH-RNA3. First, it was digested with SpeI endonuclease and religated. This way 5 0 RAS1as was removed and the 5 0 RAS cloning site (including only one restriction site SpeI) was created. Next, the plasmid was cut with KpnI and EcoRI to remove RNA1 3 0 -UTR and 3 0 RAS1s. Instead, a 295 nt fragment of RNA1 3 0 end (between positions 2940 and 3234) followed by the 3 0 RAS cloning site (including KpnI, MluI, BamHI and EcoRV restriction sites) was ligated into pMatNH-RNA3. The inserted sequence was obtained by PCR using primers 2, 3 and pB1TP3 as a template and digested with KpnI and EcoRI, prior to ligation.\nTo test the recombination activity of HCV-derived sequences (hypervariable region 1, abbreviated HVR and sequence X, abbreviated X) cDNA of the corresponding fragments of the virus' genome was obtained by RT-PCR method (40, 41) and cloned into the pUC19 vector. Then, both tested sequences were amplified by PCR with primers introducing an SpeI restriction site. Primers 4, 5 and primers 6, 7 were used to obtain HVR and X cDNA, respectively. PCR products and pMat0-RNA3 were digested with SpeI and ligated. Then pMat0-RNA3 derivatives bearing HVR and X in sense and antisense orientation were identified. HVR and X were amplified again by PCR involving primers introducing MluI and EcoRV restriction sites (primers 8, 9 and 10, 11 to amplify HVR and X, respectively). PCR products were cut with MluI and EcoRV and ligated into the 5 0 RAS cloning site of previously identified pMat0-RNA3 derivatives (carrying HVR and X in sense and antisense orientation). As a result four plasmids were obtained: (i) pMatH-HVR-RNA3-containing cDNA of MatH-HVR-RNA3 in which two HVRs were inserted in sense orientation; (ii) pMatNH-HVR-RNA3-containing cDNA of MatNH-HVR-RNA3 possessing two HVRs, 5 0 HVR in antisense and 3 0 HVR in sense orientation; (iii) pMatH-X-RNA3containing cDNA of MatH-X-RNA3 in which two Xes are in sense orientation; (iv) pMatNH-X-RNA3-containing cDNA of MatNH-X-RNA3 carrying two Xes, 5 0 X in sense and 3 0 X in antisense orientation. Their structure was confirmed by sequencing.\nTo test the recombination activity of the BMV mutants, the previously described procedure was applied (30, 33) . Infectious BMV genomic RNAs were obtained by in vitro transcription for which EcoRI linearized plasmids pB1TP3, pB2TP5, pMag1-RNA3, pMagH-RNA3, pMatNH-RNA3, pMatH-RNA3, pMat0-RNA3, pMatH-HVR-RNA3, pMatH-HVR-RNA3, pMatHN-X-RNA3 and pMatH-X-RNA3 were used. Five-leaf C.quinoa plants (local lesion host for BMV) were mechanically inoculated with mixtures containing BMV RNA1, RNA2 and one of the RNA3 derivatives. Two weeks post-inoculation, the number of lesions developed on each inoculated leaf was counted to establish the infectivity of the tested BMV mutant. Then, individual local lesions were excised and total RNA was extracted separately from every lesion. The isolated RNA was subjected to RT-PCR involving primer A (the first strand primer) and primer B (the second strand primer) specific for RNA3 3 0 fragment amplification (the region where recombination crossovers occur). As a control identical reactions involving either parental RNA3 transcript (positive control) or water (negative control) were carried out. RT-PCR products were analyzed by electrophoresis in a 1.5% agarose gel. The formation of 800 nt or shorter 500 nt products indicated that parental or recombinant RNA3 accumulated in the analyzed lesion, respectively. Next, RT-PCR products were cloned into the pUC19 vector and sequenced to determine the location of recombinant junction sites. Finally, the presence of recombinants in the selected local lesions was additionally confirmed by northern blot analysis.\nThe main question that we had to answer during our studies was how to design a vector that could be used for examining the recombination activity of any RNA sequences in vivo. As a result, the idea arose to construct a BMV-based homomolecular recombination system. In such a system, both tested sequences are supposed to be present within the same segment of the BMV genome (either in RNA1, RNA2 or RNA3). Thus, a new vector should possess two separately located RAS cloning sites, be replicable and stable during infection. It has to be capable of generating viable recombinants, which have selective advantage over parental RNA molecules. Consequently, recombinants ought to be able to out compete the vector with inserted RASes. Assuming that RNA recombination occurs according to a copy choice mechanism, we decided that RNA3, being dispensable for BMV replication, is the best candidate for a new vector. Any changes in RNA1 and RNA2, which encode BMV replicase proteins, would strongly affect the studied process. The next important question was whether the location of RASes within the same (homomolecular system) or within two different segments of the BMV genome (heteromolecular system) influences the recombination activity of the examined RNA sequence.\nTo address both issues, we decided to construct a so-called mixed system, homo-and heteromolecular at the same time.\nTo this end two RNA3 molecules, prototypes of a new vector carrying two RASes, were prepared. To obtain them we used PN0-RNA3, described earlier, and a well-characterized recombinationally active sequence from BMV RNA1 (RAS1, see Figure 1 ). The 137 nt RAS1 corresponding to RNA1 between positions 2856 and 2992 was inserted into the PN0-RNA3 RAS cloning site, in antisense (RAS1as) and sense (RAS1s) orientations ( Figure 2 ). As a result, we obtained Mag1-and MagH-RNA3 derivatives (30) . Then the 356 nt portion of Mag1-and MagH-RNA3 3 0 end was replaced with a 379 nt sequence representing the wtRNA1 3 0 end (fragment encompassing the entire 3 0 -UTR and RAS1s sequence) ( Figure 2 ). In addition, a marker mutation (called DXho) was introduced within the RNA1-derived fragment to make it distinguishable from an analogous region present in wtRNA1. To this end, the XhoI restriction site (2988-2994) was disrupted by a 4 nt insertion (GATC) between C-2991 and G-2992. Resultant RNA3 derivatives, called MatNH-and MatH-RNA3, have unchanged 5 0 -UTR, intergenic and coding regions and a highly modified 3 0 -UTR. The latter includes 3 0 -UTR coming from wtRNA1 and two RAS1 sequences (3 0 RAS1 and 5 0 RAS1) separated by a 338 nt spacer (sequence CCMV and B1). In MatNH-RNA3, 3 0 RAS1 is located in sense and 5 0 RAS1 in antisense orientation, while in MatH-RNA3 both RAS1 sequences are in sense orientation ( Figure 2 ).\nHaving these two RNA3 derivatives, we were able to construct two variants of the mixed system: one for homologous (MatH-BMV mutant) and the other for non-homologous (MatNH-BMV mutant) recombination studies. The MatH-BMV genome is composed of wtRNA1, wtRNA2 and MatH-RNA3 and the MatNH-BMV genome of wtRNA1, wtRNA2 and MatNH-RNA3 (Table 1 ). In genomes of both BMV mutants three copies of RAS1 are present: two in the RNA3 derivative (RAS1s-RAS1s or RAS1as-RAS1s in MatH-and MatNH-RNA3, respectively) and one in wtRNA1 (RAS1s). Thus, in the mixed systems two identical RASes or RAS and its complementary counterpart were capable of supporting, respectively, homologous or non-homologous (heteroduplex-mediated) recombination between the same or between different BMV genomic RNAs. As a result, we could directly compare homo-and heteromolecular recombination systems in one in vivo experiment and examine whether our presumptions concerning the new recombination vector are correct.\nHomologous recombination in the mixed homo-heteromolecular system Earlier, Nagy and Bujarski (33) demonstrated that the 66 nt portion of RAS1 did not support homologous recombination in heteromolecular system. We repeated this experiment using MH-BMV mutants. Recombinants also did not form although the entire RAS1 sequence was present in wtRNA1 and MagH-RNA3 molecules (Table 1 and Figure 3 ). To test RAS1 activity Figure 1 , white, black and gray boxes represent coding, noncoding and recombinationally active sequences, respectively, in sense (RAS1s) or antisense (RAS1as) orientation, dashed line squares encompass replaced parts of wtRNA1 and modified RNA3 molecules. Mag1-and MagH-RNA3 were created by inserting the RAS1 sequence from wtRNA1 into the RAS cloning site of PN0-RNA3 in antisense (Mag1-RNA3) or sense (MagH-RNA3) orientation. To construct MatNH-RNA3 and MatH-RNA3, the 356 nt very 3 0 end of Mag1-RNA3 or MagH-RNA3 (between KpnI and EcoRI sites) was replaced with a 379 nt portion of the wtRNA1 3 0 end (fragment containing the entire 3 0 -UTR and RAS1). Thus, both constructs contain two copies of RAS1 sequence-MatNH-RNA3 includes 5 0 RAS1as and 3 0 RAS1s, while MatH-RNA3 comprises 5 0 RAS1s and 3 0 RAS1s. Furthermore, a marker mutation DXho (marked as a white dot), removing the XhoI restriction site, was introduced into the 3 0 end of MatNH-and MatH-RNA3, to make it distinguishable from an analogous region present in wtRNA1. in the mixed homologous recombination system, a previously used, well-established procedure was applied (30, 33) . C.quinoa plants (local lesion host for BMV) were inoculated with a mixture containing in vitro transcribed wtRNA1, wtRNA2 and MatH-RNA3. After 2 weeks, when infection symptoms were well developed, the number of lesions formed on every inoculated leaf was counted to determine the infectivity of the MatH-BMV mutant. Individual local lesions were excised and total RNA was extracted separately from each of them. Then, the 3 0 -portion of RNA3 progeny accumulating in examined lesions was selectively amplified by RT-PCR involving RNA3 specific primers A and B (for their location see Figure 1 ). Reaction products were separated in a 1.5% agarose gel and their length was determined. The formation of an 800 or 400-500 nt DNA fragment indicated that the lesion contained parental or recombinant RNA3, respectively. In this way, we were able to determine the number of lesions in which a viable RNA3 recombinant was generated. The presence of recombinants in analyzed lesions was confirmed by standard northern blot hybridization. DNA fragments obtained during selective RT-PCR amplification of RNA3 were cloned and sequenced. Finally, the results obtained with our new mixed homologous recombination system were compared with analogous data previously got using the heteromolecular system (33) (see Table 1 and Figure 3 ). The data presented in Table 1 indicate that the exchange of MagH-RNA3 (carrying a single RAS1) into MatH-RNA3 (bearing two RAS1 sequences) did not affect the infectivity of the BMV mutants. The average numbers of lesions appearing on the leaves inoculated with MH-and MatH-BMV were similar: 18 and 17, respectively. Interestingly, although RAS1 did not support homologous crossovers in the heteromolecular system represented by MH-BMV, it was very active in the mixed system. About 85% of the local lesions developed during MatH-BMV infection accumulated the RNA3 recombinant instead of parental MatH-RNA3. In all of them, one RAS1 and a spacer were deleted. This indicates that crossovers occurred either within 3 0 end 5 0 RAS1 present in MatH-RNA3 (inter-or intramolecular crossovers) or within RAS1 and 5 0 RAS1 located in wtRNA1 and MatH-RNA3, respectively. Recombinant junction sites were placed in identical regions; therefore, their location could not be precisely established.\nThe data presented till now also could not answer which molecules, exclusively MatH-RNA3 or wtRNA1 and MatH-RNA3, participated in recombination. In the heteromolecular system, only RAS1-mediated crossovers between wtRNA1 and MagH-RNA3 were permitted. The situation seems to be more complicated in the mixed homologous system where three copies of RAS1 are present, all in sense orientation: two of them in MatH-RNA3 (3 0 RAS1 and 5 0 RAS1) and one in wtRNA1. Consequently, RAS1-mediated homologous recombination may happen according to four different scenarios ( Figure 3 ). It can engage MatH-RNA3 only and occur as intra-or intermolecular process or it can involve wtRNA1 and MatH-RNA3. In the latter case, recombination can be mediated by RAS1 present in wtRNA1 and either 5 0 -or 3 0 RAS1 located in MatH-RNA3.\nTo learn according to which scenario homologous recombination occurred, we checked whether mutation DXho introduced into MatH-RNA3 (just behind 3 0 RAS1) is still present in RNA3 recombinants. In this way, we were able to determine if their 3 0 -UTR was derived from MatH-RNA3 or wtRNA1 molecules. The undertaken analysis revealed that the mutation was present in 20% of recombinants. This result suggested that homologous crossovers preferentially occur between wtRNA1 and MatH-RNA3.\nHowever, there are other explanations why DXho was absent in a large fraction of recombinants. It is possible that the mutation was removed either from MatH-RNA3, due to homologous recombination between its 3 0 RAS1 and wtRNA1, or from the RNA3 recombinant (carrying a single copy of RAS1) because it could also have recombined with wtRNA1. To examine the first possibility, progeny RNA3 extracted from the local lesions accumulating MatH-RNA3 (lesions in which recombinant was not generated) was analyzed. About 800 nt RT-PCR products obtained during selective amplification of RNA3's 3 0 -portion were cloned and sequenced. In all of 20 analyzed clones DXho was present. To test the second possibility, a full-length cDNA clone of RNA3 recombinant containing DXho (RNA3-DXhoR) was obtained. It was inserted into the pUC19 vector under the T7 polymerase promoter. The resultant plasmid named pRNA3-DXhoR was used after linearization to produce an infectious RNA3-DXhoR molecule by in vitro transcription. Then, RNA3-DXhoR was used together with wtRNA1 and wtRNA2 to inoculate C.quinoa plants. After 2 weeks, total RNA was extracted from individual lesions and a 3 0 -portion of the progeny RNA3 was amplified by RT-PCR. Obtained products were cloned and sequenced. As described previously, DXho was present in all of the analyzed 20 clones. These two experiments proved that homologous recombination between either 3 0 RAS1 of MatH-RNA3 or RAS1 present in RNA3 recombinant and wtRNA1 does not occur frequently enough to explain why most recombinants lack DXho. Altogether, these results supported our initial thesis that DXho was removed from 80% of homologous recombinants, since most of the crossovers occurred within 5 0 RAS1 from MatH-RNA3 and RAS1 from wtRNA1.\nEarlier we showed that RAS1 can effectively support nonhomologous recombination if inserted into PN0-RNA3 in antisense orientation (30, 33) . The heteromolecular system used in our experiment was composed of wtRNA1, wtRNA2 and Mag1-RNA3 (M1-BMV mutant). Crossovers occurred within the local double-stranded region (local heteroduplex), which wtRNA1 and Mag1-RNA3 were capable of forming. In order to test RAS1 activity in the mixed non-homologous recombination system, C.quinoa plants were inoculated with the MatNH-BMV mutant (its genome is composed of wtRNA1, wtRNA2 and MatNH-RNA3). Two weeks later progeny RNA3 were analyzed as described above. The number of lesions developed on each leaf was counted and total RNA was extracted from individual local lesions. After RT-PCR amplification, the 3 0 -portion of BMV RNA3 accumulating in each lesion was analyzed in an agarose gel, cloned and sequenced. The presence of recombinants was confirmed by a standard Northern blot. The results obtained were compared with analogous data we had got using the heteromolecular system (30) (Table 1 and Figure 4) .\nAs described previously, we observed that the exchange of Mag1-RNA3 (with a single RAS1as sequence) for MatNH-RNA3 (with two sequences: RAS1as and RAS1s) did not influence the infectivity of the BMV mutants. The average numbers of lesions developed on each leaf during infection with M1-BMV and MatNH-BMV were 19 and 18, respectively. There was also no difference between the recombination activity of M1-BMV and MatNH-BMV. RAS-1 (in fact RAS1s and RAS1as) supported non-homologous recombination . Non-homologous recombination in the heteromolecular and mixed homo-heteromolecular systems. As in Figure 3 , light and thicker lines represent viral genomic RNAs and nascent recombinant RNA, respectively. WtRNA1 is red (the recombinationally active sequence RAS1s which it contains is additionally boxed), wtRNA2 is black and modified RNA3 is blue. When inserted into RNA3 the wtRNA1-derived RAS1s sequence is also shown as a red box, whereas the complementary RAS1as sequence is shown as a green box. The portion of the RNA3 recombinant synthesized on wtRNA1 is red, the portion synthesized on the RAS1as sequence is green and the fragment synthesized on RNA3 is blue. The black dot symbolizes the DXho mutation present in MatNH-RNA3. RF, recombination frequency. Dashed line squares encompass the region identical in both systems [the region where crossovers occur, shown in detail in (E)]. (A) Heteromolecular system (M1-BMV). A detailed description of the M1-BMV genome is presented in Figure 1 . All nascent RNA3 molecules accumulating in M1-BMV infected plants were recombinants (RF = 100%). (B) Mixed system (MatNH-BMV). Three copies of RAS1 are located in the MatNH-BMV genome, two in MatNH-RNA3 (3 0 RAS1s and 5 0 RAS1as) and one in wtRNA1 (RAS1s). The recombination frequency observed during infection with MatNH-BMV was 95%. Of the identified recombinants, 10% were without the DXho marker, and 90% with the DXho marker. (C) Putative scenario of RAS1s/RAS1as-mediated non-homologous recombination in the heteromolecular system. Owing to the presence of RAS1s and RAS1as sequences in wtRNA1 and Mag1-RNA3, respectively, they are capable of forming a local double-stranded structure supporting non-homologous crossovers (for details see Figure 1 ). (D) Putative scenarios of RAS1s/RAS1as-mediated non-homologous recombination in the mixed system. The presence of 3 0 RAS1s and 5 0 RAS1as sequences in MatH-RNA3 and RAS1s in wtRNA1 creates several opportunities of heteroduplex formation: between wtRNA1 RAS1s and MatNH-RNA3 5 0 RAS1as (intermolecular), between two pairs of RAS1s/RAS1as sequences of two different MatNH-RNA3 molecules (intermolecular) and between 5 0 RAS1as and 3 0 RAS1s of the same MatNH-RNA3 molecule (intramolecular). Recombinants are generated if BMV replicase initiates nascent strand synthesis at the 3 0 end of wtRNA1 or MatNH-RNA3 and then switches to MatNH-RNA3 within the local double-stranded region. (E) Recombinants identified during M1-and MatNH-BMV infection. Boxed fragments of recombining wtRNA1/Mag1-RNA3, wtRNA1/MatNH-RNA3 and MatNH-RNA3/MatNH-RNA3 molecules are practically identical in both systems (except for the DXho mutation present in MatNH-RNA3). The locations of the junction sites are marked with arrows and letters. The numbers indicate how many recombinants of the same type were isolated. Upper case letters refer to M1-BMV, lower case letters refer to MatNH-BMV. equally in both systems. Recombination events occurred with a similar frequency (100 and 95% for M1-and MatNH-BMV, respectively) and recombinant junction sites were located within the same region of the heteroduplexes, which recombining molecules were capable of forming.\nIn the heteromolecular system, only one type of heteroduplex supporting non-homologous crossovers could possibly form: between wtRNA1 and Mag1-RNA3. In the mixed system, recombining molecules were capable of forming three types of heteroduplexes: (i) intermolecular, between 5 0 RAS1as from MatNH-RNA3 and RAS1s from wtRNA1, (ii) intermolecular, between 5 0 RAS1as and 3 0 RAS1s located in two MatHN-RNA3 molecules and (iii) intramolecular, between 5 0 RAS1as and 3 0 RAS1s located in the same MatNH-RNA3 molecule (Figure 4) . To determine the molecules that participated in non-homologous recombination, the RT-PCR amplified 3 0 -portions of RNA3 were checked for DXho. It was present in 90% of recombinants. This result clearly showed that non-homologous crossovers almost always involve one (intramolecular recombination) or two (intermolecular recombination) MatNH-RNA3 molecules.\nThe results presented above indicated that the homomolecular system can provide new interesting data concerning the mechanism of RNA recombination, especially if it could be used for testing the recombination activity of RNA sequences derived from other RNA-based viruses. Consequently, we attempted to construct a universal BMV RNA3-based recombination vector called Mat0-RNA3 (for details see Materials and Methods and Figure 5A ). In Mat0-RNA3, as in the former PN0-RNA3 vector, only 3 0 -UTR was modified. It is composed of the 295 nt very 3 0 end of RNA1 followed by the 3 0 RAS cloning site, a 338 nt spacer and the 5 0 RAS cloning site.\nTo determine the infectivity and stability of the new vector, C.quinoa plants were inoculated with a mixture containing wtRNA1, wtRNA2 and Mat0-RNA3 (Mat0-BMV mutant). After 2 weeks, the number of lesions developed on inoculated leaves was counted and then standard analysis of progeny RNA was carried out. Twenty separate lesions were excised, the total RNA was isolated and used for the selective RT-PCR amplification of the 3 0 -portion of progeny RNA3. The length of RT-PCR products was established by electrophoresis in a 1.5% agarose gel. In addition, reaction products were cloned and sequenced. This demonstrated that the Mat0-BMV mutant is infectious (usually 20 lesions were developed on each leaf, see Table 2 ) and Mat0-RNA3 is stable during the whole period of infection and thus it can be used as a recombination vector.\nIn order to demonstrate that Mat0-RNA3 can be used as an effective tool in recombination studies, we applied it to examine the recombination activity of two specific sequences derived from the HCV genome. The first, 101 nt sequence is placed within the 5 0 -portion of the HCV genome (within the fragment encoding E2 protein) and is named HVR (40, 42) . The second, called the sequence X (X) constitutes a 98 nt 3 0 end of HCV genomic RNA. It has been shown that X represents the most conservative fragment of HCV genome (43) . Both sequences were obtained by a standard RT-PCR method involving viral RNA isolated from the blood of infected patients as a template (40, 41) . Amplified fragments were inserted into the 5 0 -cloning site of Mat0-RNA3 in two different orientations (sense and antisense), then only in sense orientation into the 3 0 -cloning site (for details see Materials and Methods). As a result, four different Mat0-RNA3 derivatives were generated: (i) MatH-HVR-RNA3, possessing two copies of HVR in sense orientation (3 0 and 5 0 HVRs); (ii) MatH-X-RNA3, with two copies of X in sense orientation (3 0 and 5 0 Xs); (iii) MatNH-HVR-RNA3, with two copies of HVR, the 3 0 -copy in sense and the 5 0 in antisense orientation (3 0 HVRs and 5 0 HVRas); (iv) MatNH-X-RNA3, with two copies of X located in different orientation (3 0 Xs and 5 0 Xas) ( Figure 5B ). The former two were applied to test HVR's and X's ability to support homologous crossovers while the latter two to examine Xs/Xas' and HVRs/HVRas' capacity to induce nonhomologous, heteroduplex-mediated recombination. Unlike previously tested mutants (MatNH-and MatH-BMV), in MatH-HVR-, MatNH-HVR-, MatH-X-and MatNH-X-BMV, the examined sequences were present only in the recombination vector. They were absent in the two other genomic RNAs, so that recombination crossovers could involve only RNA3 molecules.\nTo determine the recombination activity of HCV-derived sequences, C.quinoa plants were inoculated with four BMV mutants: MatH-HVR-BMV, MatNH-HVR-BMV, MatH-X-BMV and MatNH-X-BMV. Their genomes were composed of wtRNA1, wtRNA2 and one of the newly generated Mat0-RNA3 derivatives (either MatH-HVR-, MatNH-HVR-, MatH-X-or MatNH-X-RNA3) ( Table 2) . After 2 weeks, the standard procedure of BMV RNA3 progeny analysis was applied. The number of lesions developed during each infection was counted. The 3 0 -portion of progeny RNA3 was amplified by the RT-PCR method. The length of RT-PCR products was established (by electrophoresis in a 1.5% agarose gel), then they were cloned and sequenced.\nWe found that BMV mutants carrying HVRs/HVRs and HVRs/HVRas sequences are as infectious as Mat0-BMV; usually they developed 14-18 lesions on each inoculated leaf. The two others, MatH-X-and MatNH-X-BMV mutants, are visibly less infectious and developed 3-5 and 4-8 lesions/leaf, respectively. Despite differences in their infectivity, BMV mutants carrying 3 0 HVRs and 5 0 HVRas as well as 3 0 Xs and 5 0 Xas supported non-homologous, heteroduplex-mediated crossovers very efficiently. An RNA3 recombinant was generated in 100 and 90% of lesions developed during infection with MatNH-HVR-and MatNH-X-BMV, respectively. Recombinant junction sites were located within the left portion of the local double-stranded region that could potentially be formed either by HVRs and HVRas or by Xs and Xas. As a result, both sequences supporting non-homologous crossovers were almost entirely deleted, together with the whole spacer ( Figure 5D and E) .\nInterestingly, homologous recombinants were generated only during infection involving MatH-HVR-BMV. Fiftyfive percent of analyzed lesions contained the RNA3 recombinant. In all sequenced recombinants, one HVR and the spacer were deleted ( Figure 5C ). Their 3 0 -UTR was composed of a 295 nt RNA1-derived sequence and HVRs followed by To test the recombination activity of HCV-derived sequences X and HVR the following Mat0-RNA3 derivatives were prepared: MatH-X-RNA3-with two copies of X in sense orientation (3 0 and 5 0 Xs), MatNH-X-RNA3-with two copies of X located in different orientation (3 0 Xs and 5 0 Xas), MatH-HVR-RNA3-containing two copies of HVR in sense orientation (3 0 and 5 0 HVRs) and MatNH-HVR-RNA3-with two copies of HVR, 3 0 -copy in sense and 5 0 in antisense orientation (3 0 HVRs and 5 0 HVRas). MatH-X-and MatH-HVR-RNA3 were applied to test X's and HVR's ability to support homologous crossovers while MatNH-X-and MatNH-HVR-RNA3 were applied to examine X's and HVR's competence to induce non-homologous, heteroduplex-mediated recombination. RF, recombination frequency observed during infection involving each RNA3 derivative. (C). Homologous recombinants generated during infection involving MatH-HVR-BMV. RNA3 recombinants were only formed during MatH-HVR-BMV infection. In all of them, one recombinationally active sequence HVRs and a spacer were deleted and their 3 0 -UTR was composed of RNA1 derived sequence and HVRs followed by the CP coding region. Because crossovers occurred within identical regions, the location of recombinant junction sites could not be precisely The presence of homologous and non-homologous recombinants in the examined lesions was always confirmed not only by RT-PCR but also by northern blot analysis ( Figure 6 ). This revealed the same tendency as that observed earlier using the heteromolecular system (30, 33) . BMV accumulated to a very low level in lesions containing parental RNA3 (original molecules with duplicated sequences- Figure 6A , lane 5 and Figure 6B , lane 2). However, this changed in lesions where a recombinant was generated ( Figure 6A , lanes 1-4 and Figure 6B, lanes 1 and 3) .\nEarlier it was shown that the BMV-based heteromolecular system can be used as an effective tool for investigating the mechanism of homologous and non-homologous recombination, although it is only suitable for testing the recombination activity of the sequences derived from the 3 0 -portion of BMV RNA1 or RNA2 (30, 33) . To overcome this problem, we attempted to create a new universal recombination in vivo system. The collected data suggested that a BMV RNA3-based homomolecular system would best fulfill our expectations. To confirm the correctness of the above presumption, to determine the efficacy of the homomolecular system and to compare it with the heteromolecular one, two mixed homoheteromolecular systems were constructed-one to study homologous (MatH-BMV) and the other non-homologous (MatNH-BMV) recombination.\nThe mixed systems were prepared in such a way that two identical or two complementary sequences were capable of supporting homologous or non-homologous crossovers, respectively, either between molecules representing the same segment of the BMV genome (modified RNA3) or between molecules representing two different segments of the BMV genome (wtRNA1 and modified RNA3). Experiments involving MatH-and MatNH-BMV showed that recombination can occur both in homo-and heteromolecular systems and proved that the former should be at least as effective as the previously utilized heteromolecular one. Interestingly, the RAS1s sequence did not support homologous recombination during infection with MH-BMV (heteromolecular system) and it was Infectivity was defined as the average number of lesions per leaf. b Recombination frequency was defined as the ratio between the number of lesions that developed recombinants and the total number of analyzed lesions. quite active in the mixed system. This clearly demonstrates that not only primary and secondary structure but also the location of RAS within the viral genome affects its ability to mediate homologous crossovers. The undertaken experiments also revealed that homologous recombination occurs more often between two different RNAs (RNA1 and RNA3), while non-homologous recombination usually involves molecules representing the same segment of the BMV genome (RNA3). The obtained results constitute yet another piece of evidence that the mechanisms of homologous and nonhomologous recombination are different. The same conclusion was reached by us earlier while studying the influence of specific mutations in BMV-encoded protein 2a (26, 27) . We identified among other the mutation in the 2a protein, which inhibits non-homologous crossovers without affecting the frequency of homologous ones. At present, it is difficult to judge at which stage of the recombination process the observed differences occur. One can only suppose that the structural requirements of transfer of the replicase-nascent strand complex from the donor to the acceptor molecule must be different in homologous and nonhomologous recombination. In the case of the former, a basic factor facilitating this process is complementarity between the acceptor and the nascent strand. Consequently, there is no necessity for the replication complex to be stable during homologous crossovers (22) . Replicase can leave the donor template alone or together with a nascent strand. Then, the 3 0 end of the newly synthesized RNA molecule can function as a guide; it can find a complementary sequence in the acceptor RNA, hybridize and serve as a primer allowing viral replicase to reinitiate RNA synthesis. In non-homologous heteroduplexmediated recombination, a factor enhancing crossover seems to be the interaction between the donor and the acceptor (the formation of a local double-stranded region) (30, 33) . Considering that BMV genomic RNAs are copied within spherules (44) , intramolecular hybridization between RAS1s and RAS1as, located in MatNH-RNA3, is much more likely. Thus, the results presented here indicate that the way the virus replicates can strongly affect the recombination process. However, further detailed studies are necessary in order to explain this phenomenon.\nBased on results obtained using the heteromolecular (30, 33) and mixed systems, we constructed a new homomolecular one. Its most crucial element is the Mat0-RNA3 vector, into which both tested sequences can be introduced. In order to show the usefulness of this system, we employed it to test the recombination activity of two distinctly different sequences deriving from the genome of an RNA virus not related to BMV. Our choice was the 98 nt sequence X and an HVR both from HCV genome. There are many reasons as to why the two sequences can be deemed drastically different. The most important of them are (i) sequence X is placed in a noncoding region, while HVR in a coding one, (ii) sequence X is the least variable and HVR the most variable fragment of the HCV genome (42, 43, 45) , (iii) unlike to HVR, sequence X possesses a very stable and well-defined secondary and tertiary structure (42, 45) .\nWe ascertained that the introduction of HVR into the Mat0-RNA3 vector (in sense/sense and antisense/sense orientation) does not influence BMV infectivity. The latter was, however, reduced if HVR was replaced with sequence X. We found that HVR supports homologous recombination and HVRs and its complementary counterpart HVRas mediate non-homologous crossovers. The frequency of homologous recombination amounted to 55% and of non-homologous to 100%. Sequence Xs did not support homologous crossovers but Xs and complementary sequence Xas were capable of inducing non-homologous ones (their frequency reaching 90%).\nThe obtained results testify that the local double-stranded structures induce non-homologous recombination crossovers very efficiently. This may reflect the capacity of RNA viruses to remove inverted repeats from their genomes. Viruses lacking such ability would be an easy target for double-stranded RNA-induced RNA silencing, which is known as the plant antiviral mechanism (46) . Moreover, the data presented suggest that sequence X, which adopts a very compact and stable structure (45), is not able to mediate homologous recombination. It occurs efficiently within AU-rich HVR sequences whose structure is more labile and dynamic (42) . Earlier research on homologous recombination in BMV led to similar conclusions. It was shown that homologous recombination occurs effectively in AU-rich regions (47) and is not observed within highly structured 3 0 -and 5 0 -UTR (48) . These observations concur with the proposed mechanism of homologous RNA recombination (22) . It assumes that AU-rich regions facilitate the detachment of the polymerase-nascent strand complex from donor RNA. On the other hand, it is thought that the stability of RNA structure makes the hybridization of the nascent strand and/or replicase to the acceptor difficult.\nCurrently, it is becoming increasingly clear that RNA recombination plays a very complex role in a virus' life cycle. Not only does it permit the exchange of genetic material between viruses (3, 4, 22) , frequent homologous crossovers between molecules representing the same segment of the virus genome also stabilize genetic information (48) . Moreover, here we showed that homologous and non-homologous recombination might control the organization of the virus genome by removing direct or inverted repeats, which affect the virus' ability to replicate or accumulate in the infected cells. Interestingly, we observed that complementary sequences are more effectively deleted than homologous ones. It seems that some of the latter can prevail in the viral genome probably due to their compact stable structure that prevents recombination events.\nAltogether, the data presented here prove that the newly created BMV-based homomolecular recombination system can be used to examine in vivo recombination activity of various RNA sequences derived from the genomes of related or unrelated viruses. However, there are other factors which, in addition to RNA structure, can affect the course of the studied process. Specific properties of the viral replicase and the host proteins that are necessary for recombination events can be of equally great importance. Therefore, there is a need to create similar universal recombination systems in other viruses. We believe that these systems will be very helpful in finding some general rules in RNA recombination and will provide us with knowledge which is indispensable to understand how new RNA viruses or retroviruses are generated." }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: Mammalian reoviruses are the prototypic members of the Reoviridae family, which also includes the pathogenic rotaviruses, coltiviruses, seadornaviruses and orbiviruses. These viruses share elements of their replication cycle as well as structural features, including a non-enveloped multi-layered capsid that surrounds a segmented dsRNA genome. In humans, mammalian reoviruses are typically associated with mild and self-limiting enteric and respiratory infections. However, studies in neonatal mice reveal that reoviruses can spread to distant tissue sites in immunocompromised hosts (reviewed in [1] ). The factors that determine reovirus cellular host range are poorly understood. Because reovirus attaches to cells through interactions with broadly expressed receptors, one or more subsequent steps in the viral life cycle must help to regulate host range and pathogenesis. Our recent studies suggest that one such step is proteolysis of the capsid protein σ3 [2, 3] .\nIn cell culture, the first step in infection is attachment to cellular receptors through interactions with the viral protein σ1 [4, 5] . σ1 interacts with two known receptors: sialic acid and junctional adhesion molecule 1 [6] [7] [8] . Following binding, virions are internalized by receptor-mediated endocytosis [9] . Endocytosis is an essential step in the viral life cycle under standard infection conditions [10] . Within the endosomal and/or lysosomal compartment, proteases convert virions into particles that resemble in vitro-generated intermediate subvirion particles (ISVPs) [10] [11] [12] [13] [14] . These uncoating intermediates, typically prepared using chymotrypsin or trypsin, lack σ3 and have a cleaved form of µ1. Studies using ISVPs and ISVPs recoated with recombinant outer capsid proteins reveal that σ3 plays a key role in regulating reovirus cell entry by interacting with, protecting, and controlling the conformational status of the underlying penetration protein µ1 [15] [16] [17] [18] . In cells that cannot efficiently mediate σ3 degradation during uncoating, reovirus infection is slow or blocked; these cells can be productively infected by particles that lack σ3 [2] . In vitro, ISVP-like particles can be generated by a variety of proteases in addition to chymotrypsin and trypsin, including proteinase K, thermolysin, endoproteinase lys-C, Cat L, Cat B and Cat S [3, [19] [20] [21] .\nRecent work has provided insight into the cellular determinants of reovirus uncoating. In murine fibroblasts, where reovirus entry has been best studied, the cysteine proteases Cat L, and to a lesser extent Cat B, are required for σ3 removal, whereas the aspartyl protease Cat D is not [14, [21] [22] [23] [24] [25] . Virion disassembly in murine fibroblasts also requires acidic pH [10, 26, 27] . Recently, we demonstrated that reovirus uncoating in the macrophage-like cell line P388D is mediated by the acid-independent lysosomal cysteine protease Cat S [3] . This finding revealed that in different cell types, distinct proteases can facilitate reovirus uncoating. Our results suggested a model in which infection in some cells is acid-dependent because the proteases that mediate σ3 removal in those cells require acidic pH for maximal activity. Thus, in fibroblasts or other cells in which the acid-dependent proteases Cat L and Cat B mediate σ3 removal, infection is acid-dependent [21, 23, 28] , whereas in Cat S-expressing cells it is not [3] , because Cat S maintains its activity at neutral pH [29] . Insight from the analysis of reovirus cell entry facilitated the recent discovery that activation of the Ebola virus glycoprotein also depends on the activity of the acid-dependent endosomal proteases Cat B and Cat L [30] .\nThe role that specific intracellular and extracellular proteases play in regulating reovirus tropism, spread, and disease in animals is largely unknown, except in the murine intestinal tract where pancreatic serine proteases have been shown to mediate σ3 removal [31, 32] . Reovirus also naturally infects hosts via the respiratory tract [33] [34] [35] . One protease with well-described effects in the respiratory tract is elastase 2 (GenBank NM_001972), an inflammatory serine protease of the chymotrypsin family, which is predominantly expressed by neutrophils [36] . NE plays a prominent role in wound repair [37] [38] [39] and in controlling microbial infections [38] [39] [40] . NE expression can also promote pathogenesis; it has been implicated in smokeinduced emphysema [41] , respiratory syncytial viral bronchiolitis [42] and in the respiratory syndrome ARDS [4] . The fact that reovirus replication in the rodent lung causes an influx of neutrophils [35, 43] and that reovirus infection can recapitulate ARDS [44] , led us to ask whether NE could mediate productive reovirus uncoating. We investigated reovirus infection in the monocyte-like cell line U937, because it is known to express NE [45] . Experiments described in this report demonstrate that reovirus infection in U937 cells does not require cysteine protease activity and is not blocked in the presence of agents that raise vesicular pH. Studies using protease inhibitors suggest that, in the absence of cysteine protease activity, NE is largely responsible for productive infection of U937 cells. NE can directly mediate σ3 removal from reovirus virions; the resultant particles are infectious and do not require additional intracellular proteolysis. Our data raise the possibility that NE is involved in reovirus replication in the respiratory tract.\nAnalysis of viral replication in L929 and U937 cells treated with E64 Figure 1 Analysis of viral replication in L929 and U937 cells treated with E64. A. 3 × 10 6 L929 and U937 cells were untreated (-; black) or treated (+; grey) with 300 µM E64 for 3 h or 3 d. Cysteine protease activity was assessed using the fluorogenic substrate Z-Phe-Arg-MCA (Sigma) and plotted in arbitrary units. Activity levels in treated cells were so low (in L939 cells, 254 units at 3 h and 231 units at 3 d; in U937 cells, 200 units at 3 h and 115 units at 3 days) that they cannot be visualized on this graph. B. L929 (L; black bars) and U937 (U; grey bars) cells were treated with 300 µM E64 for 3 h prior to infection. Cells were then infected with reovirus strain Lang virions or ISVPs at an MOI of 3. Infectious virus present at 3 d p.i. was determined by plaque assay on L929 cell monolayers. Each time point represents the mean (+/-SD) derived from three independent samples.\nVirion ISVP we first established conditions under which lysosomal cysteine protease activity was inhibited. Cells were treated with 300 µM E64, a broad-spectrum cysteine protease inhibitor [46] , and protease activity was assessed using the Cat L and Cat B-specific fluorogenic substrate Z-Phe-Arg-MCA. We analyzed enzyme activity at two time points: first after 3 h of treatment, because we typically pre-treat cells with inhibitors for 3 h prior to infection, and second at 3 d, the time point at which viral yield would be quantified. As shown in Fig. 1A , treatment with 300 µM E64 completely abolished cysteine protease activity in U937 cells. Consistent with our previous findings [3] , E64 also completely blocked cysteine protease activity in L929 cells. Raw values are provided, to illustrate the relative difference in Cat L/B enzyme activity levels between U937 cells and L929 fibroblasts. In the absence of inhibitor, Cat L and B activity was significantly lower in U937 cells than in L929 cells. This may be a consequence of high expression in U937 cells of cystatin F, an intracellular cysteine protease inhibitor with specificity for Cat L and papain [47] .\nNext, we compared reovirus replication in E64-treated U937 and L929 cells. Cells were pre-treated for 3 h and infected with Lang virions or ISVPs at a multiplicity of infection (MOI) of 3. The results of a representative experiment are shown in Fig. 1B . In the absence of E64, both L929 and U937 cells supported reovirus replication, consistent with the fact that these cells express Cat L. As expected, E64 blocked virion infection of L929 cells; however, viral yields in E64-treated U937 cells were only slightly reduced relative to untreated cells. ISVPs, which lack capsid protein σ3, replicated efficiently in treated cells, indicating that 300 µM E64 was not toxic to either cell type. These results demonstrate that productive infection of U937 cells by Lang virions does not require the activity of E64-sensitive, papain-like cysteine proteases.\nAcidic pH is required for productive reovirus infection of murine L929 fibroblasts [10, 27] , in which the aciddependent proteases Cat L and Cat B mediate uncoating [21, 23] . Serine proteases, including NE, and metalloproteases function over a broader pH range. Therefore, to gain insight into the nature of the protease(s) that can promote reovirus uncoating in U937 cells, we investigated the requirements for acidic pH. L929 and U937 cells were left untreated or pre-treated with E64 in the presence or absence of bafilomycin A1 (Baf) or NH 4 Cl. These latter agents raise vesicular pH by blocking the vacuolar H + -ATPase pump or by acting as a weak base, respectively [48] [49] [50] . After pre-treatment, cells were infected with Lang virions at an MOI of 3 and viral yields were determined at 3 days post infection (d p.i.). A representative experiment is shown in Fig. 2 . Treatment with either Baf or NH 4 Cl did not inhibit viral replication in U937 cells; yields reached 2.9 and 2.7 logs, respectively. Furthermore, these agents had little effect on viral replication in U937 cells even when the cells were also treated with E64 to inhibit cysteine protease activity. In contrast, Baf or NH 4 Cl alone completely blocked reovirus replication in L929 cells, consistent with the requirement for Cat L/B-mediated σ3 removal in these cells. Given that reovirus uncoating is an essential step in the viral life cycle [10] , these findings revealed that a non-cysteine protease that functions at neutral pH can facilitate this step in U937 cells.\nTreatment of the promonocytic U937 cells with phorbol ester derivatives results in their differentiation into macrophage-like cells [51, 52] . This differentiation is characterized by several major phenotypic changes, including increases in expression of urokinase plasminogen activator receptors, upregulation of collagenase activity and a significant decrease in the expression of NE and Cat G [51, 52] . We predicted, therefore, that PMA treatment might decrease the capacity of reovirus virions to replicate in U937 cells when cysteine proteases were inhibited. To confirm that there was a significant decrease in NE expression in U937 cells differentiated with PMA, U937 cells were treated with 150 nM PMA for 72 h and expression of NE was analyzed by immunoblotting. As shown in Fig. 3A , NE was expressed in untreated U937 cells, but its expression was dramatically reduced following PMAinduced differentiation.\nTo examine the effect of U937 cell differentiation on reovirus infection, PMA-treated and untreated U937 cells were left untreated or were treated with E64 for 3 h and infected with Lang virions or ISVPs at an MOI of 3. Yields were measured at 3 d p.i. and the results of a typical experiment are shown in Fig. 3B . In the absence of E64, PMAtreated U937 cells were permissive to infection by virions. PMA treatment only decreased yields by ~0.5 log relative to untreated cells. In contrast, when PMA-differentiated U937 cells were treated with E64 to inhibit cysteine protease activity, they no longer supported productive infection by Lang virions. Because these results could be explained if E64 was toxic to PMA-treated U937 cells, we examined the replication of ISVPs. In the presence of E64, ISVPs replicated to high yields in both undifferentiated and differentiated U937 cells. Since PMA-induced differentiation of U937 cells caused a substantial decrease in NE expression, these results are consistent with the hypothesis that NE or another similarly regulated neutral protease facilitates productive reovirus infection in promonocytic (pre-differentiated) U937 cells.\nEffects of agents that raise vesicular pH on reovirus replication in U937 and L929 cells A.\nAnalysis of reovirus replication in U937 cells differentiated with PMA Figure 3 Analysis of reovirus replication in U937 cells differentiated with PMA. A. Lysates generated from 10 5 U937 cells that were untreated (-) or treated with 150 nM PMA for 72 h were resolved on SDS-12% polyacrylamide gels and electrophoretically transferred to a nitrocellulose filter. The filter was subsequently incubated with a polyclonal goat antibody against human NE (1:400) (Santa Cruz Biotechnology). The filter was washed and incubated with a secondary anti-goat antibody conjugated to horseradish peroxidase (1:5000) (Santa Cruz Biotechnology). Protein bands were detected using reagents that generate a chemiluminescent signal (Amersham). B. U937 cells that were undifferentiated (-; black bars) or differentiated (PMA; grey bars) with 150 nM of PMA for 72 h were left untreated (-) or were treated with 300 µM E64. Following pre-treatment with the protease inhibitor, cells were infected with Lang virions or ISVPs at an MOI of 3. Viral yield was quantified at 3 d p.i. as described in the legend to Fig. 1B. A.\nB. \nWe directly examined the capacity of NE to facilitate reovirus infection by using the irreversible elastase inhibitor, N-(methoxysuccinyl)-Ala-Ala-Pro-Val-chloromethyl ketone [53] . This inhibitor is highly specific for NE and does not inhibit the activity of the related serine protease, Cat G [53] . First, we established the efficacy and specificity of inhibitor treatment under our experimental conditions. U937 cells were treated with the NE inhibitor, E64, Baf or NH 4 Cl for either 3 h or 2 d and the activity of NE in cell lysates was examined using a colorimetric substrate. As shown in Table 1 , the NE inhibitor was active at both time points. In cells treated with the specific inhibitor, NE activity was less than 9% of that in untreated U937 cells.\nIn contrast, in U937 cells treated with E64, Baf or NH 4 Cl, NE activity was only modestly reduced, remaining above 80% even after 2 d. These results are consistent with the capacity of NE to function at neutral pH. To verify the specificity of the NE inhibitor, we also examined its effect on Cat L/B activity using the fluorogenic substrate Z-Phe-Arg-MCA. As expected, Cat L/B activity was completely inhibited by E64 but largely unaffected by the NE inhibitor.\nTo examine the effect of the NE inhibitor on reovirus replication in U937 cells, we pre-treated them for 3 h with E64 in the presence or absence of the NE inhibitor, infected them with Lang virions or ISVPs at an MOI of 3, and quantified viral yields at 2 d p.i. A representative experiment is shown in Fig. 4 . Consistent with the results shown in Fig 1, virion replication was not blocked in E64treated U937 cells. However, in the presence of both E64 and the NE inhibitor, yields were significantly reduced. ISVPs replicated to high yields in treated cells, indicating that the combination of inhibitors was not toxic to U937 cells. These results demonstrate that NE plays a critical role in reovirus infection of U937 cells when cysteine proteases are inhibited.\nNE, like many cellular proteases, is expressed as a proenzyme that becomes activated only after its pro-region is removed [54] . We envisioned two models by which NE could facilitate reovirus infection of U937 cells. In the first, NE could directly mediate σ3 degradation, leading to the generation of an ISVP-like particle. In the second, NE could act indirectly by activating another protease. To try to distinguish between these models, we examined the capacity of purified NE to directly mediate σ3 removal from Lang virions in vitro. Purified Lang virions were treated with NE for 1 and 4 h and the treated virus particles were analyzed by SDS-PAGE. As shown in Fig. 5A , NE efficiently removed σ3 from Lang virions; after 1 h very little intact σ3 remained on viral particles. After 4 h of NE treatment, σ3 was completely removed and the underlying µ1C was cleaved to the δ and φ fragments (φ was not retained on the gel). When we assayed the infectivity of the resultant particles by plaque assay we found that NE treatment did not negatively affect the titer of Lang particles (data not shown).\nTo determine if NE-generated SVPs required further proteolytic processing of σ3, L929 cells were pre-treated with E64 to block cysteine protease activity and infected at an MOI of 3 with Lang virions, ISVPs or NE-generated subviral particles (NE-SVPs). Viral yields were determined at 1 d p.i. As expected, E64 blocked infection of L929 cells by virions. In contrast, both ISVPs and NE-SVPs replicated efficiently in the presence of the cysteine protease inhibitor (Fig. 5B) . Because virion disassembly in L929 cells requires acidic pH [10] , we also examined the capacity of NE-SVPs to infect L929 cells treated with Baf, NH 4 Cl or monensin, three agents that raise vesicular pH by distinct mechanisms. Cells were treated with these agents and then infected with virions, ISVPs or NE-SVPs at an MOI of a U937 cells were treated with the indicated inhibitors for 3 h or 2 d. b NE activity was assessed using the colorimetric substrate MeOSuc-Ala-Ala-Pro-Val-ρNA and percent activity relative to untreated cells was calculated. c Cathepsin L and B activity were assessed using the fluorogenic substrate Z-Phe-Arg-MCA and percent activity relative to untreated cells was calculated.\n10. At 18 hours post infection (h p.i.), cell lysates were harvested and expression of the reovirus non-structural protein µNS was analyzed by immunoblotting (Fig. 5C ). As expected, when treated cells were infected with virions, viral protein expression was blocked. In contrast, µNS expression was evident even in the presence of agents that raise pH when infections were initiated with ISVPs or NE-SVPs (Fig. 5C ). Together, these results demonstrate that NE can directly mediate σ3 removal from virions to generate infectious particles that do not require further proteolytic processing by acid-dependent cysteine proteases in L929 cells.\nSerine proteases are involved in reovirus infection in the mammalian intestinal tract [31] and in this report we pro-vide evidence that they can mediate uncoating and promote infection in U937 cells. This expands the range of proteases that promote reovirus infection in cell culture to include NE as well as the cysteine proteases Cat L, Cat B, and Cat S. Several lines of evidence now support the notion that protease expression is a cell-specific host factor that can impact reovirus infection. For example, some reovirus strains are inefficiently uncoated by Cat S and thus do not replicate to high yield in P388D macrophages [3] . In this report we demonstrate that PMA-induced differentiation influences the type of protease that mediates reovirus uncoating in U937 cells. In these cells, PMA treatment is reported to increase Cat L expression [55] and decrease expression of the serine proteases NE and Cat G [56, 57] . Accordingly, when we used PMA to induce U937 cell cultures to differentiate, reovirus infection became sensitive to the cysteine protease inhibitor E64. We suspect that Cat L is largely responsible for uncoating in these PMA-differentiated cells, but the acid-independent protease Cat S may also play a role. We are currently addressing this question by analyzing infection in PMAdifferentiated cells treated with either Baf or NH 4 Cl.\nOur data do not completely resolve this question. Cat G is expressed by U937 cells and, like NE, it is down-regulated by PMA treatment. Furthermore, we found that in vitro treatment of reovirus virions with purified Cat G generates SVPs that behave like NE-SVPs in that they are infectious in the absence of further proteolytic processing (data not shown). Results of our experiment with the NE-specific inhibitor suggest that NE is largely responsible for the E64-resistant infection in U937 cells. While this inhibitor is reported not to inhibit Cat G [53] , we have not independently confirmed this. Another approach to assess the role of Cat G in reovirus infection of U937 cells would be to examine the effect of Cat G-specific inhibitors on infection. We tried one such inhibitor, Cathepsin G Inhibitor I (Calbiochem) [58] , but found that it was cytotoxic to U937 cell cultures. Given that both NE and Cat G can generate infectious reovirus SVPs, more work needs to be done in order to understand the role that these two proteases play in infection in these cells.\nPreviously, we reported that virion uncoating mediated by Cat S does not require acidic pH [3] . These results were consistent with the acid-independence of Cat S activity [37] . Together, the results in Fig. 2 and Fig. 4 reveal that, like Cat S, NE-mediates infection in an acid-independent manner. This finding thus provides further support for a model in which the requirement for acidic pH during reovirus infection of some cell types reflects the requirement for acid-dependent protease activity in those cells rather than some other requisite acid-dependent aspect of cell entry. The small effect of Baf and NH 4 Cl on E64-resistant reovirus growth (Fig. 2 ) may reflect the participation of one or more acid-dependent proteases (such as Cat D) in the activation of NE.\nElastase is stored in azurophilic granules that are the major source of acid-dependent hydrolases in neutrophils [59] . Although these granules do not contain LAMP-1 or LAMP-2 [60] they contain the lysosomal markers LAMP-3 [61] and CD68 [62] and are accessible to endocytosed fluid-phase markers under conditions of cellular stimulation [63] . NE can be released from neutrophils during degranulation [64] and its cell surface expression can be induced upon PMA treatment [65] . However, studies in U937 cells have shown that NE is predominantly retained intracellularly and that little if any activity is present in the extracellular medium [45] . Consistent with this, we have been unable to generate ISVP-like particles by treatment of virions with U937 culture supernatants (data not shown). This observation, together with our finding that PMA treatment decreases the capacity of E64-treated U937 cells to support reovirus infection, leads us to favor a model in which NE-mediated virion uncoating in U937 cell cultures occurs intracellularly.\nIn vivo, a number of viruses, including dengue and respiratory syncytial virus, induce the release of IL-8, a cytokine that serves as a chemoattractant for neutrophils and promotes their degranulation [66, 67] . Reovirus replication in the rat lung results in neutrophilic invasion [35, 43] and studies in cell culture indicate that reovirus infection can induce IL-8 expression [68] . Thus, the capacity of reovirus to induce IL-8 secretion in vivo might facilitate the release of neutrophilic lysosomal hydrolases, including NE, into the extracellular milieu. In this report, we have shown that mammalian reovirus can utilize this acid-independent serine protease for uncoating. Our data suggest that, in vivo, one consequence of reovirus-induced IL-8 expression would be the generation of infectious NE-SVPs. Like ISVPs, these particles would be predicted to have an expanded cellular host range because they can infect cells that restrict intracellular uncoating [2] . Thus, inflammation might be predicted to exacerbate reovirus infection by promoting viral spread. Future studies using mice with deletions in the NE gene will be required to elucidate the role this protease plays during reovirus infection in the respiratory tract and other tissues. Finally, given the recent finding that endosomal proteolysis of the Ebola virus glycoprotein is necessary for infection [30] , our results raise the interesting possibility that NE or other neutrophil proteases may play a role in cell entry of other viruses. [Furlong, 1988 #81] . ISVPs were prepared by treating purified virions with chymotrypsin as described elsewhere [Nibert, 1992 #95] .\nCysteine protease activity was measured as described previously [23] Samples were frozen and thawed three times and titrated by plaque assay on L929 cells as described elsewhere [69] . Viral yields were calculated according to the following formula: log 10 (PFU/ml) t = x hrlog 10 (PFU/ml) t = 0 +/-standard deviation (SD). \nTo analyze NE expression, cell lysates were generated from U937 cells, either treated or untreated for 48 h with 150 nM PMA as described for the analysis of viral protein expression. Lysate from the equivalent of 1 × 10 6 cells was run on SDS-12% polyacrylamide gels and transferred to nitrocellulose. Membranes were blocked overnight in TBST containing 10% nonfat dry milk. NE expression was analyzed using a polyclonal antibody against NE (1:400 in TBST) (Santa Cruz Biotechnology Inc, Santa Cruz, CA). Membranes were washed with TBST and incubated with a horseradish peroxidase-conjugated anti-goat IgG (1:5000 in TBST). Bound antibody was detected by treating the nitrocellulose filters with enhanced chemiluminescence (ECL) detection reagents (Amersham) and exposing them to Full Speed Blue X-ray film (Henry Schein, Melville, NY).\nCells were plated at 10 6 /well in a 6-well plate 18-24 h prior to infection. Virus was allowed to adsorb to cells for 1.5 h at 4°C. At this temperature, virus binds to cells but is not internalized [70] . After adsorption, the cultures were incubated at 37°C in fresh medium. Prior to some infections, cells were pre-treated for 3 h with 300 µM E64, 100 nM Baf, 25 µM monensin (Sigma), or 20 mM NH 4 Cl.\nIn those instances inhibitors were also included in the post-adsorption culture medium. At the indicated times p.i., cells were collected by centrifugation at 179 × g, washed twice in chilled PBS and lysed in TLB. After centrifugation at 179 × g to remove cellular debris, samples were resuspended in sample buffer. Protein samples (representing 1 × 10 5 cells) were analyzed by electrophoresis on SDS-12% polyacrylamide gels and transferred to nitrocellulose membranes for 2 h at 100 V in 25 mM Tris-192 mM glycine-20% methanol. Nitrocellulose membranes (Bio-Rad Laboratories, Hercules, Calif.) were blocked overnight at 4°C in TBST (10 mM Tris [pH 8.0], 150 mM NaCl and 0.05% Tween) containing 5% nonfat dry milk, rinsed with TBST, and incubated with a rabbit anti-µNS polyclonal antiserum [71] (1:12500 in TBST) for 1 h. Membranes were subsequently washed with TBST and incubated for 1 h with horseradish peroxidase-conjugated anti-rabbit immunogloblin G (IgG) (1:7500 in TBST) (Amersham, Arlington Heights, Ill.). Bound antibody was detected by treating the nitrocellulose filters with enhanced chemilumescence (ECL) detection reagents (Amersham) and exposing the filters to Full Speed Blue X-ray film (Eastman Kodak, Rochester, N.Y.).\nPurified virions (1.4 × 10 11 ) were incubated with 25 µg/ ml of purified neutrophil elastase (Calbiochem) in 40 µL of VDB at 37°C for 3 h. Reactions were terminated by adding 1 mM PMSF and 200 µM NE inhibitor to the reaction mixture. 5.0 × 10 10 particles were run on SDS-12% polyacrylamide gels stained with Coomassie Brilliant Blue to confirm the removal of σ3. Viral infectivity was determined by plaque assay on L929 cell monlayers.\nPurified Lang virions (1.4 × 10 11 ) were treated with 25 µg/ ml of NE in 40 µL of VDB at 37°C for the times indicated. Reactions were terminated as described above. To verify σ3 removal, the proteins from 5.0 × 10 10 particles were separated on SDS-12% polyacrylamide gels and visualized with Coomassie Brilliant Blue staining. Viral infectivity for each time point was determined by plaque assay on L929 cell monolayers." }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: Understanding the thermodynamics of nucleic acid duplexes is important for many reasons. For example, such knowledge facilitates design of ribozymes (1), antisense and RNAi oligonucleotides (2) (3) (4) (5) (6) (7) (8) (9) , diagnostic probes including those employed on microarrays (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) and structures useful for nanotechnology (24) (25) (26) (27) . Many modified residues have been developed for such applications. Examples include propynylated bases (28) (29) (30) , peptide nucleic acids (5, (31) (32) (33) , N3 0 -P5 0 phosphoramidates (34-38) and 2 0 -O-alkyl RNA (39) (40) (41) (42) (43) . A modification that is particularly stabilizing in DNA and RNA duplexes (44) (45) (46) (47) (48) (49) (50) (51) is a methyl bridge between the 2 0 oxygen and 4 0 carbon of ribose to form a 'locked nucleic acid' or LNA as shown in Figure 1 . McTigue et al. (48) have shown that the enhanced stability due to a single LNA residue in a DNA duplex can be predicted from a nearestneighbor model.\nHybridization of oligonucleotides to RNA is important for applications, such as antisense therapeutics (4, 8, 21, 46, (52) (53) (54) , diagnostics (32, 33, 42, 55) , profiling gene expression with microarrays (18) (19) (20) 56) , identifying bands by Northern blots of gels (57, 58) and probing RNA structure (1, 3, 15, (59) (60) (61) . Oligonucleotides with 2 0 -O-alkyl modifications can be particularly useful for these applications because they are easily synthesized (39, 43) , chemically stable and bind relatively tightly to RNA (39) (40) (41) (42) . However, for many applications, it is desirable to modulate the binding affinity. For example, sequence independent duplex stabilities would benefit applications that involve multiplex detection, such as microarrays.\nHere, we show that introduction of LNA into 2 0 -O-methyl RNA oligonucleotides can increase stabilities of 2 0 -O-methyl RNA/RNA hybrid duplexes and that the enhancements in stability can usually be predicted with a simple model.\nHigh-performance liquid chromatography (HPLC) was performed on a Hewlett Packard series 1100 HPLC with a reverse-phase Supelco RP-18 column (4.6 · 250 mm). Mass spectra were obtained on an LC MS Hewlett Packard series 1100 MSD with API-ES detector or on an AMD 604/402. Thin-layer chromatography (TLC) was carried out on Merck 60 F 254 TLC plates with the mixture 1-propanol/ aqueous ammonia/water ¼ 55:35:10 (v/v/v).\nOligoribonucleotides were synthesized on an Applied Biosystems DNA/RNA synthesizer, using b-cyanoethyl phosphoramidite chemistry (62) . For synthesis of standard RNA oligonucleotides, the commercially available phosphoramidites with 2 0 -O-tertbutyldimethylsilyl groups were used (Glen Research). For synthesis of 2 0 -O-methyl RNA oligonucleotides, the 3 0 -O-phosphoramidites of 2 0 -Omethylnucleotides were used (Glen Research and Proligo). The 3 0 -O-phosphoramidites of LNA nucleotides were synthesized according to the published procedures with some minor modifications (44, 47, 63) . The details of deprotection and purification of oligoribonucleotides were described previously (64) .\nOligonucleotides were melted in buffer containing 100 mM NaCl, 20 mM sodium cacodylate, 0.5 mM Na 2 EDTA, pH 7.0. The relatively low NaCl concentration kept melting temperatures in the reasonable range even when there were multiple LNA substitutions. Oligonucleotide single-strand concentrations were calculated from absorbencies above 80 C and single-strand extinction coefficients were approximated by a nearest-neighbor model (65, 66) . It was assumed that 2 0 -Omethyl RNA and RNA strands with identical sequences have identical extinction coefficients. Absorbancy versus temperature melting curves were measured at 260 nm with a heating rate of 1 C/min from 0 to 90 C on a Beckman DU 640 spectrophotometer with a water cooled thermoprogrammer. Melting curves were analyzed and thermodynamic parameters were calculated from a two-state model with the program MeltWin 3.5 (67) . For almost all sequences, the DH derived from T m À1 versus ln (C T /4) plots is within 15% of that derived from averaging the fits to individual melting curves, as expected if the two-state model is reasonable.\nFree energy parameters for predicting stabilities of 2 0 -O-methyl RNA/RNA and 2 0 -O-methyl RNA-LNA/RNA duplexes with the Individual Nearest-Neighbor Hydrogen Bonding (INN-HB) model (64) were obtained by multiple linear regression with the program Analyse-it v.1.71 (Analyse-It Software, Ltd, Leeds, England; www.analyse-it. com) which expands Microsoft Excel. Analyse-It was also used to obtain parameters for enhancement of stabilities of 2 0 -O-methyl RNA/RNA duplexes by substitution of LNA nucleotides internally and/or at the 3 0 end when the LNAs are separated by at least one 2 0 -O-methyl nucleotide. Results from T m À1 versus ln (C T /4) plots were used as the data for the calculations. 3 show typical data from optical melting curves, and Table 1 lists the thermodynamic parameters for the helix to coil transition with either no or one LNA nucleotide in the primarily 2 0 -O-methyl strand of a hybrid with a Watson-Crick complementary RNA strand.\nSingle LNA substitutions at the 5 0 end of heptamer duplexes have little effect on stability\nThe effects of single LNA substitutions at the 5 0 end of the 2 0 -O-methyl strand were studied in duplexes of the form,\nwhere superscript M denotes a 2 0 -O-methyl sugar, N is A, C, G, or U with a 2 0 -O-methyl or LNA sugar, r denotes ribose sugars, and Q is the Watson-Crick complement to N. As summarized in Table 1 , 5 0 terminal LNA substitutions make duplex stability more favorable by 0.3-0.6 kcal/mol at 37 C with an average enhancement of 0.45 kcal/mol. Thus, 5 0 terminal LNA substitutions increase the binding constant for duplex formation by $2-fold at 37 C.\nThe effects of single LNA substitutions at the 3 0 ends of heptamer duplexes is idiosyncratic\nThe effects of single LNA substitutions at the 3 0 end of the 2 0 -O-methyl strand was studied in duplexes of the form, Table 1 ). If N is A, C or G, then LNA substitutions have similar effects. On average, an LNA substitution makes duplex stability more favorable by 1.2 kcal/mol at 37 C. In the two sequences with a 3 0 terminal LNA U on the 2 0 -O-methyl strand, duplex stability is, however, affected little, averaging a destabilization of 0.08 kcal/ mol at 37 C. In both cases, the terminal U is preceded by a GC pair, but both orientations of the GC pair give similar destabilization upon LNA substitution at the 3 0 terminal U.\nSingle LNA substitutions in the interior of A M C M U M A M C M C M A M enhance the stability of the duplex formed with its complementary RNA by $1.4 kcal/mol\nThe effect of interior position on the free energy increment for a single LNA substitution for a 2 0 -O-methyl RNA was studied for the duplex 5 0 A M C M U M A M C M C M A M /3 0 r(UGAUGGU). As summarized in Table 1 , a single interior LNA substitution makes duplex stability more favorable by 1.2-1.7 kcal/mol at 37 C, with an average of 1.4 kcal/mol. This corresponds to roughly a 10-fold increase in binding constant. Thus, interior and 3 0 terminal LNA substitutions usually improve binding more than 5 0 terminal LNA substitutions. Table 1 . For 13 of 16 sequences, the LNA substitution makes duplex stability more favorable by 1.0-1.5 kcal/mol at 37 C, with an average enhancement of 1.3 kcal/mol. The enhancement for the other three sequences averages 2.1 kcal/mol at 37 C.\nThe dependence on the 5 0 nearest-neighbor nucleotide of effects from substituting U L for U M was studied in duplexes of the form, neighbor that is preceded by A M and U M , respectively. In both cases, the LNA substitution enhances duplex stability by 1.14 kcal/mol at 37 C. Thus, for seven duplexes, the enhanced stability from an LNA substitution is relatively independent of the nearest-neighbor nucleotide 5 0 to the LNA. The one exception is for the nearest neighbor 5 0 G M U L /3 0 r(CA). Interestingly, this nearest-neighbor combination is also destabilized by LNA substitution at a 3 0 terminal U (Table 1) . Evidently, an LNA substitution in the middle of a 2 0 -O-methyl strand usually affects heteroduplex stability with an RNA strand by about the same amount as an LNA substitution at a 3 0 terminus.\nThe effects of LNA substitutions are approximately additive when LNA nucleotides are spaced by at least one 2 0 -O-methyl nucleotide Table 2 contains thermodynamic parameters measured for duplexes having more than one LNA substitution and Table 3 compares the stabilities at 37 C with those predicted from four simple models. The first model, labeled 'additivity', predicts the DG 37 for duplex formation in the 5 0 ACUACCA/ 3 0 UGAUGGU series by adding the free energy increments measured for single LNA substitutions in the same context to the DG 37 for duplex formation in the absence of LNA nucleotides. The second model predicts the DG 37 (kcal/mol) for duplex formation with the following equation as deduced from fitting the data in Tables 1 and 2 Here, DG 37 (2 0 -O-MeRNA/RNA) is the free energy change at 37 C for duplex formation in the absence of any LNA nucleotides, n 5 0 tL is the number of 5 0 terminal LNAs, n iAL/UL and n iGL/CL are the number of internal LNAs in AU and GC pairs, respectively, n 3 0 tU and n 3 0 tAL/CL/GL are the number of Here, T m À1 is the inverse melting temperature in kelvin, R is the gas constant, 1.987 cal K À1 mol À1 , C T is the total oligonucleotide strand concentration, and both strands have the same concentration. Table 1 . Thermodynamic parameters of duplex formation between RNA and 2 0 -O-methyl oligoribonucleotides with and without a single LNA substitution a Oligonucleotides RNA Average of curve fits 3 0 terminal LNAs that are U or not U, respectively. Both methods that use experimental data for DG 37 (2 0 -O-MeRNA/RNA) provide reasonable predictions that are within 1 kcal/mol of the measured value (Table 3) . Two other methods that use nearest-neighbor models to approximate DG 37 (2 0 -O-MeRNA/RNA) provide somewhat less accurate, but still reasonable predictions as described below. The duplex with the worst prediction, 5 0 G M U L U M C L G M G L /3 0 CAAGCC has a 5 0 G M U L /3 0 CA nearest neighbor, consistent with this motif being unusually unstable by $1.2 kcal/mol. Thus, it is likely that the DG 37 of Equation 1 should be made less favorable by 1.2 kcal/mol for every internal 5 0 G M U L /3 0 CA nearest neighbor in a duplex. Evidently, the effects of multiple LNA substitutions are approximately additive when the LNAs are spaced by at least 1 nt.\nThe data may also be fit to a nearest-neighbor model containing 30 of the LNA enhancement parameters associated with duplexes of RNA strands bound to 2 0 -O-methyl RNA/ LNA chimeras. These parameters are listed in Supplementary Material. The number of occurrences for each nearest neighbor is limited, however, so the values are only roughly determined.\nPredictions for RNA/RNA duplexes at 1 M NaCl can be used to approximate stabilities of 2 0 -O-methyl RNA/RNA duplexes at 0.1 M NaCl\nThe stabilities of RNA/RNA duplexes at 37 C and 1 M NaCl are predicted well by an Independent Nearest-Neighbor Hydrogen Bonding (INN-HB) model (64) . In this model, the stability of an RNA/RNA duplex is approximated by:\nHere, DG init is the free energy change for initiating a helix; each DG j NN ð Þ is the free energy increment of the jth type nearest neighbor (see Table 4 ) with n j occurrences in the sequence; m term-AU is the number of terminal AU pairs; DG termÀAU is the free energy increment per terminal AU pair; DG sym is 0.43 kcal/mol at 37 C for self-complementary duplexes and 0 for non-self-complementary duplexes. \nÀ0.73 ± 0.26 5 0 AU3 0 À1.10 ± 0.08 Similar sequence dependent parameters may also be applicable to 2 0 -O-methyl RNA/RNA heteroduplexes because they are expected to have A-form conformations similar to those of RNA/RNA homoduplexes (68) . This was tested by comparing the predicted stabilities of RNA/RNA duplexes in 1 M NaCl at 37 C with those measured for 2 0 -O-methyl RNA/RNA duplexes in 0.1 M NaCl at 37 C. The predicted thermodynamics are listed in parentheses in Tables 1 and 2 . On average at 37 C, the RNA/RNA duplexes in 1 M NaCl are 0.12 ± 0.01 kcal/mol of phosphate pairs more stable than the 2 0 -Omethyl RNA/RNA duplexes in 0.1 M NaCl. Presumably, much of this difference is due to a sequence independent effect of salt concentration, which would primarily affect the DS for duplex formation (22, 69) . Thus, a reasonable approximation for the first term on the right hand side of Equation 1 is:\nNote that DG sym from the RNA/RNA calculation is subtracted because a 2 0 -O-methyl RNA/RNA duplex cannot be selfcomplementary because the backbones differ. For the duplexes studied here, the number of phosphate pairs is one less than the number of base pairs. The effects of LNA substitutions are likely not very dependent on salt concentration. Thus, it is probable that in 1 M NaCl or in the presence of Mg 2+ (70) that DG 37 (2 0 -O-MeRNA/ RNA) can be approximated by DG 37 (RNA/RNA, 1 M NaCl). Table 3 compares measured values for duplexes with more than one LNA to predictions from combining Equation 1-3. The measured DG 37 values average À10.5 kcal/mol and the root-mean-square difference between measured and predicted DG 37 values is 0.6 kcal/mol with the largest difference being 1.7 kcal/mol. Again, the sequence with the largest difference contains a 5 0 G M U L /3 0 CA nearest neighbor so the prediction would be improved if Equation 1 was corrected for the apparent instability of this motif.\nThe results for 2 0 -O-methyl RNA/RNA duplexes provide preliminary nearest-neighbor free energy increments for predicting stabilities of such duplexes\nThe comparison of predicted RNA/RNA stabilities with those measured for 2 0 -O-methyl RNA/RNA duplexes suggests that the INN-HB model will also be applicable to 2 0 -O-methyl RNA/RNA duplexes (71) . The results in Tables 1 and 2 Table 4 ). Three nearest neighbors are only represented once or twice in the database, and these parameters are in parentheses. The parameters for 2 0 -O-methyl RNA/RNA and RNA/RNA duplexes are similar, especially if the RNA/RNA Watson-Crick nearest-neighbor parameters are each made less favorable by 0.12 kcal/mol, which largely accounts for the difference in salt concentration as suggested above. Evidently, the first term on the right hand side of Equation 1 can also be approximated by: Table 3 compares predictions from combining Equations 1 and 4 with measured values for duplexes with more than one LNA. The root-mean-square difference between measured and predicted DG 37 values is 0.6 kcal/mol with the largest difference being the 1.7 kcal/mol associated with the duplex containing a 5 0 G M U L /3 0 CA nearest neighbor. Undoubtedly, this model can be expanded and refined by more measurements, but it appears sufficient to aid sequence design for many applications.\nComplete LNA substitution is no more stabilizing than substitution at every other nucleotide starting at the second nucleotide from the 5 0 end\nThe effect of complete LNA substitution for a 2 0 -O-methyl RNA backbone was studied for the sequences 5 0 A L C L U L A L C L C L A L /3 0 r(UGAUGGU) and 5 0 G L C L U L A L C L U L G L / 3 0 r(CGAUGAC). As summarized in Table 2 , the stabilities of these duplexes at 37 C are within experimental error of those measured for 5 0 A M C L U M A L C M C L A M /3 0 r(UGAUGGU) and 5 0 G M C L U M A L C M U L G M /3 0 r(CGAUGAC), respectively. Evidently, the most effective use of LNA nucleotides is to space them every other nucleotide with the first LNA placed at the second nucleotide from the 5 0 end.\nInternal mismatches make duplex formation less favorable Table 5 contains thermodynamic parameters measured for the formation of duplexes containing single mismatches and the difference in stabilities relative to completely Watson-Crick complementary duplexes (Tables 1 and 2 ). All internal mismatches make duplex formation less favorable by at least 2 kcal/mol at 37 C corresponding to at least a 25-fold less favorable equilibrium constant for duplex formation. In general, terminal mismatches destabilize much less than internal mismatches. In fact, when the 3 0 terminal U L of 5 0 A M C M U M A M C M C M U L makes a GU pair, the duplex is stabilized by 0.14 kcal/mol at 37 C relative to a terminal AU pair.\nFor four cases, the effect of a mismatch with an LNA nucleotide was compared with that for the equivalent 2 0 -O-methyl nucleotide. In each case, the mismatch penalty for the LNA was less than that for 2 0 -O-methyl RNA. However, for an A M -G mismatch flanked by LNAs in the context 5 0 A L C M U L A M C L C M A L /3 0 r(UGAGGGU), the LNAs enhanced the mismatch penalty by $1 kcal/mol relative to a completely 2 0 -O-methyl RNA strand. Thus, oligonucleotides containing LNA may discriminate best against mismatches flanked by LNAs.\nOligonucleotide hybridization to RNA has many applications, ranging from quantifying gene expression (18) (19) (20) 56) to designing therapeutics (4, 8, 21, 46, (52) (53) (54) . LNA nucleotides have characteristics useful for these purposes. For example, LNA usually stabilizes duplexes (4, 44, 48, 51) and is more resistant than RNA and DNA to nuclease digestion (4, 6, 51) . The results presented here provide insights that are useful for designing 2 0 -O-methyl RNA/LNA chimeric oligonucleotides for various purposes. Some trends may be general for RNA A-form helixes and thus may also be relevant to other chimeras with nucleotides that favor A-form conformations. The results suggest several principles for the design of 2 0 -O-methyl RNA/LNA chimeras for hybridization to RNA\nThe database in Tables 1 and 2 is too small to The magnitude and sequence dependence of the stabilization due to LNAs are surprising. Ribose and therefore probably 2 0 -O-methyl ribose sugars in single strands are typically found in roughly equal fractions in C2 0 -endo and C3 0 -endo conformations. If the methylene bridge of an LNA only locks the sugar into the C3 0 -endo conformation, then the expected stabilization due to preorganization would be: DDG ¼ ÀRT ln 2, which is À0.4 kcal/mol at 37 C (310.15 K). The stabilization observed for a 5 0 terminal LNA is roughly À0.4 kcal/ mol, but the average stabilizations for internal LNAs and 3 0 terminal A L , C L and G L are more favorable at À1.3 and À1.2 kcal/mol, respectively. Moreover, if stabilization was only due to preorganization of an LNA sugar, then the effect would not saturate when alternate sugars are LNA. Evidently, the LNA substitution also affects the 5 0 neighboring base pair in a way that enhances the stabilization beyond that expected from preorganization of a single sugar. Interestingly, NMR structures of DNA/LNA chimeras bound to RNA show that only the DNA sugar 3 0 of the LNA is driven to a C3 0endo conformation for the sequence d(5 0 CTGAT L ATGC)/ 3 0 GACUAUACG, but all non-terminal DNA sugars are C3 0 -endo when all three Ts are LNAs (76) . The free energy increments at 37 C for LNA substitutions ranged from +0.83 to À1.90 kcal/mol with an average of À0.55 kcal/mol. This compares with a range from +0.18 to À2.17 kcal/mol and an average of À1.32 kcal/mol for the single internal LNA substitutions in Table 1 . The comparision suggests that single LNA substitutions are on average more stabilizing to 2 0 -O-methyl RNA/RNA duplexes than to DNA/DNA duplexes. This may reflect the expectation that LNA substitutions do not have a large effect on the conformations of 2 0 -O-methyl RNA/RNA duplexes, but alter the conformations of DNA/DNA duplexes.\nLNA substitutions should be useful for probing RNA with short 2 0 -O-methyl RNA oligonucleotides RNA structure can be probed with short oligonucleotides on microarrays (3) . To optimize such methods, it is necessary to have tight binding that is sequence independent and that discriminates against mismatches. It appears that LNA nucleotides can be used to achieve this. For example, free energy increments for 2 0 -O-methyl RNA/RNA nearest neighbors range from À0.7 to À3.5 kcal/mol, corresponding to 5 0 A M U M /3 0 UA and 5 0 G M C M /3 0 CG, respectively ( Table 4 ). The average increment of À1.3 kcal/mol of internal and 3 0 terminal LNA can help compensate for such less favorable stability of AU relative to GC pairs. The stability enhancement from LNA can also allow the use of shorter oligonucleotides.\nThe potential disadvantage to LNA substitutions in 2 0 -Omethyl RNA oligonucleotides is that discrimination against mismatches containing an LNA may be less than with a complete 2 0 -O-methyl RNA backbone. This was clearly true for three of the four cases where such direct comparisons were made. Nevertheless, internal mismatches with LNA nucleotides are considerably destabilizing, averaging a penalty of 4.1 kcal/mol at 37 C (Table 5) , which translates to almost a 1000-fold weaker binding due to a single mismatch. When LNAs flanked an A M -G mismatch, the mismatch penalty at 37 C was 4.4 kcal/mol compared with 3.3 kcal/mol in the absence of LNAs. Such an effect may reflect enhanced rigidity due to LNA, which thereby prevents a mismatch from adopting a favorable conformation. Thus, it may be advantageous to use LNAs to flank nucleotides likely to give small mismatch penalties." }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: Draft sequencing requires that the order of base pairs in cloned fragments of a genome be determined usually at least four times (4· depth of coverage) at each position for a minimum degree of draft accuracy. This information is assembled into contigs, or fragments of the genome that cannot be joined further due to lack of sequence information across gaps between the contigs. To generate high-quality draft, usually $8· coverage is optimal (1). Finished sequence, without gaps or ambiguous base calls, usually requires 8· to 10· coverage, along with additional analyses, often manual, to orient the contigs relative to one another and to close the gaps between them in a process called finishing. In fact, it has been stated that 'the defining distinction of draft sequencing is the avoidance of significant human intervention' (1) , although there are computational tools that may also be capable of automated finishing in some circumstances (2) .\nWhile some tabulate the cost differential between highquality draft versus finished sequences to be 3-to 4-fold, and the speed differential to be >10-fold (1), others state that the cost differential is a more modest 1.3-to 1.5-fold (3) . In either case, draft sequencing is cheaper and faster. Experts have debated whether finished sequencing is always necessary, considering the higher costs (1, 3, 4) .\nThus, here we set out to determine whether draft sequence data are adequate for the computational prediction of DNA and protein diagnostic signatures. By a 'signature' we mean a short region of sequence that is sufficient to uniquely identify an organism down to the species level, without false negatives due to strain variation or false positives due to cross reaction with close phylogenetic relatives. In addition, for DNA signatures, we require that the signature be suitable for a TaqMan reaction (e.g. composed of two primers and a probe of the desired T m s). Limited funds and facilities in which to sequence biothreat pathogens mean that decision makers must choose wisely which and how many organisms to sequence. Money and time saved as a result of draft rather than finished sequencing enables more target organisms, more isolates of the target and more near neighbors (NNs) of the target to be sequenced. However, if draft data do not facilitate the generation of highquality signatures for detection, the tradeoff of quantity over quality will not be worth it.\nWe used the Sequencing Analysis Pipeline (SAP) (5, 6) to compare the value of finished sequence, real draft sequence and simulated draft sequence of different qualities for the computational prediction of DNA and protein signatures for pathogen detection/diagnostics. Marburg and variola viruses were used as model organisms for these analyses, due to the availability of multiple genomes for these organisms. We hope that variola may serve as a guide for making predictions about The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact [email protected] bacteria, in which the genomes are substantially larger, and thus the cost of sequencing is much higher than for viruses. Variola was selected as the best available surrogate for bacteria at the time we began these analyses because:\ni. it is double-stranded DNA; ii. it has a relatively low mutation rate, more like bacteria than like the RNA or shorter DNA viruses that have higher mutation rates and thus higher levels of variation; iii. it is very long for a virus, albeit shorter than a bacterial genome; iv. we have access to many genomes, which were sequenced by our collaborators at the US Centers for Disease Control and Prevention in Atlanta, GA; v. there are finished genomes available, so we can compare actual finished data with simulated draft data. Only recently have a fairly large number of Bacillus anthracis genomes become available to us. However, since only some of these are finished, currently we cannot compare finished with draft results for this bacterial genome.\nThe sequencing analysis pipeline uses the DNA and protein signature pipelines\nThe draft SAP simulations are nearly identical to those using finished genomes, described previously (5, 6) . The SAP ( Figure 1 ) performs stochastic (Monte Carlo) simulations and includes our DNA and Protein Signature Pipelines as components, which will be summarized briefly below. It is necessary to describe what the signature pipelines do before the SAP can be clearly described, so signature pipelines will be discussed first. As a step within the DNA and Protein Signature Pipelines, DNA sequence alignments of multiple draft genomes are required. For this we use the WGASA software, also summarized below. Once each of these components of the SAP has been presented, the SAP itself will be described.\nThe DNA Signature Prediction Pipeline, described in detail elsewhere (7) (8) (9) (10) , finds sequence regions that are conserved among target genomes by creating a consensus based on a multiple sequence alignment. WGASA is the software used in the analyses here to create an alignment and will be discussed below. Next, the DNA Signature Pipeline identifies regions that are unique in the target sequence consensus relative to all other non-target bacterial and viral sequences that we have in a >1 Gb database, which is frequently updated from the NCBI GenBank sequence database (11) and other sources (e.g. our collaborators at the CDC, USDA and other public sources, such as TIGR, Sanger Institute and the Joint Genome Institute). From the conserved, unique regions, signatures are selected based on the requirements of a particular technology, in this case, TaqMan PCR. These signature candidates may then proceed for further in silico screening (BLAST analyses to look for undesired inexact matches) before undergoing laboratory screening. For an SAP run, first a pool of target genome and a pool of NN genomes are collected. Then many random subsamples of target and NN genomes are selected from the pool, and each subsample is run through either the DNA signature pipeline or the protein signature pipeline, which identify regions conserved among target genomes and unique relative to non-target genomes, where unique regions are evaluated by comparing to a large sequence database of all currently available bacterial and viral complete genomes or the non-redundant protein database, excluding NNs from the NN pool that are not in that random subsample. Thus, each run of the SAP requires many runs of the DNA or protein signature pipelines with different random samples, generating a range of outcomes that are plotted on range plots.\nProtein signature prediction and SAP methods have previously been described in detail (6) . The following briefly describes the procedure. First, target genomes are aligned using WGASA. A set of gene (start, end) pairs for both the plus and minus strands relative to the reference genome is required. This implies that coding frames for the translation of nucleic acid codons into amino acids for each protein of the target organism's genome have been correctly determined. From the aligned genomes, nucleotide codons are translated into amino acid sequence based on the gene locations, and conserved strings of six or more amino acids among all the target genomes are recorded. These conserved fragments are then compared with the NCBI GenBank non-redundant (nr) database of amino acid sequences, unveiling peptides that are unique to the target species. For our computations, we require that if a peptide signature is longer than six amino acids, then every sub-string of length six amino acids is also conserved and unique. There may be many conserved and unique peptide signatures on the same and on different proteins. The resulting conserved, unique peptides that are at least six amino acids long from open reading frames are considered to be protein signature candidates.\nSignature peptides may be used as targets for antibody or ligand binding and may be developed for use in detection, therapeutics or vaccines (12, 13) . Since the signature regions are highly conserved within a species, it is likely that they are functionally important to the organism's survival or reproduction. Those signatures that land on or near protein active sites may be developed into therapeutics, since antibody or ligand binding may interfere with protein function. Signature regions may even be considered as vaccine targets, since these unique peptides may evoke a specific response in the host (14, 15) .\nFor draft genomes, WGASA, or Whole Genome Analysis through Scalable Algorithms, is used to align multiple sequences. This is the only available tool that enables multiple sequence alignment of draft genomes and that is capable of aligning large or many genomes. WGASA requires at least one finished reference genome and the others may be draft.\nOnly recently has it become possible to use the DNA Signature Pipeline to predict signature candidates for draft genomes. This capability is due to the invention of software for multiple sequence alignment of draft genomes with at least one completed full genome. WGASA was developed by David Hysom, Chuck Baldwin and Scott Kohn in the Computations directorate at Lawrence Livermore National Laboratory. They designed the software in close communication with members of our bioinformatics team, and it is tailored for our needs of generating diagnostic and forensic pathogen signatures.\nWGASA can efficiently align large (e.g. bacterial) genomes. In addition, the developers have created a parallel version that runs in minutes, allowing the SAP simulations, involving thousands of calls to WGASA, to complete in a feasible time frame. In addition to the SAP analyses, this tool has enabled us to revisit signature predictions for several important organisms, such as the food-borne pathogen Listeria, that were previously problematic because some of the sequences were available only in draft format.\nThe tool requires that there be one or more complete, finished genome, and any number of draft sequences. It is based on suffix-tree algorithms (16) . It requires that anchors, identical sequence fragments of user-specified length, be found in each of the genomes to be aligned. Thus, there must be some level of sequence conservation among the genomes in order to discover anchors of sufficient length (e.g. 35-60 bp) that are present in all the genomes. Then the regions between the anchors are aligned using a tool, such as clustalw or HMMer. The algorithm functions most efficiently if anchors are frequent and dispersed across the genomes to provide even coverage. If substitutions, deletions, insertions or gaps in sequence information (e.g. between contigs) result in an anchor's absence in one or more of the genomes, then those regions must be aligned using clustalw, which is slower and more memory intensive for large amounts of sequence data. Similar to all anchor-based alignment algorithms, WGASA is dependent upon a high degree of co-linearity across all input genomes.\nThe SAP for DNA signature analyses operates as follows. First, all available complete genomes of target were gathered into a pool with the total genome count called T. A second pool was created of all available NN complete genomes, with the total count of sequences called N. Next, we selected 10 random samples of size t targets and n NNs, for all t ranging from 1 to T and all n ranging between 1 and minimum(10,N). We ran the DNA Signature Prediction Pipeline for each sample, with signature prediction based on conservation among the t target strains and uniqueness relative to a >1 Gb database minus those NNs in the NN pool that were not chosen in that sample. Thus, for each sample, signature candidates were predicted as though we had only t target and n NN sequences, as well as the rest of the less-closely related organisms in our database that are not considered NNs. In addition to the number of TaqMan signature candidates, the fraction of the genome that is conserved among the t target sequences was also calculated. Based on the combined results of the many signature pipeline runs using random target samples of size t and n, we assessed how much sequence data, that is, the values of t and n, was required to approximate the number of signature candidates c that were predicted when the full data set (all target and NN sequences, t ¼ T and n ¼ N) was analyzed with the signature pipeline. Using the full data set will yield the fewest signatures, because lack of conservation or uniqueness will winnow away all unsuitable candidates.\nThus, the SAP performs Monte Carlo sampling from the target and NN genomes, runs each sample through the signature pipeline and summarizes the results of the hundreds of signature pipeline runs in a single plot. On our 24 CPU Sun server, up to seven signature pipeline simulations may be run in parallel, each requiring $15-22 min for viral genomes. All of the SAP analyses of dozens of bacteria and viruses to date have used a total run time of 6.26 years (operating in parallel), with an average pipeline run time of 0.522 h, and a process time span of 2.32 years.\nThe span of predictions generated by different random samples of genomes is illustrated using range plots (Figures 2-8 ). Along the y-axis, whole numbers represent the number of target strains t and the incremental values between the integers represent the number n of NN genomes. Only Figures 3, 5 and 6 have the incremental n values, because for the other plots of target sequence conservation only, the number of NNs was not relevant, and for the protein analyses NN comparisons were not made (described below). Outcomes of the number of signature candidates or the fraction of the target genome that is conserved are plotted along the x-axis as horizontal lines spanning the range (of predicted numbers of signatures or fraction conserved) for the s random samples of size (t,n) with the median and quantiles of the range indicated by colored, short vertical lines. If a random sample of t target strains and n NN strains were sequenced, there would be a 90% chance that the number of signature candidates for that sample would be less than or equal to the 90% quantile mark. The expected outcome is a reduction in the number of signature candidates or the fraction of the genome that is conserved as the number of target and NN sequences used in the simulations increases, due to a reduction in conservation from additional targets and a reduction in uniqueness from additional NNs.\nThe SAP analyses for proteins proceed much like that for DNA signatures. Random samples of size t target sequences are generated, where t ranges from 1 to T, the total number of target sequences in the pool. Either finished data, actual draft data, or draft data simulated as described below are aligned using WGASA. The protein signature prediction pipeline is run on each random sample, and the range, median, 75th and 90th quantiles of the number of protein signature candidates for the samples of a given target size t is plotted in range plots as described above.\nOur DNA SAP analyses examined the effects of both the number of target as well as the number of NN sequences, but To discriminate samples in which zero NNs were used, the range is drawn as a horizontal gray line, and when n > 0, the range is drawn as a black line. The best estimate of the true value is the quality measure determined using the entire target and NN pools, and is represented by a vertical black line. This best estimate plus a constant c ¼ 20 is at the location of the vertical dashed line and was selected to indicate a reasonable distance from the true answer. The 75% quantile for each range is shown with a black, vertical tick mark. our protein SAP analyses investigated the effects of only the number of target sequences. This is because composing the lists of NN proteins for random, temporary exclusion from the protein nr database (to estimate the value of that NN sequence data) would be difficult to automate for rapid, high-throughput computations. Thus, we compared the target proteins with all the proteins in nr, regardless of their phylogenetic relationship to the target. This was comparable with DNA SAP results using all available NN data.\nWe had sequence data for four strains of Marburg virus, both the actual draft and the finished versions of those same isolates, provided for these analyses by a colleague working at Lawrence Livermore National Laboratory. The draft sequence was of $3· to 6· coverage, which enabled us to compare SAP results using the same strains in finished form. The identities of these sequences are provided in the Appendix. For the draft Marburg analyses, we selected one finished strain, the reference strain from GenBank (gi|13489275|ref|NC_001608.2|\nMarburg virus, complete genome), as the WGASA reference genome, and then used random sub-samples from the four draft genomes. Marburg was the only organism for which we could obtain a sufficient number of draft genomes for the SAP Monte Carlo simulations. A total of 814 simulations for DNA signatures, i.e. individual runs of the DNA signature pipeline, and 48 simulations for protein signatures were performed using Marburg finished and draft data, requiring an average of 15 min per simulation.\nWe used finished sequence data generously provided by collaborators at the US CDC for 28 variola major genomes and 22 NN genomes from the Orthopox family. The sequence identities are provided in the Appendix. Since we did not have real draft data available, we developed a program to simulate draft sequence from finished sequence, based on guidance from two colleagues who have been involved in sequencing efforts and the finishing process in the Biology and Biotechnology Research Program at Lawrence Livermore National Laboratory. In outline, the draft simulator program randomly cuts a genome into contigs of a size randomly selected from an exponential distribution. Stochastic simulation also determines whether there are gaps or overlaps between contigs, as well as the size of the gap or overlap. Sequencing errors are also simulated.\nThe following paragraphs describe the draft simulation process in greater detail. First, the 5 0 end of the sequence is simulated as missing or present according to a random (Bernoulli) trial based on the probability of there being a gap in the sequence data. If simulations randomly determine that the first part of the sequence is missing, then the size of the missing segment is selected randomly from a uniform distribution ranging from the minimum gap size to the maximum gap size. The length of the first contig is selected randomly from an exponential distribution with a non-zero minimum contig size and a maximum contig size that is a fraction of the mean genome length for the species. The mean of this exponential distribution is also specified as a fraction of the mean genome length.\nNext, a random Bernoulli trial again determines whether there is a gap or overlap between the first and second contigs, and the size of the gap or overlap is chosen from the appropriate uniform distribution (range for gap size ¼ 1-2000 bases, range for overlap size ¼ 20-40 bases). The size of the contig is selected from the exponential distribution as described above. Additional contigs are simulated in a similar manner.\nWithin each contig, sequencing errors are simulated based on the size of the contig, and whether the base position is at an end (first or last 100 bases) or in the middle of the contig. For long, double-stranded DNA viruses (e.g. variola) and bacteria, the sequencing error rates are larger at the beginning and end of a contig than in the middle, and small contigs are more likely to contain sequencing errors than are large contigs. In contrast, due to differences in generating the products for Sanger sequencing that are employed for smaller RNA and DNA viruses, there are often more sequencing errors in the middle of contigs for such smaller viral draft genomes. Although we did not specifically simulate draft for RNA and short DNA viruses, our simulator should work with minor modification to a few parameters. Thus, there are four parameters that must be specified for simulating sequencing errors: (i) the size cutoff for small versus large contigs, (ii) the probability of errors in the middle portion of small versus large contigs, (iii) the length of the contig ends where sequencing is either less accurate (bacteria and long doublestranded DNA viruses) or more accurate (small viruses, RNA viruses) and (iv) the probability of sequencing errors at the contig ends. If there is a sequencing error at a particular base, we assumed that that base is randomly changed to one of the other three bases with equal probability. Although additional features could be added to the draft simulation tool, the stochastic features that we have incorporated capture the main features of draft sequence and produce data that are suitable for SAP analyses.\nWe performed six sets of analyses using simulated variola draft. Three sets of simulated variola draft runs of the SAP used the following parameters: probability of a gap between contigs ¼ 0.95; probability of overlap between contigs ¼ 0.05; minimum gap size if there is a gap (uniform distribution) ¼ 1 bp; maximum gap size ¼ 2000 bp; minimum overlap if there is overlap (uniform distribution) ¼ 20 bp; maximum overlap ¼ 40 bp; minimum contig size (exponential distribution) ¼ 2000 bp; maximum contig size ¼ 0.5 · (mean genome length) bp mean contig size ¼ 0.05 · (mean genome length) bp cutoff size for small versus large contigs ¼ 10 000 bp; probability of sequence errors inside large contigs ¼ 0.01; probability of sequence errors inside small contigs ¼ 0.05; We will refer to the above set of simulations as those with a high probability of sequencing errors, or low-quality draft. The other three simulated variola draft runs used all the same parameters as above, except that the sequencing error rates were dramatically lower, more in line with error rates of 10 À5 / base that the US Centers for Disease Control and Prevention (CDC) has indicated for their draft variola genomes: probability of sequence errors inside large contigs ¼ 10 À5 ; probability of sequence errors inside small contigs ¼ 10 À4 ; probability of sequence errors in the contig ends ¼ 10 À3 ; These runs were referred to as low error rate, or highquality draft. Finally, we performed SAP runs using high error rate (low quality) simulated draft of the NN sequences and intermediate quality simulated draft of target genomes, using the following probabilities of sequencing errors: probability of sequence errors inside large contigs ¼ 10 À3 ; probability of sequence errors inside small contigs ¼ 10 À3 ; probability of sequence errors in the contig ends ¼ 10 À3 ;\nThe intermediate quality simulated draft is consistent with error rates for draft sequencing cited in the literature (1,3) .\nFor the parameter values specified above, three SAP experiments were simulated. In the first, only the target sequences were simulated into draft, and the NN sequences remained as finished sequences. In the second, the NN sequences were converted to simulated draft and the target sequences remained as finished. In the third, both target and NN sequences were simulated into draft. In the second and third cases, all the NNs were run through the draft simulator each time they were chosen, so that the draft sequences (i.e. location and extent of gaps and sequence errors) differ for the same genome among samples. In the first and third cases, the target sequences must be aligned, and WGASA requires that one of the sequences be a finished genome for reference. Thus, for each random sample from the pool of target genomes, one genome was randomly selected to be the finished genome, and so was left as finished sequence, and the other genomes in the sample were replaced with simulated draft sequence (by running through the draft simulator) before alignment. As with NNs, target draft sequences differ for the same genome among samples due to the randomness of the draft simulation each time it is run. In addition, the target genome that is chosen to be the finished, reference genome differs between samples, and the other target genomes in the sample simulation are replaced with simulated draft versions of the actual finished sequences. Then these sequences were aligned using WGASA and the SAP process was run as described above. A total of 1101 stochastic simulations per 'experiment' were performed, requiring $18 min per simulation. Each simulation involved randomly selecting the subset of target and NN sequences to be included, simulating the draft data based on the finished genomes, aligning the target sample, and finally running the DNA Signature Pipeline. There were four combinations examined: (i) finished variola and finished NN, (ii) draft variola and finished NN, (iii) finished variola and draft NN and (iv) draft variola and draft NN, with each of the draft runs repeated for both low and high sequencing error rates. The combination (iv) was also run with intermediate quality simulated draft variola and low-quality simulated draft NNs. In total, there were eight computational experiments for the finished and simulated draft variola data.\nWe have used the following function to estimate viral sequencing costs, based on discussion with our laboratory colleagues involved in sequencing and finishing. This is merely a rough estimate, and the actual costs of sequencing any given organism may differ substantially from this rule-of-thumb calculation. In Equation 1, it is assumed that the cost of sequencing viruses does not decline for sequencing second and subsequent isolates. While this may be a false assumption in cases where isolates are similar to one another, in other cases where the new sequences are divergent, as isolates from different outbreaks or for viruses with rapid mutation rates, the cost is especially unlikely to decline. In addition, the $0.40/bp figure for draft of 6· to 8· coverage could range from $0.30 to $0.50/bp using shotgun sequencing, but may be as low as $0.10-$0.20/bp if primer walking works well (i.e. known primer sites are found in new isolates). Finishing could be 1-3 times more than the cost of draft, so we used a factor of 2 times more (draft $0.4/bp, finished $0.4 + 0.8/bp) in the equation above as a reasonable estimate. With rapidly evolving sequencing technologies and costs, these are only rough guides that may quickly become outdated.\nIt may be substantially less expensive, on the order of 3-fold, to generate draft compared with finished sequence data for an organism like Marburg virus, according to estimates using Equation 1. For example, for $45K, one could sequence either two finished genomes or one finished and three draft. However, draft sequencing of this low quality (3· to 6·) for Marburg causes a dramatic decline in the ability to computationally eliminate regions of poor conservation, and thus to exclude poor signature regions ( Figure 2 ). This occurs because gaps in the draft data of some of the sequences mask sequence variation among strains. Using the best available data, all six finished genomes, there is 75.2% sequence conservation. The deficiencies of draft data give a false impression that there is 92.6% sequence conservation (Table 1) . Each additional finished genome reduces the conserved fraction by $5%, compared with a reduction of only 2% per genome for the draft data.\nThe overestimation of conservation using draft Marburg data also results in overestimation of the number of signature candidates (Figure 3 ). Samples of four draft targets plus one finished reference yield 43 signature candidates. A smaller sample size of only two draft targets and one finished reference generate upwards of 80 candidates. These results differ from those using finished genomes, where the lack of sequence conservation is more evident and there are zero TaqMan signatures conserved among all strains. Most combinations of four finished genomes are sufficient to eliminate nonconserved signatures ( Figure 3A ). Although predictions that there are 0 signature candidates shared among all finished strains may seem to argue against TaqMan methods, in fact this information provides important guidance for the development of TaqMan signatures with degenerate bases or a set of signatures that will, in combination, pick up all sequenced strains. Other analyses indicate that there are TaqMan signatures conserved among five of the six strains, so that two signatures would form a minimal set that could detect both the one divergent and the other five strains.\nEstimated sequencing costs of draft variola and draft NNs indicate that draft may require only one-quarter to one-half the costs of finished sequencing. Simulations of high-quality draft data indicated that it is as good as finished data for diagnostic signature prediction. The conservation range plots ( Figure 4A and B) are virtually identical for finished and high-quality draft, and indicate that $98% of the genome is conserved among sequenced isolates. For intermediate quality draft ( Figure 4C ) the conservation range plot is also similar to that for finished sequence, showing that $97% of the genome appears to be conserved. The range plots for the number of TaqMan signature candidates are very similar for finished sequence data, high or intermediate quality draft target, and high or low-quality draft NNs ( Figure 5A-D) .\nIn contrast to the results using high-quality simulated draft or actual Marburg draft, simulations of low-quality variola draft target illustrate that sequence conservation may be underestimated compared with results with finished sequence data, due to sequencing errors (Table 1 and Figure 4D ). With lowquality draft target, it appears that only 58% of the genome is conserved among isolates.\nLow-quality (high error rate) draft NN data, however, yield results that are very similar to those with high-quality draft or finished NN data, as long as the target sequence information is of intermediate to high quality ( Figure 5C -D andFigure 6): At least four NN sequences are necessary to ensure that signature regions are unique, whether the NN data are low-or highquality draft or finished. That is, our simulations indicate that low-quality draft NN data are adequate for predicting DNA signatures, as long as there is good quality target sequence data. This results because errors in the NN sequences occur at random locations that differ in each NN sequence. As long as at least one of the NNs has enough correct sequence to eliminate each of the non-unique target regions, then the unique regions of the target can be determined.\nThe results illustrated in the figures are emphasized by the data in Table 1 . This table shows the fraction of the target genome that is conserved and conserved+unique, the number of conserved+unique regions that are at least 18 contiguous base pairs long and the number of base pairs in the largest of these regions, since these are the sections that are of sufficient length for one or possibly more primers to be located. The number of these regions is similar for finished data and for draft with a low error rate. Low-quality draft (with a high rate of sequencing errors) for the target data, however, gives the false impression that there are fewer and shorter regions that are conserved and suitable as signature regions than is actually the case.\nThere is an artifact in some of our results that is a consequence of the order in which we calculate conservation and then uniqueness, although this does not affect the signatures that are predicted. First, a conservation gestalt is generated from the sequence alignment, in which non-conserved bases are replaced by a dot ('.'). Then uniqueness is calculated based upon perfect matches of at least 18 bp long between the conservation gestalt and a large sequence database of non-target The percent of the target genome that is conserved varies slightly among the runs using finished target sequences because different genomes were randomly selected to be the reference strain in each multiple sequence alignment.\nsequences. Non-conserved bases in the conservation gestalt may break up a region into conserved fragments of <18 bases long, and as a result these short fragments are not tested for uniqueness. Consequently, if there is a low level of conservation, then we may overestimate the fraction of the genome that is unique. For example, in Table 1 the conserved+unique fraction is 4% with finished variola target data, but is overestimated at 58% with low-quality draft. This artifact does not, however, affect TaqMan signature prediction, since the regions suitable for primers and probes must have at least 18 contiguous, conserved bases, and all of these are tested for uniqueness, i.e. there is no underestimation of uniqueness in conserved fragments that are at least 18 bp in length, and thus no underestimate of uniqueness in the predicted signatures.\nWe are working to eliminate this issue in future versions of the software.\nProtein results show a large disparity between finished and draft data. There are 113 protein signature candidates for finished Marburg data compared with only two protein signature candidates for Marburg draft (Figure 7 ). For variola, using all available target data, 97, 14, 6 and 0 protein signatures are predicted using finished, low error, intermediate error and high error draft target data, respectively ( Figure 8 ). Thus, sequencing errors substantially reduce the detection of amino acid sequence conservation, even if sequencing errors occur at the low rate of 10 À4 -10 À5 across most of the genome. The pattern of how additional sequences reduce the number of protein signature candidates also differs for draft compared with finished sequence data. With finished data, there is a large range in the number of peptide signature candidates predicted with 17 or fewer variola genomes, and this range narrows around the lower bound with >17 genomes. With 16 genomes, the 75% quantile mark approaches the final predicted number of 97 signatures ( Figure 8A ). This pattern indicates that there is a set of 97 peptides that are highly conserved among all currently sequenced variolas, which are unlikely to be eroded even as more sequence data are obtained. In other words, additional sequence data are probably not needed at this time in order to computationally predict good peptide signature targets, and as few as 16 finished target sequences would most likely have been adequate to generate this same list of $100 peptide signatures.\nDraft data, in contrast, whether they are of low or high quality, mask the above pattern ( Figure 8B and C): the range and 75th quantile of the number of peptide signatures gradually decline with each additional target sequence (rather than a sudden, sharp drop as is seen with the finished data), suggesting that additional target sequences would continue to erode the number of peptide signatures. This occurs since sequencing errors occur at random, in different locations in each of the draft target genomes, and obscure the truly conserved peptides. One might falsely infer from peptide SAP results based on the draft data that additional sequencing (beyond the 28 variola major genomes used here) would be useful in generating peptide signature candidates. In actuality, however, SAP analyses using the finished sequence data indicate that there are already ample sequence data for peptide signature prediction.\nThe failure of draft sequencing for Marburg at 3· to 6· coverage or of simulated variola draft with a high error rate to facilitate the prediction of detection signatures highlights a need for finished viral sequences, or at least for draft of high quality such as 8·. Otherwise, a large number of signature candidates either will fail in screening because they are incorrectly designated as conserved among strains (as observed with the Marburg results), or too few regions will be classified as conserved (as observed with variola), and thus not be considered for signatures.\nThe variola simulations with intermediate to high-quality draft (that is, a low error rate, approximating what one might observe with 8· coverage) target and/or NN genomes deliver virtually the same results as finished genomes. Considering that it costs approximately three times as much to generate finished sequence as it does draft, our analyses indicate that investing in more high-quality draft target genomes is better than investing in fewer finished genomes. For our analyses, only one target strain must be finished, and the remaining target sequences and all the NNs may be provided as draft.\nOur results indicate that NN sequencing may be of low coverage, and thus of low quality, without serious detriment to signature prediction, as long as there are at least four NN draft genome sequences.\nIf high-quality draft sequence is used, and it appears that there is too little sequence conservation among target strains, one might relax specifications for 100% conservation among strains for diagnostic signature prediction. Calculations indicate that it is often possible to generate signatures if one allows a base to be considered 'conserved' if it is present in only a fraction of the genomes (e.g. 75%) rather than the standard requirement for 100% conservation when finished sequence data are used. We have used this 'ratio-to-win' option to generate signature candidates for some highly divergent RNA viruses (for which we have finished sequence), although usually our preference is to include degenerate bases, especially when there are only a few bases with heterogeneity among strains in a given signature candidate. Using a ratio-to-win approach may be particularly important for the generation of protein signature candidates, since draft target data severely compromises the ability to detect conserved strings of amino acids.\nIn summary, intermediate to high-quality draft sequencing of target genomes, combined with low-quality draft sequencing of close phylogenetic relatives, is sufficient for the prediction of DNA diagnostic signatures. Prediction of peptide/ protein signature candidates, in contrast, requires finished sequencing to avoid substantial underestimation of conserved peptide regions." }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: An epitope can be defined as the molecular structure recognized by the products of immune responses. According to this definition, epitopes are the specific molecular entities engaged in binding to antibody molecules or specific T cell receptors. An extended definition also includes the specific molecules binding in the peptide binding sites of MHC receptors. We have previously described [1] the general design of the Immune Epitope Database and Analysis Resource (IEDB), a broad program recently initiated by National Institute of Allergy and Infectious Diseases (NIAID). The overall goal of the IEDB is to catalog and organize a large body of information regarding antibody and T cell epitopes from infectious pathogens and other sources [2] . Priority will be placed on NIAID Category A-C pathogens (http://www2.niaid.nih.gov/Biodefense/ bandc_priority.htm) and emerging/re-emerging infectious diseases. Epitopes of human and non-human primates, rodents, and other species for which detailed information is available will be included. It is envisioned that this new effort will catalyze the development of new methods to predict and model immune responses, will aid in the discovery and development of new vaccines and diagnostics, and will assist in basic immunological investigations.\nThe IEDB will catalog structural and phylogenetic information about epitopes, information about their capacity to bind to specific receptors (i.e. MHC, TCR, BCR, Antibodies), as well as the type of immune response observed following engagement of the receptors (RFP-NIH-NIAID-DAIT-03/31: http://www.niaid.nih.gov/contract/archive/ rfp0331.pdf).\nIn broad terms, the database will contain two general categories of data and information associated with immune epitopes -intrinsic and extrinsic (context-dependent data). Intrinsic features of an epitope are those characteristics that can be unequivocally defined and are specified within the epitope sequence/structure itself. Examples of intrinsic features are the epitope's sequence, structural features, and binding interactions with other immune system molecules. To describe an immune response associated with a specific epitope, context information also needs to be taken into account. Contextual information includes, for example, the species of the host, the route and dose of immunization, the health status and genetic makeup of the host, and the presence of adjuvants. In this respect, the IEDB project transcends the strict boundaries of database development and reaches into a systems biology application, attempting for the first time to integrate structural information about epitopes with comprehensive details describing their complex interaction with the immune system of the host, be it an infected organism or a vaccine recipient [1] [2] [3] .\nFor these reasons, it was apparent at the outset of the project that it was crucial to develop a rigorous conceptual framework to represent the knowledge related to the epitopes. Such a framework was key to sharing information and ideas among developers, scientists, and potential users, and to allowing the design of an effective logical structure of the database itself. Accordingly, we decided to develop a formal ontology. Over the years, the term \"ontology\" has been defined and utilized in many ways by the knowledge engineering community [4] . We will adopt the definition of \"ontology\" as \"the explicit formal specifications of the terms in a domain and the relationships among them\" [5] . According to Noy and McGuinness [6] , \"ontology defines a common vocabulary for researchers who need to share information in a domain and helps separate domain knowledge from operational knowledge\". Thus, availability of a formal ontology is relevant in designing a database, in cataloging the information, in communicating the database structure to researchers, developers and users, and in integrating multiple database schema designs and applications.\nSeveral existing databases catalog epitope related data. We gratefully acknowledge that we have been able use these previous experiences in the design and implementation of the IEDB. MHCPEP [7] , SYFPEITHI [8] , FIMM [9] , HLA Ligand Database [10] , HIV Immunology Database [11] , JenPep [12] , AntiJen [13] , and MHCBN [14] are all publicly available epitope related databases. In general, these databases provide information relating to epitopes, but do not catalog in-depth information relating to their interactions with the host's immune system. It should also be noted that none of these databases has published a formal ontology, but all of them rely on informal or implicit ontologies. We have taken into account as much as possible these ontologies, inferring their structure by informal communications with database developers or perusal of the databases websites.\nThe ontology developed for IEDB and described herein complements two explicit ontologies that are presently available: the IMGT-Ontology and the Gene Ontology (GO). The IMGT-Ontology [15] was created for the international ImMunoGeneTics Database (IMGT), which is an integrated database specializing in antigen receptors (immunoglobulin and T Cell receptors) and MHC molecules of all vertebrate species. This is, to the best of our knowledge, the first ontology in the domain of immunogenetics and immunoinformatics. The GO project [16] provides structured, controlled vocabularies that cover several domains of molecular and cellular biology. GO provides an excellent framework for genes, gene products, and their sequences, but it does not address the specific epitope substructure of the gene products. The IMGT provides an excellent ontological framework for the immune receptors but lacks information relating to the epitopes themselves. Therefore it was necessary to expand the available ontologies and to create an ontology specifically designed to represent the information of immune receptor interaction with immune epitopes. Wherever possible, the IEDB ontology conforms to standard vocabularies for capturing values for certain fields. For capturing disease names, IEDB uses the International Classification for Diseases (ICD-10) [17]. The NCBI Taxonomy database nomenclature [18, 19] is used to capture species and strain names, and HLA Allele names are consistent with the HLA nomenclature reports [20] .\nThe IEDB is being developed as a web-accessible database using Oracle 10g and Enterprise Java (J2EE). Industry standard software design has been followed and it is expected that IEDB will be available for public users by the end of 2005.\nProtégé http://protege.stanford.edu was used to design and document the IEDB ontology. Protégé is a free, open source ontology editor and knowledge-base framework, written in Java. It provides an environment for creating ontologies and the terms used in those ontologies. Protégé supports class, slot, and instance creation, allowing users to specify relationships between appropriate entities. Two features that IEDB ontology effort used extensively were Protégé's support for creating ontology terms and for viewing the term hierarchies and the definitions. The support for a central repository on ontologies, along with browsing support, is key in reviewing and reusing ontologies.\nWhile there are several open source tools available [21] for developing ontologies, we selected Protégé because of its extensibility to a variety of plug-ins that are readily available for integration. It also has the ability to export to different formats including the Ontology Web Language (OWL) (http://www.w3.org/TR/owl-features/), which allows interoperability with other ontologies.\nWe have previously described some of the general concepts relating to the IEDB design [1, 2] . More information relating to various aspects of the project can be accessed at http://www.immuneepitope.org/. Herein, we report a detailed description of the novel aspects of the IEDB ontology. In designing our application architecture, we have followed the common system engineering practice of first determining the scope and nature of the data involved. A first essential step is to understand the semantics of the domain and to capture that knowledge in an agreed-upon format. Arranging the domain concepts in a taxonomy is one of the initial organizing steps in the ontology design process. The class hierarchy represents the generalization and specialization relationships between the various classes of objects in a domain [6] . Briefly, classes describe concepts in the domain. Subclasses represent concepts (classes) that are more specific than the superclass and these subclasses can have their own unique properties. Slots represent properties of the classes. For example, in Figure 1 , we see that there is a class named Reference and three more specific subclasses of Reference: Journal Article, Patent Application, and Direct Submission. Figure 1 also shows that the class Epitope has a number of properties (slots) associated with it such as \"has Epitope Structure\" and \"has Epitope Source\".\nOur approach for creating the class hierarchy was a topdown development process where we defined each class in a domain and then identified its properties before building the hierarchy. The main classes identified for IEDB are Reference, Epitope Structure, Epitope Source, MHC Binding, Naturally Processed Ligand, T Cell Response, and B Cell Response (Figure 1 ). The Epitope class is the main class that encompasses all the individual concepts that were identified. The individual concepts are related to other classes. The primary relationships use the sub-class relationship or use a property (shown in the figures by the arcs labeled \"has\") that has a restriction on the type of the value that may fill that slot.\n\"Reference\" is the class encompassing information related to the data source from which an epitope and its related information are extracted into the IEDB. We have identified three broad subclasses of References that describe where epitope information will be obtained. They are Journal Article, Patent Application, and Direct Submission. The complete listing of slots (fields) encompassed by the Journal Article, Patent Application, and Submission classes are provided in Figure 2 . The Journal Article class refers to manuscripts published in peer-reviewed journals. The Patent Application class captures all the reference fields for a patent application that contain epitope information. The Submission class captures information about sources that contribute data to the IEDB directly. Data deposited by the Large Scale Antibody and T Cell Epitope Discovery contracts [3] and those transferred from other websites fall into this class.\nThe Epitope Structure and Epitope Source classes capture intrinsic features of an epitope. The Epitope Structure class captures the physical and chemical features of an epitope. Virtually any molecular structure may provoke an immune response, such as proteins, carbohydrates, DNA, and lipids. In the Epitope Structure class, structural information relating to linear sequences and 2-D structures of the epitope, if available, are catalogued. The Epitope Source class captures the phylogenetic source of an epitope, including species of origin, gene name, protein name, and links to other databases for more detailed information about proteins and genes. Figures 3A and 3B show the listings of properties (slots/fields) encompassing the Epitope Structure and Epitope Source classes.\nThe experimental data and information about specific experiments and the methodology utilized are captured in the Assay Information class. The name of the assay used, the type of response measured in the assay, and the readout of the assay are examples of information captured in the Assay Information class. This important class is used as a superclass of several other classes (and thus its properties are inherited by those classes). A complete listing the properties (slots/fields) in the Assay Information class is shown in Figure 3C .\nAs with Assay Information, the classes Immunization, Antigen, and Antigen Presenting Cell are used in multiple other class descriptions. Features relating to the induction of the immune response are captured in the Immunization class ( Figure 4A ). It has relationships to other classes like Immunized Species, Immunogen, In vivo Immunization, and In vitro Immunization. Immunized Species contains information relating to the host that is being immunized. The Immunogen class describes the molecules that induce the immune response and an associated carrier molecule, if present. Features relating to how the immunogen was introduced to the immunized species are captured under the In vivo and In vitro Immunization classes.\nSimilarly, antigens are defined as the whole molecules that react with the products of an immune response (as opposed to the epitopes which are the specific structures, contained within the antigen that engages the immune receptor). Information relating to the antigen and any associated carrier molecule is captured in the Antigen class ( Figure 4B ). During immune responses, antigen-presenting cells process antigens and present peptide epitopes complexed with MHC molecules. This information is captured in the Antigen Presenting Cells class, which has a relationship to the MHC Molecules and the Source Species classes ( Figure 4C ). The Source Species class describes the species information from which the antigen presenting cells are derived.\nThe MHC Binding class captures the details relating to the interaction of the epitope with specific MHC molecules and information relating to the MHC molecule along with any available Epitope-MHC complex structure details. This class also has a slot that is restricted to be an instance of the Assay Information class ( Figure 5A ).\nOverview of IEDB Class Hierarchy Figure 1 Overview of IEDB Class Hierarchy.\nclasses. Extrinsic features are context-dependent attributes, being dependent upon specific experimental conditions. The Naturally Processed Ligand class captures data related to epitopes that are naturally processed and presented on the cell surface. This class has properties that are instances of classes including Antigen Presenting Cell, Antigen, and Assay Information ( Figure 5B ).\nThe Naturally Processed Ligand class differs from the MHC Binding class in that information related to the antigen that was processed and the cell types in which the processing occurred is represented. MHC Binding class captures data relating to in vitro MHC binding assays, which assess the epitope's binding capacity to the MHC molecule. Hence the MHC Binding class does requires neither the Antigen class not the Antigen Presenting Cells class. In general, naturally processed ligands are assessed in the absence of a T cell response, for example, identified by direct elution from MHC molecules extracted from infected cells or antigen presenting cells. Thus, the Immunization class is not used as a value restriction by the Naturally Processed Ligand class.\nThe T Cell Response class captures all of the T cell mediated immunity-related information ( Figure 6A ). It has properties that are of type: Immunization, Effector Cells, Antigen Presenting Cell, Antigen, Assay Information, and Epitope-MHC-TCR Complex. The Effector Cell class describes the cells that are elicited upon immunization and that acquire measurable functions as a result. The B Cell Response class describes antibody responses that are related to the epitope ( Figure 6B ). This class has properties that are of type: Immunization, Antibody Molecule, Antigen, Assay Information, and Antigen-Antibody Complex. Because B cell responses do not require MHC binding and \nThere are three classes that capture information about the 3D structure of complexes: Epitope-MHC Complex, Epitope-MHC-TCR Complex, and Antigen-Antibody Complex. The Epitope-MHC Complex, Epitope-MHC-TCR Complex, and Antigen-Antibody Complex classes are used as restrictions on properties of the MHC Binding, T Cell Response, and B Cell Response classes respectively ( Figures 5A, 6A, and 6B) . These Complex classes capture the Protein Data Bank (PDB) Identifier, which provides detailed information about 3D structures. The Protein Data Bank [22, 23] contains approximately 1600 3D structures that are of immunological interest. Other information that is not available in PDB, such as the atom pairs that are involved in the interactions between molecules, the specific residues, the contact area of the molecules, and allosteric effect, is also captured here.\nEach class has numerous slots that capture detailed information associated with epitopes. As mentioned above, a complete list of all the classes, their properties, and relationship, can be found at http://www.immuneep itope.org/ontology/index.html. One of the files provided as supplementary material contains two examples of how two literature references [24, 25] containing epitope information are extracted into the IEDB ontology (additional file 1). Along with the class hierarchy, the IEDB's data dictionary (additional file 2) provides more detailed information about the fields that are defined for the IEDB. The data dictionary contains a textual overview description and a listing of fields that are required to be completed for IEDB entries. The data dictionary also allows database users to provide comments and suggestions to IEDB team to enhance the formal ontology.\nThe IEDB will be a comprehensive resource pertaining to epitopes of the immune system. By extensively curating both intrinsic and extrinsic features associated with epitopes, the IEDB is expected to provide substantially greater detail about specific epitopes than any other databases presently available. The IEDB will be populated with data derived from three main sources, namely the peer-reviewed literature, patent applications, and direct submission. Epitope data published in the literature and patent applications are curated manually by the IEDB's curation team. Data from already existing epitope databases, whose authors have agreed to share their data, will also be imported into the IEDB. Apart from these, a main data source will be the direct submission of data from the Large-scale Antibody and T Cell Epitope Discovery programs [3] that are funded by NIAID. Presently, fourteen contracts have been awarded under this program, and all of them will submit their data to the IEDB. Direct antibody and T cell epitope submissions will also be sought from the broader research community, with an emphasis on antibody epitopes to NIAID Category A-C pathogens. Because of the large scale of the IEDB project, a formal ontology is critical to ensure consistency in the representation of data.\nCommunication between database developers, researchers, analysis tool developers, and team members is crucial, and can be performed in harmony only when a common vocabulary is established. An ontology, which is an explicit formal specification of the terms in the domain and relationships among them, is an effective way to share the knowledge contained in that domain. Accordingly, since the IEDB's domain is epitope-related data, we have created an ontology that captures detailed conceptual structure related to these data.\nThe development of this ontology has relevance for the expansion and modification of the epitope knowledge base. Our ontology design defines individual concepts as separate classes and then defined relationships between these classes and other objects in the domain. These classes serve to restrict the values that will describe properties of objects in the database. For example, the species is a separate concept defined in its own class. Depending on the context, this can refer to an immunized species or the species from which antigen presenting cells are derived. Similarly MHC Molecules is defined as a separate class, and it is used as a value restriction by concepts like MHC Binding and Antigen Presenting Cells. Defining concepts as separate classes and using them to restrict the values of properties in other classes facilitates the expansion and modification of our ontology. Adding properties (slots) to concepts is a task easily accomplished when there are well-defined class descriptions that may serve as value restrictions on the properties, and providing that High-level classification of Immunization (A), Antigen (B), and Antigen Presenting Cells (C) class these class descriptions are general enough to apply in all instances. We have ensured in our design that each concept is atomic and that it can be re-used by various classes.\nThe development of a formal ontology is valuable to database users and in particular to scientists contributing data to, and downloading data from, the IEDB. We anticipate that the availability of a formal ontology will ensure that a common language and shared understanding of concepts will inspire this type of communication, thus ensuring maximum efficiency and accuracy. The formal ontology developed will most likely require refinement and fine tuning when users provide suggestions and new technologies for performing experiments are discovered. The IEDB website will provide mechanisms for the users to provide suggestions and participate in the enhance- ment of the ontology. The IEDB Data Dictionary has a separate column for the users to provide comments on specific data fields. The IEDB website will also host web forms that will guide users to conform to the ontology definitions when submitting data. Apart from the web forms, an XML schema definition (XSD) will be available on the website for users to inspect and use in their data submission. Users will also be able to download epitope records from the website.\nIn the process of developing new ontologies, it is good practice to leverage existing community standards. In our initial analysis, we confirmed that there were no explicit ontologies that efficiently captured epitope details as per the scope of the IEDB program. As mentioned above, we have utilized, as much as possible, inferred ontologies from existing epitope databases. Among the ontologies that we analyzed, IMGT-Ontology and Gene Ontology were the only two formal ontologies that were related to the epitope domain. The IMGT-Ontology was designed for the ImMunoGeneTics database. IMGT is an integrated database specializing in antigen receptors (immunoglobulins and T-cell receptors) and the major histocompatibility complex of all vertebrate species. The ontology developed for this database has specific immunological content, describing the classification and specification of terms needed for immunogenetics. The IEDB does conform to IMGT's standards about receptors and MHC molecule chains in the sense that all the chain names follow IMGT's controlled vocabulary.\nGO provides structured controlled vocabularies for genes, gene products, and sequences annotated for many organisms. The IEDB complements GO in terms of epitopes of immunological interest since GO is incomplete in this area. Antigens, which are primary sources of epitopes, are annotated in GO. Thus, in essence, the IEDB could be utilized to provide an extension of GO for antigens that contain epitope-related information.\nPerhaps the most important element in the development of the IEDB ontology is that, to the best of our knowledge, this represents the first immunological ontology specifically designed to capture both intrinsic biochemical and extrinsic context dependent information. In this respect, it is similar in spirit, but different in approach, from other knowledge resources relating to systems biology. We anticipate that the development of this type of ontology and associated databases might lead to completely new methods for describing and modeling immune responses. Accordingly, this new program might represent a novel tool to assist in the design, testing, and development of new ways to combat infectious diseases and other immune related pathologies such as cancer and autoimmune diseases.\nA complete listing of IEDB's class hierarchy and its properties is available at http://www.immuneepitope.org/ ontology/index.html " }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: In real-time RT-PCR, the expression levels of the target genes of interest are estimated on the basis of endogenous controls. Various housekeeping genes, ribosomal RNA (rRNA) and total RNA are most commonly used as references in gene expression analysis today. The purpose of these controls is to remove or reduce differences due to sampling, i.e. differences in RNA quantity and quality.\nThe ideal endogenous control should be expressed at a constant level among different tissues of an organism, at all stages of development and should be unaffected by the experimental treatment. It should also be expressed at roughly the same level as the RNA under study [1] . However, data normalization in real-time RT-PCR remains a real problem, especially for absolute quantification [1] . Numerous studies have revealed that no single universal gene has a constant expression level under all developmental or experimental situations. The best choice of reference gene to use as an endogenous control varies, depending on the tissues of interest in the experiment. A large number of genes have for this reason been selected for normalization of mRNA expression data [2, 3] . If the selected reference gene fluctuates randomly between samples, small differences in expression between the genes of interest will be missed. Gene expression coefficient of variation (CV) between different groups of individuals should ideally be as low as possible [4] . In general, the stability of several potential reference genes should be tested in every examined tissue or cell, and under different experimental design [5, 6] . An increasing number of papers are discussing the selection of reference genes in real-time RT-PCR analyses [3, 7] .\nTwo of the most commonly used reference genes are those encoding glyceraldehyde-3P-dehydrogenase (GAPDH) and β-actin. Recently, the use of these two genes as endogenous controls has been scrutinized, and several studies have documented that the GAPDH and β-actin genes should be used with caution as controls [2, 8, 9] . GAPDH in mammals is known to play a role in a broad range of cellular mechanisms (for review see Sirover [10] ), including being a key enzyme in glycolysis. Overall, GAPDH mRNA levels might be regulated under a large number of physiological states, and its use as a reference is inappropriate for most experimental conditions. Actin is a major component of the protein scaffold that supports the cell and determines its shape, and is the most abundant intracellular protein in eukaryotic cells. Even though commonly used as a reference, the application of the β-actin gene has recently been characterized as a historical carryover from northern blots and conventional RT-PCR (for a general discussion on the use of 'classic' reference genes like GAPDH and β-actin, see Huggett et al. [7] ). Eukaryotic elongation factor 1A (eEF1A, formerly elongation factor 1 alpha) plays an important role in translation by catalyzing GTP-dependent binding of aminoacyl-tRNA to the acceptor site of the ribosome. However, the protein is involved in a broad diversity of functions and constitutes 1-3% of the total cytoplasmic protein content of the cell. In human, cDNAs of two actively transcribed isoforms have been cloned (eEF1A-1 and eEF1A-2) (for review see Thornton et al. [11] ). Two paralog EF1A genes (A and B) have recently been applied as references in real-time qRT-PCR of Atlantic salmon [12] . It is plausible to assume that the presence of these highly similar genes is a result of a tetraploidization event that occurred in a salmonid ancestor in the comparatively recent past [13, 14] .\nPreviously, the 18S rRNA gene was considered to be an ideal internal control in qRT-PCR analysis (Ambion [15] ). Ribosomal RNA constitutes up to 80-90% of total cellular RNA, and several studies have shown that rRNA varies less under conditions that affect the expression of mRNAs (discussed in Bustin & Nolan [16] ). However, questions have been raised against the use of ribosomal RNA genes as references. Vandesompele et al. [5] have stressed the fact that there sometimes might be imbalances in rRNA and mRNA fractions between different samples, making genes encoding ribosomal RNAs unsuitable as references.\nTo meet these challenges of accurate interpretation of realtime qRT-PCR data, the authors suggested that an index of the most stable housekeeping genes should be used for normalization, and developed the geNorm VBA applet for Microsoft Excel in this regard [5] . A similar software tool, the BestKeeper, has been developed by Pfaffl et al. [6] . These tools can be used to find the most stable reference genes under different experimental conditions. We used the geNorm software which determines the individual stability of a gene within a pool of genes [5] . The stability is calculated according to the similarity of their expression profile by a pair-wise comparison, using their geometric mean as a normalizing factor. The gene with the highest M, i.e. the least stable gene, is then suggested excluded in a stepwise fashion until the most stable genes are determined, and an index suggested, based on the best genes. geNorm has been used to select the most stable reference genes in several recent studies (e.g. [4, 17, 18] ).\nThe aim of this work was to evaluate the usefulness of six potential reference genes in the Atlantic salmon. Salmonid fish are among the most widely studied model fish species in general, and extensive basic information on many different aspects of their biology has been collected [19] . Large-scale DNA-sequencing projects on salmon have been initiated in several laboratories http://www.sal mongenome.no/cgi-bin/sgp.cgi; http://web.uvic.ca/cbr/ grasp/; http://www.abdn.ac.uk/sfirc/salmon/; http:// www.bcgsc.ca/gc/salmon. In this work we selected the two 'classic' reference genes encoding GAPDH and β-actin, two genes encoding 18S rRNA and S20 ribosomal protein and two paralog genes encoding elongation factor 1A (EF1A A and EF1A B ). To evaluate their usefulness as reference genes, RNA from eight tissues of six adult salmon were subjected to real time PCR. The relative transcription levels of the genes were also estimated in four phases of young salmon going through smoltification, in order to check their stabilities under physiological stressful conditions.\nThe ranking of the six examined genes analyzed by geNorm is shown in Table 3 . In six tissues (muscle, liver, gills, head kidney, spleen and thymus), the EF1A B gene emerged as the most stable, whereas the EF1A A gene was ranked number one in brain and the β-actin gene was ranked number one in intestine. The 18S rRNA and S20 genes were ranked among the worse genes in all tissues. Not surprisingly, the GAPDH gene was ranked worse in five tissues (liver, head kidney, spleen, brain and thymus), confirming the general skepticism against the use of this gene as reference [7, 16, 20] . Combined, the total ranking reads EF1A B >EF1A A >β-actin>18S rRNA>S20>GAPDH. We did not analyze our data with the Bestkeeper software. Analyzing reference genes in virus infected cells, Radonic et al. [4] concluded that the Bestkeeper tool gave results that slightly deviated from, but nevertheless corresponded to, those obtained using geNorm.\nTo be able to evaluate gene stability under stressful conditions, mRNA expression of the selected genes was examined in gills of salmon going through smoltification. Prior to seawater entry, juvenile anadromous salmon undergo a parr-smolt transformation, characterized by behavioral, morphological and physiological changes, known to be challenging for the fish. Physiological alterations include increased seawater tolerance, olfactory sensitivity, metabolic rate, scope for growth and changed hemoglobin and visual pigments [21] . We selected to examine the gills during smoltification, because this tissue plays a major role in ionic and osmotic regulation during adaptation to hyperosmotic seawater. Figure 1 shows the raw Ct values of the studied genes in gills before, during and after smoltification (smoltified in seawater and desmoltified in freshwater). In Figure 2 the same data are presented, but now normalized against an index calculated by geNorm of the three most stable genes (β-actin, EF1A A and EF1A B ). Based on the M values, geNorm ranks the stability of the six genes from 24 fish going through smoltification in the following order: EF1A B >EF1A A >β-actin>S20>18S rRNA>GAPDH ( Figure 3 ). In Figure 1 it can be seen that the 18S gene had the lowest individual raw Ct variation. Most individual raw Ct variation of the studied genes is seen in the presmolt and smolt groups. A characteristic drop in expression can be seen for all genes in the smolt group, compared to the presmolt group. After transfer to seawater, the individual raw Ct variation decreased for all genes. Overall, the raw Ct data suggest that the physiological challenging smoltification process affected the expression of all six genes. When the same data were normalized against an index of the three most stable genes, β-actin, EF1A A and EF1A B , the relative expression levels were altered for all genes. Now the ribosomal 18S gene emerges as the second worse, whereas the two paralog EF1A genes became the most stable. This might have to do with the fact that geNorm will top-rank co-expressed genes [22] , a weakness that has to be considered when evaluating paralog genes likely to be co-regulated. Even though the eEF1A-2 gene has been identified as an important oncogene and has been shown to be differently expressed in human tissues [11] , Hamalainen et al. [23] found the eEF1A-1 gene to be a good reference gene in real-time RT-PCR examinations. A similar finding was reported by Frost and Nilsen [24] in salmon louse, where they showed that the eEF1A and S20 genes were valid candidate references, whereas the 18S rRNA and GAPDH genes were unsuitable. The current findings based on geNorm evaluation question the recommended application of ribosomal genes as references (as suggested for example by Ambion (see reference [15] ), and are in line with earlier warnings against the use of rRNA genes as references [5, 6] . To avoid the normalization of the genes for β-actin, EF1A A and EF1A B against an index partly based on their own expression, the S20 gene was included in the index instead, and the mean normalized expression for these three genes calculated with the new index. The patterns of expression, however, were approximately the same for the three genes as seen in Figure 2 , suggesting that the gene-stability measure M can be used to find the most appropriate reference genes.\nWe see a correlation between the A260/230 absorbance on the NanoDrop and the PCR efficiency (data not shown). We tend to get PCR efficiencies that are too high in some samples with low A260/230 ratios. When the samples are treated with DNase solution, the A260/230 ratio usually drops. After DNase treatment, the A260/280 ratio increased from 1.8 to 2.1 (n = 45 samples). At the Table 3 : Evaluation of the usefulness of six potential reference genes in eight tissues of Atlantic salmon ranked by the geNorm software. 1 = best, 6 = worst. Six individuals were analyzed for six genes in eight tissues. qRT-PCR analysis of six genes in gills of six Atlantic salmon going through smoltification; presmolt (before smoltification), smolt (during smoltification), smoltified (finished smoltified in seawater) and desmolt (desmoltification in freshwater) Figure 1 qRT-PCR analysis of six genes in gills of six Atlantic salmon going through smoltification; presmolt (before smoltification), smolt (during smoltification), smoltified (finished smoltified in seawater) and desmolt (desmoltification in freshwater). Numbers indicate raw Ct values.\nqRT-PCR analysis of six genes in gills of six smoltifying Atlantic salmon Figure 2 qRT-PCR analysis of six genes in gills of six smoltifying Atlantic salmon. The same data as in Figure 1 , but now normalized against an index of the three best genes (β-actin, EF1A A and EF1A B ) calculated with the geNorm software. The four groups were analyzed with Kruskal-Wallis test, and if significant, the overall p-value is given in the graphs. For β-actin, there were significant differences between the presmolt and the smoltified group (p < 0.05), the presmolt and the desmolt groups (n<0.01) and between the smolt and desmolt groups (p < 0.05). For EF1A A , there was a significant difference between the presmolt and the desmolt groups (p < 0.01). For EF1A B , there was a significant difference between the smolt and the smoltified groups (p < 0.05). An asterisk denotes significant differences between the groups.\nsame time, the A260/230 ratio dropped from 2.4 to 2.1. The DNase treatment therefore adds substances to the RNA solution that increases the absorbance at 230 nm more than it decrease the 260 nm absorbance. The added substance (salt or some other component) may inhibit the RT reaction or the PCR reaction, sometimes called PCR poisoning. We have seen that the A260/230 ratios are quite low in samples that give inadequate PCR efficiency slopes, especially with RNA from head kidney, thymus and intestine tissues, in which the gradient of the standard curve is less than -3.3 ( Table 2 ). The reason one obtain better amplification rate efficiencies with the more diluted samples is because the inhibitor has been diluted below its effective level. The obvious way around this problem is to dilute the amount of cDNA put into the PCR reaction. Alternatively, cleanup columns can be used to purify and concentrate the RNA.\nTranscription levels of the examined genes and the coefficient of variance (CV) in different tissues varied considerably. mRNA levels in tissues are regulated by numerous endogenous and exogenous stimuli [16] . Transcription rates in metabolic active tissues might be up-regulated compared to those of less active tissues, whereas inter-tissue variation in degradation rates of mRNAs, for example, might affect mRNA stability [25] . The results revealed that muscle had the lowest CVs of the studied genes, compared to higher CVs in more active tissues like thymus, head kidney and spleen. In thymus, intestine, head kidney, gills, brain, liver and spleen, the 18S and S20 genes had the lowest CVs, based on raw Ct values. In all tissues, except intestine, the GAPDH gene had the highest CV. Except for thymus, the two elongation factor genes had relatively similar expression in all eight examined tissues. Their expression are most likely co-expressed in the examined tissues, and therefore favored in geNorm calculations [22] . The results also demonstrated that assays optimized for one tissue of an organism do not necessarily work equally well in other tissues. Of the tissues studied in this work, intestine, head kidney and spleen were the most troublesome.\nOur data, based on geNorm calculations, suggest that the Atlantic salmon EF1A genes that have been tested in the present study may be good candidate reference genes. The GAPDH gene seems unsuitable as a reference in quantitative real-time RT-PCR. With regard to the 18S rRNA gene, this must be applied with caution. Tools like the geNorm applet for Microsoft Excel can be useful to help select the most stable genes in various experiments.\nTissues from 15 individuals were collected (852 ± 702 g, ranging from 254 to 1898 g). These individuals were not separated based on sex, size or sampling time, but treated as one heterogeneous group to examine the width of mRNA expression of the studied genes in eight different organs. This group of fish was handled and fed according to normal aquacultural management, and none of these individuals were exposed to any particular treatment. To examine if physiological stress may alter the gene expression in the gills, a total of 24 individuals were collected before (termed presmolt, 18.3 ± 0.9 g), during (termed smolt, 28.7 ± 3.7 g) and after smoltification. After smoltification, one group was kept and desmoltified in freshwater (termed desmolt FW, 30.0 ± 3.8 g), while the other group was transferred to seawater (termed smoltified SW, 30.2 ± 4.3 g) (n = 6 in each group). The Atlantic salmon examined during smoltification were from the anadromous population \"Vosso\" of the river Vosso in Southwestern Norway (see Nilsen et al. [27] for details on how these fish were treated). All fish were treated and euthanized according to Norwegian national legislation for laboratory animals.\nSamples from eight organs, i.e. gills, liver, brain, head kidney, spleen, thymus, white muscle and posterior intestine, were dissected out and immediately frozen in cryo tubes in liquid N and stored at -80°C before RNA extraction. The RNA extracted from three spleen and four head kidney tissue samples were of low quality, and we had to redo the sampling from four individuals, These tissue samples Stability of six genes in gills of Atlantic salmon during smoltifi-cation calculated with the geNorm software were stored on RNA later (Ambion) at -20°C before further processing.\nRNA was isolated with phenol-chloroform extraction as described by Chomczynski and Sacchi [28] , and stored in 100 µl RNase-free MilliQ H 2 O. Total RNA was extracted using Trizol reagent (Invitrogen, Life Technologies), according to the manufacturer's instructions. Genomic DNA was eliminated from the samples by DNase treatment according to the manufacturer's description (Ambion). The RNA was then stored at -80°C before further processing. The quality of the RNA was assessed with the NanoDrop ® ND-1000 UV-Vis Spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) and the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). A 260/280 nm absorbance ratio of 1.8 -2.0 indicates a pure RNA sample. The RNA 6000 Nano LabChip ® kit (Agilent Technologies, Palo Alto, CA, USA) was used to evaluate the integrity of the RNA. We used the RNeasy MinElute Cleanup kit from Qiagen to purify our most troublesome samples. With this kit the A260/230 ratio increased on average by 5 % (n = 10).\nThe PCR primer and TaqMan MGB probe sequences used for quantification of the genes encoding 18S rRNA, S20 ribosomal protein, β-actin, GAPDH, EF1A A and EF1A B , are shown in Table 1 . Four of these genes, 18S, β-actin, EF1A A and EF1A B , have also been used as references in real-time RT-PCR analyses of Atlantic salmon in other recent studies [12, 28] . The primers amplify PCR products between 57-98 basepairs (bp) long, which is within the range of 50-150 bp as suggested by Applied Biosystems for their Taq-Man assays. qPCR assays were designed using Primer Express 2.0 software (Applied Biosystems, Foster City, CA, USA) to select appropriate primer and probe sequences from known Atlantic salmon genes. The mRNA sequences encoding S20 ribosomal protein and GAPDH were obtained from GenBank accession numbers BG936672 and BU693999, respectively (exon-exon borders were not considered). The EF1A A assay was based on a cDNA clone that we reported to the GenBank previously (AF321836), whereas the EF1A B assay was based on the EST BG933853.\nAn alignment with zebrafish indicated the exon-exon borders [29] . The chosen primers were subsequently used to confirm that the salmon genes contained an intron between the same sites as deduced from the alignment with zebrafish. The PCR products containing the introns were cloned into TOPO vector (Invitrogen) and sequenced (sequences can be provided upon request). PCR primers for β-actin were based on Atlantic salmon BG933897 and designed to span exon-exon borders of this gene, as deduced from corresponding genes in human and zebrafish (NW633959). For 18S rRNA the PCR primers and probe were designed from the Atlantic salmon sequence AJ427629, and placed in a conserved region of the gene based on comparison with the human gene. RNA samples were subjected to DNase treatment to avoid genomic DNA contamination. Amplified PCR products of all actual cDNAs were sequenced to ensure that the correct mRNA sequences were quantified. The fragments were sequenced with BigDye version 3.1 fluorescent chemistry (Applied Biosystems) and run on an ABI PRISM ® 377 DNA apparatus at the University of Bergen Sequencing Facility.\nA two-step real-time RT-PCR protocol was developed to measure the mRNA levels of the studied genes in eight tissues of Atlantic salmon. For evaluation of the potential reference genes, raw Ct values are presented. The geNorm VBA applet for Microsoft Excel was used to determine the most stable genes from the set of tested genes [5] . The Ct values were transformed to quantities using standard curves, according to the geNorm manual. The gene expression stability (M) was calculated with the geNorm applet, and the genes were ranked from best to worst, based on the M value.\nThe GraphPad Prism 4.0 software (GraphPad Software, Inc.) was used for the statistical analyses in this work. Linear regression was used to determine PCR efficiency based on dilution curves. Non-parametric Kruskal-Wallis test was used to compare differences among four groups of salmon going through smoltification.\nPAO was responsible for the experiment, data analysis and drafted the manuscript. KKL conducted the real-time RT-PCR analysis, and contributed throughout the experimental process. AEOJ constructed the qPCR assays for two of the genes. TON provided the cDNA from the smoltification experiment. IH participated as a supervisor in the study design, analyses and writing. " }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: Respiratory viruses play an important role in exacerbations of COPD and this has been increasingly recognised since the application of molecular detection methods [1, 2] . The most prevalent viruses detected by polymerase chain reaction so far were respiratory syncytial virus (RSV), Influenza A, Rhinovirus and Parainfluenza 3. Human metapneumovirus (hMPV) is a recently discovered respiratory virus first isolated from a dutch child with lower respiratory tract infection (LRTI) [3] . World wide distribution is probable since it has been isolated in North HMPV has been recognized as a member of the Paramyxoviridae like RSV and it is not only associated with bronchiolitis in most cases, but also with pneumonia, croup and exacerbations of asthma [14, 15] , diseases which share some features with COPD. Up to date reports about hMPV in adults are scarce. In a general Canadian population 14.8% of patients of all age groups with acute respiratory tract infections were hMPV positive. Thirty-three percent of hMPV-infected patients were hospitalized and the hospitalization rates were significantly higher among patients below 5 years and those over 50 years of age [16] . In another prospective cohort of adults, hMPV was detected in 4.5% of all illnesses but also in 4.1% of asymptomatic subjects. HMPV was most prevalent in young adults with children and in frail elderly [17] . HMPV infection can be severe since the virus was isolated from the lungs from a previously healthy man who died from acute pneumonia [18] . The role of hMPV in acute exacerbations of COPD (AE-COPD) has been studied recently in outpatients and only low frequencies have been observed [17, 19] . Up to now the prevalence of hMPV in patients hospitalized with AE-COPD is unknown. Our aim was therefore to investigate the frequency of detection of hMPV in a prospective cohort of hospitalized patients with AE-COPD and to compare these results to patients with stable COPD and to smokers without COPD.\nThree different groups were studied. The first group consisted of hospitalized patients with an acute exacerbation of COPD (AE-COPD), the second group were subjects with stable COPD and the third group was composed of smokers without COPD. The groups were defined as previously published [20] . Briefly AE-COPD patients suffered from COPD as defined by GOLD [21] . Acute exacerbation was characterized by worsening in dyspnea, cough, and expectoration. A routine posterior-anterior chest radiograph was evaluated on admission by expert radiologists to exclude other other reasons for increased symptoms as pneumonia, tuberculosis, pulmonary fibrosis, bronchiectasis, bronchial carcinoma or congestive heart failure.Stable COPD patients did not have an exacerbation within the last 30 days prior to hospital admission and had no changes in therapy within the last 14 days (including inhaled and oral medication) and had been admitted for other medical reasons into departments of internal medicine other than pulmonary care. COPD subjects were recruited in a 2:1 ratio each month in order to prevent seasonal selection bias. Smokers have been smoking more than 10 pack-years, could have chronic symptoms like cough and phlegm but did not report dyspnea and did not have bronchial obstruction (FEV 1 /FVC>70%, FEV 1 >80% predicted). None of the smokers had a history of COPD or asthma, nor was using systemic or topic pulmonary medication. The smokers were recruited either from our smoking cessation initiative or by newspaper advertisement.\nThe study was approved by the ethical committee of the Ruhr-University of Bochum, Germany. Written informed consent was obtained from all patients and control subjects before inclusion in the study.\nClinical evaluation, spirometric tests, nasal lavage, induced sputum, specimen processing and viral ribonucleic acid (RNA) extraction were carried out as described by Rohde et al [2] . Elution volume was 100 µl. cDNA was generated with random-hexamer primers as previously published[2].\nA hMPV-specific real-time RT-PCR designed and evaluated by Maertzdorf et al was used [22] . Primers and probe are localized within the nucleoprotein gene (NL-N) and the presence of a degenerate base within the probe allows detection of all four genetic lineages of hMPV.\nThe assays were performed using the TaqMan ® PCR Core Kit. The final volume was 25 µl containing 500 nM of the forward primer (NL-N-forward (5'-CATATAAGCAT-GCTATATTAAAAGAGTCTC-3')), 250 nM of the reverse primer (NL-N-reverse (5'-CCTATTTCTGCAGCATATTTG-TAATCAG-3')) and 500 nM of the probe (NL-N-probe (5'-FAM-TGYAATGATGAGGGTGTCACTGCGGTTG-TAMRA-3', in which Y is either a C or a T residue). Nuclease-free water was used as negative control and a plasmid containing the N gene of hMPV (kindly provided by James Simon, VIRONOVATIVE, EUR Holding, Erasmus University Rotterdam) was used as a positive control in all PCR runs. Cycling parameters were as follows: 5 min at 95°C, 45 cycles of 30 s at 95°C and 1 min at 60°C. Amplification and detection of RNA from virus isolates or clinical specimens were performed using the GeneAmp ® 5700 Sequence Detection System (Applied Biosystems). The real-time PCR product was cloned with the QIAGEN ® PCR cloning kit (QIAGEN, Hilden, Germany) and this standard plasmid DNA was used for absolute quantification of hMPV viral load. Calculations were performed as previously described for absolute quantification of RSV viral load [23] .\nThe primary objective of this study was to compare the frequency of hMPV detection in respiratory specimens between COPD patients with or without an acute exacerbation and smokers without COPD.\nContinuous data were checked for normal distribution using the Kolmogorov-Smirnov test. The data were of non-parametrical distribution and results were expressed as median and range. Differences between groups were assessed by Kruskal-Wallis test. To further analyse significant differences between two individual groups a pair wise comparison by two-sided Mann-Whitney U-test was performed. All significance levels were set to 5%. Data were analysed and processed using SPSS Version 12.0 on a Windows XP operating system.\nA total of 229 subjects were investigated between October 1999 and June 2004: 130 patients with AE-COPD, 65 patients with stable COPD and 34 smokers without COPD. The clinical characteristics and lung function measurements are summarized in table 1. FEV 1 , FEV 1 in % of predicted value and FEV 1 /FVC were normal in smokers, significantly decreased in stable COPD patients (all) and further significantly decreased in AE-COPD patients (all p < 0.05 compared to stable COPD and all p < 0.001 compared to smokers).\nHMPV could be detected in three subjects. All these subjects were AE-COPD patients. The prevalence of hMPV in AE-COPD patients was 2.3%. The virus was simultaneously detected in nasal lavage and induced sputum in one patient only. The viral load was about 100 times higher in nasal lavage than in induced sputum in this patient. Overall the viral load in nasal lavage was about 3.5 times higher compared to induced sputum (for details see table 2). The hMPV positive patients did not differ significantly from other AE-COPD patients when clinical parameters and lung function were analysed. hMPV was detected in the winter season only.\nThe main finding of this controlled study investigating the incidence of hMPV in subjects with COPD and smokers without COPD is that this recently discovered respiratory virus was detectable only during exacerbation of COPD. The frequency of detection was very low but in positive cases the viral load was considerable. There was no detection in patients with stable COPD or smokers without COPD.\nRecently Vicente et al [19] reported about the incidence of hMPV in 89 COPD patients. Five patients (5.5%) were hMPV positive. Two of these patients had to be transferred to hospital. Although this was not a controlled study and not all details of the study are available due to the fact that the data were published in form of a letter, these results support our findings. The incidence of hMPV in this and in our study is low compared to other respiratory viruses. In a similar previous study we found that Picornaviruses were detectable in 36% of AE-COPD patients, Influenza A in 25% and Respiratory syncytial virus in 22%[2]. There is another prospective cohort study of adults in which hMPV was detected in 4.5% of all illnesses. HMPV was most prevalent in young adults with children and in frail elderly from long term care facilities [17] . Unfortunately this report does not specify how many of the elderly patients suffered from COPD. In our asymptomatic smokers without COPD hMPV could not be detected. A recent study investigating nasal secretions from adults with and without respiratory illnesses found hMPV in 5 of 146 ill patient and in none of 158 control subjects, strongly supporting our data [24] . A further recent study found hMPV in two out of 111 adult patients (1.8%) who presented to the emergency department for AE-COPD during 2 winter/ spring seasons in Quebec, Canada, also in support of our findings [25] . In a US American study investigating clinical samples collected between 1991 and 1995, hMPV could not be detected at all in 196 patients indicating important geographical and seasonal differences in hMPV prevalence [26] . Taken together the results presented here are in keeping with other studies in adults and add important information on the prevalence of hMPV in hospitalized AE-COPD.\nTo our knowledge this is the first study analysing the viral load of hMPV in COPD patients. We found a mean of 6.5 × 10 5 viral copies/ml in nasal lavage and 1.88 × 10 5 viral copies/ml in induced sputum. These values indicate that hMPV may have been the infectious agent triggering exacerbation in these patients. Viral load cut-off values for infectivity in COPD exacerbations have not been studied in detail yet and need further investigation. However, viral loads between 1120 copies/ml in Cytomegalovirus infection in lung-transplant patients [27] and 5.8log 10 copies/ ml in SARS [28] have been considered to indicate infectious disease. Moreover hMPV was only found in acute exacerbation and not in stable disease or in smokers without COPD supporting a triggering role in AE-COPD.\nHMPV infection can be severe since it was isolated from the lungs from a previously healthy man who died from acute pneumonia [18] . Our hMPV positive patients did not differ in their clinical characteristics or lung function from the other AE-COPD patients which does not indicate a more severe course of AE-COPD in these patients.\nTaken together this is the first controlled study on the relevance of hMPV in hospitalized AE-COPD. HMPV was detected in a very low frequency but with noticeable viral load in AE-COPD patients. Given that asymptomatic carriage of hMPV is very unlikely it should be considered as another possible trigger of AE-COPD. Since every AE-COPD has considerable impact on the course of the disease and regional outbreaks of hMPV are possible it should be included into future diagnostic and therapeutic considerations. \nThe author(s) declare that they have no competing interests." }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: T he term \"globalization\" was popularized by Marshall McLuhan in War and Peace in the Global Village . In the book, McLuhan described how the global media shaped current events surrounding the Vietnam War [1] and also predicted how modern information and communication technologies would accelerate world progress through trade and knowledge development. Globalization now refers to a broad range of issues regarding the movement of goods and services through trade liberalization, and the movement of people through migration.\nMuch has also been written on the global effects of environmental degradation, population growth, and economic disparities. In addition, the pace of scientifi c development has accelerated, with both negative and positive implications for global health. Concerns for national health transcend borders, with a need for shared human security and an enhanced role for international cooperation and development [2] . These issues have signifi cant bioethical implications, and thus a renewed academic focus on the ethical dimensions of public health is needed.\nFuture developments in science and health policy also require a fi rm grounding in bioethical principles. These core principles include benefi cence; nonmalefi cence (to do no harm); respect for persons and human dignity (autonomy); and attention to equity and social justice. According to the World Health Organization [3] , global ethical approaches should (1) monitor and update ethical norms for research, as necessary; (2) anticipate ethical implications of advances in science and technology for health; (3) apply internationally accepted codes of ethics; (4) ensure that agreed standards guide future work on the human genome; and (5) ensure that quality in health systems and services is assessed and promoted.\nThe Bioethical Implications of Globalization (BIG) Project is a 42-month dialogue funded by the European Commission that involves a series of expert panel discussions on four specifi c globalization and health subject areas: (1) mobility of people;\n(2) technological globalization; (3) liberalization of trade; and (4) new global health threats (bioterrorism). In addition, BIG includes a multipleround Delphi Process (Box 1) to solicit input on these issues from a broad, interdisciplinary audience.\nThe project's purpose is both to raise short-term, practical considerations about globalization and health and\nDelphi is a group communication technique designed to obtain opinions from a panel of selected experts on specifi c issues through the sending of questionnaires to be completed within a specifi ed time. The experts are contacted individually and they do not know other group participants and their opinions-the aim is to submit the group participants to the same conditions. Participants do not meet personally, thereby avoiding undue infl uence. The process foresees the following points: The process is repeated a number of times, until a convergence of all group members is obtained. The process ends with analysis of the answers and formulation of the fi nal report.\n(Adapted from http://www.bigproject. org/dephi.htm) Thomas E. Novotny*, Emilio Mordini, Ruth Chadwick, J. Martin Pedersen, Fabrizio Fabbri, Reidar Lie, Natapong Thanachaiboot,\nto provide a longer-term, strategic perspective on the four selected public health-related issues. The fi nal conclusions will be presented to a high-level meeting of European Union (EU) policy makers in 2006; these conclusions may then inform future research directions and stimulate additional critical thinking about globalization and its bioethical implications for health policy. This article presents preliminary results from the BIG Project.\nMobility results from the increasing ease of domestic and international travel as well as from instantaneous access to information through the Internet and other electronic resources. Mobility may involve the pursuit of a better quality of life, development of markets for traded goods and services, return of resources to home countries, and improvement of professional and business networks.\nHowever, migration may also affect psychological and physical health as a result of confl ict, famine, poverty, and the insuffi cient cultural or economic integration of migrants within their new home society. It may contribute to the spread of infectious diseases across borders ( Figure 1 ). The recent epidemic of SARS was a classic example of an infectious disease propagated through the movement of people across borders; it required attention from the original site to control migration (quarantine) as well as vigilance by secondary sites to protect their populations ( Figure 2 ) [4] . For these and other reasons, the International Organization for Migration is increasingly concerned with migratory patterns and their health consequences in a globalizing world (for an illustration of the emerging confl ict of ideas, see http:⁄⁄www.iom. int and http:⁄⁄www.noborder.org/ iom/index.php).\nCross-border health commerce is related to mobility. In Europe, this commerce is likely to increase as the EU enlarges to include Eastern and Central European nations. Such commerce may include the movement of health providers from East to West as well as \"medical tourism\" in pursuit of less costly or more accessible high-quality health care. In addition, international trade in illegal health products and inconsistent regulatory and safety standards for exports may threaten public health, especially in unregulated pharmaceutical markets.\nEthical concerns may also result from the vast growth in international tourist travel. Such travel now accounts for a twelfth of world trade, supporting an economy the size of a middle-income country [5] . Tourism may provide substantial economic benefi ts to many developing countries, and it may improve cultural understanding among travelers. However, these benefi ts require an ethical concern for the environment and for persons employed in the tourist industry.\nThe rights of nations to protect against infectious disease and unsafe medical practices, as well as the rights of human beings displaced by war, traffi cking, and economic and cultural disruption, are critical concerns for health policy makers. Poverty and social disparities are key factors in the growth of global migration. Therefore, it is timely to consider whether mobility is a human right, and whether those who migrate have rights to health care in their new country. These questions should be considered by health policy makers within the ethical contexts of individual autonomy, social justice, nonmalefi cence, and benefi cence.\nTechnology drives globalization and in turn is driven by globalization. However, there is considerable ambiguity as to the value of technological globalization, especially for health in low-income countries. The \"digital divide\" may be important in improving health or income disparities as the electronic revolution provides scientists and health workers in both the developed and developing world with unprecedented access to information. Much could be done to reduce information inequities for the developing world through collective international action, but new global governance mechanisms may be needed to achieve this goal for information technology [6] .\nInterestingly, the Internet is a structural necessity for fi nancial and corporate globalization, but the same technology is used by nongovernmental organizations, political groups, and cultural movements to support grassroots social justice and human rights campaigns against these globalizing corporations. Neither side in this struggle would advocate limitations to the expansion of Internet technology, but both sides need to consider the bioethical implications of increased information access.\nOn the other hand, the ethical issues surrounding genomics (with both environmental and human concerns) are quite ambiguous. While there may be signifi cant benefi ts to identifying genetically benefi cial products or genetic determinants of disease, there are also concerns about altering natural environments and about collecting routine genetic information from general populations [7] .\nFor example, some experts assert that genetically modifi ed (GM) crops will signifi cantly increase crop yields without requiring any additional farmland, thus preserving valuable rain forests and animal habitats. herbicides, and pesticides. Farmers are not allowed to trade or save GM seed from one harvest to the next, and \"terminator technology\" (producing grains that are genetically modifi ed so that they cannot be used to generate new crops) is under development. (See http:⁄⁄www.globalissues.org/ EnvIssues/GEFood/Terminator.asp for more information on this technology.) Thus, ethical considerations of distributive justice and benefi cence must be considered in the debate about the global applicability of GM crops.\nFor the pharmaceutical and health care industries, genetic testing could provide information about the shape of future markets and the possible tailoring of specifi c pharmacotherapy to genomic susceptibility. For governments, genetic testing may provide predictive information on a population basis that could aid future health care planning. Genetic information might also be similarly used by the insurance industry, but the identifi cation of genetically \"high-risk\" individuals would likely interfere with their autonomy, in that they may not be able to purchase health insurance. For example, the Apolipoprotein E test may indicate that an individual has two copies of one form (allele e4) of the gene that leads to Alzheimer disease [9] . Could this information be used by insurance companies or possible employers to deny insurability, despite no current adverse health effects?\nIn the post-genomic era there is potential to both reduce and increase health inequities, but much will depend on how ethical issues are addressed. If interventions to increase the life span for those with access to high quality health care must compete with expensive investments in genetic research on infectious diseases (which affect the poor most of all), health inequalities may be amplifi ed between those with access and those without access to health care. If research participants or patients in low-income countries have unequal access to information, they may not be properly informed about genetic testing and the counseling needed if adverse genetic information is found. Population-based genomic research may characterize groups of people in such a way that encourages discrimination. Such research may also lead to disputes about ownership of genetic resources from participant populations. Health professionals must have a solid grounding in bioethical issues as they make clinical decisions based on genetic information. However, health policy makers and global governance structures must also be accountable for the potential adverse consequences such decisions might engender.\nOne may ask: will genomic science really help developing nations? To what extent can benefi ts be shared? Will pharmaceutical and biotechnology companies invest in poor countries if they can make more money working on therapeutics for high-income countries? Thus, concern for the bioethical issues of social justice and benefi cence arises. Genomics has the potential to be a global public good, but there is considerable uncertainty as to its bioethical justifi cation in all cultures [10] .\nIn general, globalization helps liberalize trade through removal of import restrictions and tariffs, through removal of restrictions on trade in services, and through linkage of trade sanctions to the protection of intellectual property rights. All these activities may have an impact on population health.\nDefenders of trade liberalization claim that this process is one of the most effective means of increasing a country's wealth and, by extension, population health. Even if this were always true, there may be specifi c policies that have particularly detrimental effects on health (such as opening markets to trade in manufactured tobacco products). Further, there may be an ethical argument based on social justice against some trade liberalization policies. If, for example, trade liberalization between rich and poor countries produces proportionally more wealth in rich countries compared with poor countries, this may suggest a socially unjust result of liberalization; poor countries' economies may not grow as fast as rich countries' economies in this situation.\nThe relationship between wealth and health is actually somewhat controversial: the so-called Preston curves demonstrate a dramatic relationship between health and economic prosperity up to about a Purchasing Power Parity of US$3,000 per capita per year [11] . However, there are cheap, cost-effective approaches to population health (such as vaccination, clean water, and sewage disposal) that may not be affected by the increase in Purchasing Power Parity. These approaches were relatively more important than economic development per se in early 20thcentury interventions in developed countries, and they are likely to be more important for infl uencing health among developing country populations today than simple economic growth. On the other hand, high-intensity technological improvement rather than economic growth may be more important to health in rich countries compared with developing countries.\nThe concern for intellectual property rights in trade has been an extraordinarily contentious issue in recent years. Newer drugs that are effective against diseases in resourcepoor but highly impacted countries, such as antiretroviral drugs against HIV, have been prohibitively expensive in these countries, in part because of patent protections. With the Trade-Related Intellectual Property agreement, patent protection became linked to trade policy; if countries in need of cheaper essential drugs did not conform to patent rules, trade retaliation from exporting countries might ensue. However, restrictions on poor countries' responses to legitimate public health emergencies may be unethical on the basis of distributive justice, nonmalefi cence, and benefi cence. Exceptions for public health emergencies (such as HIV/AIDS) under the Trade-Related Intellectual Property agreement include the right to compulsory licensing (local companies produce patented medicines in exchange for a royalty payment to the patent holder) or parallel importing (importing patented drugs sold more cheaply elsewhere) that will make essential medicines more available to highly impacted countries without fear of trade retaliation from the originating country [12] .\nThe General Agreement on Trade in Services is a relatively new treaty that covers trade in health services [13] . The agreement has been severely criticized by some, who claim that it increases privatization of health care services and undermines public health care systems. However, given its ambiguities, the actual impact of the agreement on the health sector will be largely determined by the way in which the agreement is further specifi ed in multinational commitments [14] . Social justice, equity, benefi cence, and nonmalefi cence will all come into play in the implementation of this treaty.\nConcerns for security against biological weapons have recently arisen among both poor and wealthy nations. Some, however, question the enormous sums now being spent to address the perceived threats due to bioterrorism even without strong evidence for actual threats. Even without such evidence, global bioethical principles at least suggest the need for a framework for consideration of distributive justice in this arena.\nFor example, should a nation with a limited supply of a vaccine against weaponized smallpox offer its stockpiles to a neighboring country that is under direct attack? This case is complicated by the fact that the infection could spread to its own territory. In the case of widespread biological attacks, which global governing agency, country, or other entity would be responsible for global resource allocation? Clearly, risks from bioweapons are trans-border, but resources may be unevenly and inequitably distributed, requiring a bioethically based policy determination on a global basis [15] .\nA further concern with respect to biomedical research is the issue of dual-use technology development for health benefi ts as well as for possible bioweapons. Governments must balance the secrecy necessary for security with the need for disclosure of information that is essential for research and development in health. It is very diffi cult to sequester new knowledge that might be applied to building biological weapons without simultaneously impeding research on defense against those bioweapons and on other benefi cial biomedical advances. Most BIG Project scientists agree that the benefi ts of releasing scientifi c information in general outweigh the risk of its misuse. However, the scientifi c community needs to consider whether new codes of conduct are necessary or whether existing governance is suffi cient to support a bioethical approach to research on possible dualuse technologies.\nGlobal bioethical challenges require careful theoretical deliberation and practical considerations for international health policies [16] . The BIG Project seeks to guide these processes in four selected areas of interest to the EU, so that the project results may be helpful to policy makers at local, national, and international levels.\nThe BIG Project has found that bioethical principles are important in considerations of migration, trade, information technology, genomics, and bioweapons threats. Globalization in these arenas is neither a right nor a wrong process, but it demands careful consideration of bioethical principles including social justice, benefi cence, nonmalefi cence, and individual autonomy. These concerns may not be immediately obvious to health policy makers, and thus the BIG Project results may help clarify the larger goals and purposes of bioethically based health policy development within the EU and elsewhere. More information about the BIG Project can be found at http:⁄⁄www. bigproject.org/project.htm." }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: In Japan, cancer has been recognized as a major component of the overall pattern of disease for decades. Thus, the importance of cancer prevention by lifestyle modification should now be strongly acknowledged.\nInternationally, several studies have estimated the proportion of total cancer deaths attributable to various risk factors based on epidemiologic evidence [1, 2] , and various international guidelines and recommendations derived from these have appeared [3] [4] [5] [6] . Not surprisingly, domestic guidelines and recommendations for cancer prevention in Japan such as the 'Twelve recommendations for cancer prevention [7]' and 'Healthy People Japan 21 [8] ' have been significantly influenced by these reports.\nPublic awareness of risk factors in relation to cancer prevention has been surveyed in only a few countries [9, 10] , and results have demonstrated poor awareness. Other studies focusing on specific cancers only have also appeared [11] [12] [13] [14] . However, none of these studies quantitatively evaluated public awareness of the attributable fraction of individual risk factors.\nIn Japan, it appears that most people are aware of the major risk factors of cancer. Although we are unaware of any published evidence, however, public knowledge and information on cancer prevention now seems influenced largely by the mass media and other sources, rather than by information provided directly by health professionals, resulting in a distorted picture of causation. Cancer control policy therefore urgently requires a clarification of the discrepancies which now exist between ideal levels of public concern about risk factors and the current reality, particularly public health policy makers in their formulation of cancer control measures. To address this need, the present study was designed to provide information on awareness of the attributable fraction of cancer causes among the Japanese general population. Since we are interested in quantitatively estimating the awareness of preventability, we placed special emphasis on gauging awareness by attributable fraction of cancer.\nThe study was conducted as a part of an omnibus survey in December, 2003, by commission to a polling agency. The omnibus survey is a monthly multipurpose cross-sectional survey which includes public opinion research, social research, scientific research, market research, and others. Using a stratified two-stage sampling method, a total of 2,000 people aged 20 or older were randomly selected as study subjects, from 160 districts selected from area units representing 12 geographical blocks (Hokkaido, Tohoku, Kanto, Keihin, Koshinetu, Hokuriku, Tokai, Kinki, Hanshin, Chugoku, Shikoku, Kyushu) and 3 types of city scale (14 metropolises, other cities, towns and villages) in proportion to the population distribution as at March 2002. After an initial visit to obtain oral informed consent and schedule a visit for the interview, the survey was conducted by face-to-face interview using trained interviewers in each district. The omnibus survey does not collect any personally identifiable information such as name, date of birth or address details at interview. For the present report, we obtained the electronic data file for the relevant interview component, with no personal identifiers. Ethical approval was not applicable to the present study under the Japanese ethical guidelines for epidemiologic studies, which comply with the declaration of Helsinki.\nAmong the 2,000 people selected for survey (977 men, 1,023 women), interviews were successfully obtained The questionnaire of this survey comprised questions on the awareness of various environmental and genetic risk factors in relation to cancer prevention by enquiring about the attributable fraction of cancer. Fractions were: 1) 12 risk factor candidates, namely alcoholic beverages, unbalanced diet, use of food additives and pesticide chemicals, charred fish and meat, tobacco smoking, obesity, physical inactivity, endocrine-disrupting chemicals, air pollution such as diesel emissions, occupational exposure, cancer-causing viral and bacterial infection, and stress; 2) genetic factors in general; and 3) the preventable fraction of cancer occurrence by lifestyle modification [see Additional file 1].\nThe first question asked about the preventable fraction of cancer which would result in Japan if each factor were completely and totally eliminated, using the fine categories of <5%, 5 to <10%, 10 to <15%, 15 to <20%, 20 to <25%, 25 to <30%, 30 to <40%, 40 to <50%, 50 to <60%, 60 to <70%, 70 to <80%, 80 to <90%, and 90 to 100%. These categories were exhibited together on a pie chart.\nThese risk factor candidates were selected with reference to previous international and domestic recommendations and guidelines [1] [2] [3] [4] [5] [6] [7] [8] . The second question asked about the fraction of cancer genetically predetermined using the same categories as the first, while the third asked about the preventable fraction of cancer by modification of lifestyle using estimation of an actual percent value. In addition to these questions, subjects were also asked about their smoking and drinking practices, and occupational and educational status.\nMean values of the attributable fractions were calculated for each risk factor of cancer and compared by demographic and habitual smoking and drinking status. For analyses, the mid-values of each category were assigned for categorical variables. All analyses were performed using Stata statistical software, S/E Version 8 [15] .\nA total of 1,355 (67.8%) subjects responded to the survey, with a higher response rate in women (72.9%) than in men (62.3%). Response rate was lower in the 20s age strata than in the other age groups, but no trend to an increase in response rate with increasing age was observed. Overall, no significant difference in area and age distribution was seen between the sampled population and survey respondents. Response rate tended to be lower among subjects who reside in the Kanto region and in cities other than the 14 metropolises than among other subjects ( Table 1) .\nCharacteristics of the 1,355 respondents (609 men, 746 women) are presented in Table 2 . The proportion of current smokers was 44% in men and 15% in women, and decreased with age in both genders. In female subjects aged in their 20s, 26% currently smoke and 49% drink alcohol beverages at least 4 times a week.\nAwareness of the attributable fraction of cancer causes among the Japanese general population is presented in Table 3 . Among the 12 risk factor candidates, the attributable fraction was considered highest for cancer-causing viral and bacterial infection (51.3%), followed by tobacco smoking (43.0%), stress (39.0%), and endocrine-disrupting chemicals (37.1%). In contrast, the attributable fraction of charred fish and meat (21.4%) and alcohol drinking (21.7%) were considered low compared with other risk factor candidates. The attributable fraction of other risk factor candidates such as occupational exposure, air pollution, food additives and pesticides, unbalanced diet, obesity and physical activity ranked between the high and low fractions. The attributable fraction responses tended to be higher in women than in men, and were increased among inhabitants of larger cities and in homemakers and decreased in those engaged in agriculture, forestry and fisheries. In contrast, risk factor candidate rankings were similar by gender, age group, city scale, and educational and occupational status. In men, those who neither smoke nor drink tended to consider the preventive fraction of the risk factors higher than those who both smoke and drink, whereas in women, the former subjects considered the values lower than the latter.\nThe speculated fraction of cancer which is genetically determined was 31.5% as an average (Table 3 ). This fraction was higher in current heavy smokers and former drinkers, and lower in homemakers and students. On the other hand, an average 35.5% of cancer were considered preventable by lifestyle improvement, with this ratio being higher in homemakers, former smokers, and never and former drinkers.\nThe present survey, targeted at the Japanese general population, showed that the attributable fraction of cancer among Japanese tended to be higher for cancer-causing infection, occupational exposure, air pollution and food additives than major lifestyle factors such as dietary factors. In addition, the attributable fraction of cancer estimated by the Japanese general population was higher than that derived from epidemiologic evidence in the West, which is frequently quoted as 30% for tobacco smoking and 30% for food as a whole [1, 2] .\nSome of the major cancers in Japan, including gastric and liver cancers, are known to be related to cancer-causing viral and bacterial infection, and a higher level of concern about such infection among Japanese than in Western populations would therefore be understandable [9] . Notwithstanding the validity of such concern, however, the high level of concern for infection, as well as for endocrine-disrupting chemicals, identified in the present survey was most likely due to the severe acute respiratory Likewise, a high level of concern for tobacco smoking was also observed, in spite of a relatively dull reduction in the rate of male current smokers in past decades compared with the U.S. This was probably due to recent enactment of the Health Promotion Law, which curbs passive smoking in public spaces.\nRespondent estimates for attributable fractions were generally high. This may be in part due to anchoring and adjustment effects of the response categories used and the tendency of people to respond near the middle of the scale. Given that responses tended to be generally high, concern over the present results should probably be focused on rankings rather than absolute values per se. Although tobacco smoking ranked among the top factors, risk factor candidates whose actual contribution is considered to be low, such as endocrine-disrupting chemicals, occupational exposure, air pollution such as diesel emissions and the use of food additives and pesticide chemicals ranked higher than previous estimates of the attributable fraction of cancer causes [1, 2] . In contrast, this should be compared with the results for unbalanced diet, which ranked at only 8th among the 12 risk factor candidates despite an actual ranking which is estimated to be as high as that for tobacco smoking. Particularly in light of findings on long-term exposure to common lifestyle factors such as diet as a cause of cancer, these results suggest that public awareness of cancer prevention is still insufficient.\nWe are unaware of any previous studies aimed at determining public awareness of the attributable fraction of cancer as a whole or at gauging the level the awareness of cancer prevention by attributable fraction. Accordingly, to our knowledge, this is the first attempt to discover the level of awareness for each risk factor candidate, and the questionnaire used has hence not been fully validated. In addition, as indicated above, responses to this type of cross sectional survey are subject to social conditions such as information from the mass media and other sources on disease epidemics and other putative risk factors. Thus, the results might not necessarily reflect actual public awareness. However, the study subjects were recruited from among a nationally representative random sample, and the response rate was similar to that of recent omnibus surveys in other countries [16] [17] [18] [19] . Nevertheless, the exclusion of non-respondents may have distorted the results.\nIn conclusion, awareness of the attributable fraction of cancer causes among the Japanese general population tended to be dominated by infection, occupational exposure, air pollution and food additives rather than dietary factors. The results of the present survey provide valuable clues and perspectives toward the formulation of relevant cancer prevention strategies in Japan." }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: Loop-mediated isothermal amplification (LAMP) is a unique gene amplification method in which DNA can be isothermally amplified using only one enzyme [1] [2] [3] .\nSince the advent of the LAMP method, many researchers have been engaged in basic research from a variety of perspectives. As a result, it is currently being put to practical use in the reagents for detecting various pathogens such as SARS [4] and the West Nile virus [5] and reagents for identifying the sex of fertilized eggs in cow in vitro fertilization [6] . Furthermore, LAMP is a gene amplification method with a variety of characteristics and applications in a wide range of fields, including SNP typing [7] and quantification of template DNA [8] . In particular, LAMP is considered to be effective as a gene amplification method for use in gene point-of-care testing (g-POCT) devices, which are used for simple genetic testing whenever and wherever necessary. First, since LAMP can amplify genes isothermally, the amplification reaction can be carried out with a simple heater. There is no need for the special device used Pattern diagram of LAMP reaction and hybridization of fluorescently labeled oligo DNA probes Figure 1 Pattern diagram of LAMP reaction and hybridization of fluorescently labeled oligo DNA probes. The LAMP reaction takes place in three steps (starting material production step, cycling amplification step, and elongation and recycling step) by the primers depicted in the enclosure. In the starting material production step, the starting material (6) is generated by primers (forward inner primer (FIP) and backward inner primer (BIP)). A complementary strand (11) of the starting material (6) is synthesized from the starting material (6) by a reaction that uses itself as a template and by a reaction from an FIP annealed to the loop segment, thus making up the cycle amplification step. During this step, probes (probe F and probe B, respectively) designed for the region between the F1 and F2 region or the B1 and B2 region can hybridize to the loop segment. As the cycle reaction progresses, an elongation and recycling step takes place, during which elongated products (8, 13, etc.) with an inverted repeat structure are generated. Numbers 14 and 15, which have a cauliflower structure, are also generated. They have many loop structures to which probes can hybridize.\nfor polymerase chain reaction (PCR) to rapidly control the temperature [3] . Next, a large amount of DNA (10-30 µg/25 µl) can be synthesized in a short time (15-60 min) while maintaining high specificity. This characteristic greatly facilitates detection of the LAMP reaction [9] . Moreover, since the LAMP reaction progresses by generating a characteristic stem-loop structure, LAMP products have a single-stranded segment in the molecule (loop segment; see Figure 1 and reference No. 1). By using oligo DNA probes designed to recognize the sequence of the single-stranded segment, it is possible to carry out hybridization assay without performing heat denaturation after amplification. This means that all processes, from the amplification reaction to the detection reaction, can be carried out completely isothermally.\nIf these characteristics of the LAMP method are used effectively, we believe it will be possible to develop simple genetic testing devices that have not been realized yet despite a strong awareness of their necessity, in a wide range of fields, including infectious disease testing, food inspection, and environmental testing. The key to developing such simple devices will be figuring out how to simply and clearly present the final amplification results. The objective of this research is to establish new techniques for sequence-specific visual detection of amplification results by means of the LAMP method. To that end, we made use of a reaction that has been known for a long time, i.e., cationic polymers like polyamines form an insoluble complex with DNA [10] . It is well known that one of such the polyamine, polyethylenimine (PEI), strongly interacts with DNA. PEI is widely used as a nucleic acid precipitant for nucleic acid purification [11] and as an in vivo and in vitro non-viral vector [12, 13] . We discovered that an insoluble PEI-LAMP product complex was generated under certain optimized conditions when PEI was added to LAMP reaction solution. Using this precipitation titration, we investigated whether it was possible to perform bound/ free separation of fluorescently labeled probes. In this paper, we report the novel visual detection methods of the presence of sequences of HBV and HCV cloned to plasmid as a model experiment using this method and the results of an investigation of the basic reaction conditions required to achieve this.\nThe primers used for LAMP reaction is schematically depicted in enclosure of Figure 1 . Forward Inner Primer (FIP) consists of F2 and the complementary sequence of F1, and Backward Inner Primer (BIP) contains of B2 and the complementary sequence of B1 when each sequences (F1, F2, B1, and B2) are defined on the template sequence as shown in Figure 1 . In some references such as Notomi et al. [1] or Parida et al. [5] , a spacer of few thymidines was inserted between F1c or B1c and F2 or B2 in the inner primer (FIP or BIP) so that one and two thymidine spacers were inserted in FIP and BIP of HBV, respectively. However, the spacer was not used in this study because the LAMP reaction can progress with the use of inner primers without the spacer as shown by Hong et al. [4] , Hirayama et al. [6] , and Iwasaki et al. [7] . The LAMP reaction takes place isothermally in the three steps shown in Figure 1 , i.e., starting material production step, cycling amplification step, and elongation and recycling step, by using of polymerase with strand displacement activity. First, the starting structure (structure 6) is generated from the template nucleic acid in the starting material production step. Next, the starting structure becomes structure 7 with a stem-loop structure by self-primed DNA synthesis. When the forward inner primer is hybridized to the loop segment and strand displacement synthesis takes place, structure 11, which is a complementary strand of structure 6, is generated. This means that an auto cycle reaction was established between structure 6 and structure 11. In addition, products bound by an inverted repeat with two amplified regions, like structures 8 and 13, are generated in association with the auto cycle reaction (cycling amplification step). Then, with these structures as starting points, products elongated to a length of several kbp and products with complex structures with cauliflower-like structures (14, 15) are ultimately generated.\nSince the LAMP products have a loop structure, oligo DNA probes (green arcs in the figure) in the reaction solution can sequentially hybridize to products as the LAMP reaction proceeds. The cauliflower structures, in particular, contain two or more loops to which the probes can hybridize. This characteristic of the LAMP reaction plays an important role in the detection method described here.\nPlasmid DNA cloned with HBV or HCV sequence was added to a LAMP reaction solution containing both HBV primers and the probe (FITC-labeled) and HCV primers and the probe (ROX-labeled) and amplified, after which low molecular weight PEI (Mw = 600; 0.2 µmol as a monomer) was added. As shown in Figure 2A , precipitate emitting green fluorescence characteristic of FITC was obtained in LAMP reaction solution containing the HBV template, precipitate emitting red fluorescence characteristic of ROX was obtained in LAMP reaction solution containing the HCV template, and precipitate emitting a color (orange) that was a combination of FITC green and ROX red fluorescence was obtained in LAMP reaction solution containing both templates. They could be observed using an ordinary UV illuminator or UV-LED (light emitted diode). When LAMP products (lambda DNA) not related to HBV and HCV were present, precipitate with no fluores-Sequence-specific visual detection method that utilizes precipitation titration of LAMP products by adding PEI Figure 2 Sequence-specific visual detection method that utilizes precipitation titration of LAMP products by adding PEI. (A) Results of sequence-specific visual detection after adding PEI to LAMP reaction solution. After LAMP reaction in the presence of both FITC-labeled HBV probes and ROX-labeled HCV probes followed by addition of the prescribed amount (0.2 µmol as monomer) of PEI (Mw = 600), it was centrifuged for several seconds using a desk-top, low-speed centrifuge. The tube was then visually observed as is on a UV illuminator (365 nm). It was possible to differentiate the LAMP reaction by visualizing the presence of precipitate fluorescence and the color of the fluorescence. 1, LAMP reaction negative. 2, When LAMP reaction with PSA amplification (unrelated LAMP reaction) occurred. 3, When it contained HBV template nucleic acid. 4, When it contained HCV template nucleic acid. 5, When it contained both HBV and HCV template nucleic acids. (B) Diagram of principle of sequence-specific visual detection method that utilizes precipitation titration of LAMP products by adding PEI. First, a LAMP reaction is carried out using a LAMP primer set for two types of template nucleic acid and fluorescently labeled probes, which can hybridize to loop segments of each LAMP products. When a LAMP reaction corresponding to a certain fluorescently labeled probe progresses, the probe will sequentially hybridize to the loop segment generated during the reaction. On the other hand, an unrelated probe remains free in the solution. When an optimized amount of PEI is added after reaction for a set length of time, the positive charge of PEI neutralizes the negative charge of the DNA to form an insoluble LAMP product-PEI complex. At this stage, fluorescently labeled probes hybridized to LAMP products are taken up by the LAMP product-PEI complex together with the LAMP products. Since most of the PEI added is used for formation of the LAMP product-PEI complex, free oligo DNA probes cannot form a complex with PEI. When the generated insoluble complex is pelletized by centrifugation and the pellet is irradiated with excitation light, the labeled fluorescent dye hybridized to LAMP products produces fluorescence. Figure 3 Precipitation titration of LAMP products by addition of PEI. (A) Effect of amount of PEI on sequence-specific incorporation of ROX-labeled lambda DNA recognition probes by DNA-PEI complex (Mw of PEI is 600). When 0.2 µmol to 1.0 µmol of PEI was added as a monomer, almost 100% of labeled probes hybridized to the LAMP products for lambda DNA was taken up by the DNA-PEI complex. On the other hand, when 0.4 µmol to 0.8 µmol of PEI was added as a monomer to a reaction solution in which an amplification reaction did not take place, a small amount (<20%) of labeled probes precipitated. Precipitation of this nonspecific probe did not take place when unrelated LAMP products (PSA) were present. (B) Effect of the Mw of PEI on sequence-specific incorporation of ROX-labeled lambda DNA recognition probes by DNA-PEI complex. When PEI with Mw 600 was used, the fluorescence intensity of supernatant decreased only when LAMP product for lambda DNA was present. As the Mw of the PEI used increased from 1,800 to 10,000, the fluorescence intensity of the supernatant decreased in the absence of a LAMP reaction since the formation of insoluble PEI-oligo DNA probe complex occurred. (C) Effect of amount of KCI on sequence-specific incorporation of ROX-labeled lambda DNA recognition probes by LAMP product-PEI (Mw = 600) complex. Normal LAMP reaction solution (control) contains 10 mM of KCI. Since formation of the PEI-LAMP product complex was inhibited by an increase in the amount of KCI added, the fluorescence intensity of supernatant increased regardless of the sequence of the LAMP product. (D) Effect of amount of LAMP product for lambda DNA on sequence-specific incorporation of ROX-labeled lambda DNA recognition probe by LAMP product-PEI complex (Mw of PEI = 600). Even when the amount of LAMP product was 1 µg per 25 µL, almost 100% of labeled probes (1 pmol) was taken up by PEI-DNA complex.\ncence was obtained, and visible precipitate was not generated in a sample not containing a template (LAMP reaction negative). This means that it was possible to assess whether the HBV template nucleic acid was in the reaction solution, HCV template nucleic acid was in the reaction solution, or both were in the reaction solution by visualizing the fluorescent color of the precipitate. The LAMP method is a nucleic acid amplification method that is so sensitive that it is possible to create an amplification reaction from only six copies of template nucleic acid [1]. Therefore, a combination of LAMP amplification and this detection method makes possible sequence-specific visual presentation of the presence of trace amounts of nucleic acid, i.e., only several copies, found in a sample.\nA model that represents the principle of this method is shown in Figure 2B . Oligo DNA probes labeled with fluorescent dye are hybridized to a specific LAMP product. When an optimized amount of low-molecular-weight PEI (Mw = 600) is then added, the positive charge of PEI neutralizes the negative charge of the DNA, which results in formation of an insoluble DNA-PEI complex. When this solution is left to stand for a few minutes or it is centrifuged with a small, desk-top centrifuge for a few seconds, the complex is deposited at the bottom of the tube. When the precipitate is observed on an illuminator (365 nm), the fluorescence of the dye from the probe taken up by the precipitate is visualized. On the other hand, probes unrelated to the sequence of the amplified LAMP product are not taken up by the LAMP-PEI complex because they are not hybridized to the LAMP product. When the molecular weight of the PEI used is small, oligo DNA probes and PEI cannot interact sufficiently to form an insoluble complex. Therefore, unrelated probes remain in the supernatant. Since the fluorescent probes in the supernatant are dispersed, the fluorescence cannot be visualized. Consequentially, bound/free separation of labeled oligo DNA is achieved as a result of insolubilization by PEI of LAMP products.\nThe following experiments were conducted to confirm this principle. First, we investigated the effect of the added amount of PEI on this precipitation titration ( Figure 3A ). Oligo DNA probes for lambda DNA were captured in the precipitate when 0.2 to 1 µmol of PEI was added to 25 µL of LAMP reaction solution for lambda DNA. If the amount of PEI added is too high or too low relative to the optimal range, the PEI-DNA complex precipitate is not formed, resulting in oligo DNA probes remaining in the solution. This phenomenon is characteristic in ionic interaction between cationic polymers and anionic polymers [14] . Namely, when the amount of the cationic polymer PEI is too low, the PEI-DNA complex becomes anionic. In contrast, when the amount of PEI is too high, the PEI-DNA complex becomes cationic. In both cases, the PEI-DNA complex is solubilized as a result. This characteristic, which is shown in Figure 3A , indicates that this precipitation titration is based on neutralization of the negative electric charge of the DNA by the cationic polymer PEI. In the case of LAMP negative, a small amount of free probe was deposited under certain conditions when 0.4 µmol to 0.8 µmol of PEI was added as a monomer (< 20%). However, the amount of precipitate in this case was so small that it was impossible to confirm it visually. On the other hand, when LAMP products (PSA) unrelated to the probe sequence were present, no free probe at all was deposited. This is because almost all of the PEI molecules added were consumed in the precipitation of unrelated LAMP products, an excess of which was present relative to the amount of probe. In other words, more reliable detection is achieved by first confirming whether the LAMP reaction has occurred by checking whether white DNA-PEI complex precipitate is generated as a result of addition of PEI and then determining for which nucleic acid template the LAMP reaction occurred based on the fluorescent color of the precipitate. Because of the above results, the amount of PEI added was fixed at 0.2 µmol for the following experiments in order to avoid generation of free probe precipitate as much as possible.\nWe investigated the effect of the Mw of PEI on this detection system ( Figure 3B ). When PEI with different Mw was added to the LAMP solution so that the amount per monomer of each was the same, we found that sufficient BF separation occurred if LAMP amplicons were present, even if the Mw of PEI increased up to 10,000. That is, the fluorescence intensity of the supernatant of LAMP solution with specific amplification (lambda DNA) is lower than that with unrelated amplification (PSA). As the Mw of PEI increased, however, almost all probes in LAMP reaction negative solution formed an insoluble PEI-oligo DNA complex. This result means that the LAMP reaction negative solution cannot be distinguished from the LAMP reaction positive solution, which successfully amplifies the targeting sequence if PEI with a high molecular weight is used. As was also observed in Figure 3A , when LAMP amplicons are present, almost all of the added PEI reacts with LAMP amplicons, so there is little opportunity for interaction with free oligo DNA probes, but when LAMP amplicons are not present, all of the added PEI interacts with oligo DNA probes. Under conditions where a large amount of PEI with a high molecular weight can strongly interact with oligo DNA, an undesirable insoluble PEIoligo DNA probe complex forms because the molecular weight of PEI is high. Therefore, the average molecular weight of PEI in this detection system should be about 600. We conducted a similar experiment using spermine, which is a polyamine with a lower molecular weight. In that experiment, an adequate amount of insoluble complex was not generated compared with PEI of an average molecular weight of 600 under conditions used for the present research (data not shown). It is well known that spermine can also make DNA insoluble, as indicated in many other reports [10] . Therefore, optimal conditions in the case of spermine as a precipitant might exist, but since further investigation would have gone beyond the scope of this paper, no further investigation was carried out.\nInsolubilization of LAMP products by PEI was inhibited by addition of an excessive amount of KCl to the LAMP reaction solution after amplification ( Figure 3C ). This was because excessive amounts of potassium ions and chloride ions inhibited the electrostatic interaction between DNA and PEI. This finding is further indication that this detection method is based on neutralization of the negative electric charge of DNA by the positive electric charge of PEI. The effect of ionic strength on the LAMP reaction has been investigated and found that the presence of 200 mM or more of KCl markedly delayed the LAMP reaction (data not shown). Therefore, it can be said that this detection method will function without trouble if the solution used has a composition that is optimized for the LAMP reaction.\nWe investigated the sensitivity of this detection method ( Figure 3D ). Almost all probes were taken up by the precipitate in the case of up to 1 µg of LAMP product. Moreover, we were able to visualize the fluorescence in the precipitate even if the specific LAMP product was 0.2 µg. We found that this detection system was sensitive enough as a detection system for visual assessment to be used in simple g-POCT devices. We can see from the results shown in Figure 3C that all of the 1 pmol of probe added hybridized to 1 µg (= 3 nmol nucleotide) of LAMP product. In other words, one molecule of probe bound to every 1,500 base pairs of LAMP products. The LAMP product is a mixture of products of several different sizes, with an average molecular size of 2 kbp [1]. This finding that one molecule of probe binds to every 1,500 base pairs reflects well the fundamental characteristic of this LAMP reaction.\nThe new detection method described above utilizes the unique nature of low-molecular-weight PEI, i.e., it cannot form an insoluble complex with a single-stranded anionic polymer with a low molecular weight such as an oligo DNA probe, but it can form an insoluble complex with DNA with a high molecular weight such as LAMP product. Until now, not much attention appears to have been paid to the complex consisting of oligo DNA and PEI with a molecular weight of 1,000 or lower, which was used in this study. This is probably because the interaction between low-molecular-weight DNA and oligo DNA is very weak and because its practical utility as a vector or nucleic acid precipitant is low. However, Kunath et al. reported that the complex formed by PEI (5 kDa) and plasmid DNA was more unstable than that formed by PEI (25 kDa) [15] . Osland and Kleppe reported that spemidine, which has a structure similar to that of PEI, formed an aggregate with double-stranded oligo DNA, but it did not form an aggregate with single-stranded DNA [10] . Drawing upon these results, the low-molecular-weight PEI used in this study is thought to interact with DNA with high selectivity to differences in DNA concentration and structure (number of strands and molecular weight). The fact that we took advantage of this nature of lowmolecular-weight PEI as a nucleic acid precipitant for detection led to the establishment of this detection method.\nThis detection method effectively utilizes the characteristics of the LAMP method, i.e., a large amount of amplification product can be synthesized in a short time while maintaining high specificity. Since a large amount of amplification product is created by the LAMP reaction, precipitate of a size that can be easily confirmed with the eyes is generated when PEI is added to the LAMP reaction solution. Moreover, the fact that the amplification is highly efficient means that the amount of labeled probe for detection that can be added is large. As a result of these characteristics, the LAMP reaction followed by addition of PEI yields precipitate with a clear color and in a size that can be identified visually, as shown in the photograph in Figure 2 . Furthermore, the fact that the amount of the amplification product is large means that the range of the optimum amount of PEI necessary to generate the insoluble complex is wide. Thus, a precise system for adding the PEI solution is not necessary. This will contribute to simplification of g-POCT devices that use this detection method. In the case of PCR, the most widely used gene amplification method, the amount of amplification product is usually 1/20 or less that of LAMP [9] . Consequently, in order to apply this method to PCR, a device for rapidly cycling the temperature to perform PCR, a high-luminance fluorogenic reagent, a system for washing unreacted probes, and a system for accurately dispensing PEI would be necessary. This would likely be an obstacle to putting to practical use a simple, inexpensive g-POCT device for genetic testing.\nThere are several ways to make LAMP products insoluble besides addition of a cationic polymer like PEI. We tried carrying out the BF separation using chilled ethanol. Since the addition of chilled ethanol lowered the temperature of the LAMP solution, there was a strong tendency for nonspecific probes to weakly hybridize to LAMP products and precipitate (data not shown). Moreover, isopropanol and PEG are believed to be inferior to a PEI solution because it is necessary to add several times the amount of LAMP reaction solution and they are not as easy to handle. Therefore, we believe that low-molecular-weight PEI, which is efficiently insolubilizes amplicons with only a small amount, is the best option for high BF separation, as was shown in this study.\nIf the 5' end of the inner primer is fluorescently labelled, the LAMP product should be visible as in the current study. However, visualization using inner primers fluorescently labelled at the 5' end is not preferred, because the possibility of false positives from self-extension of the labeled primer cannot be excluded. There is no risk of false positives with the oligo DNA probes fluorescently labeled at the 3' end as in this study, so that highly accurate genetic testing can be established.\nIt is necessary to add PEI to the LAMP reaction solution after the LAMP reaction takes place since PEI strongly inhibits the LAMP reaction. However, opening the reaction tube after amplification is generally avoided to prevent carry-over contamination. Therefore, development of a technique for adding PEI in a closed system is needed to put this method to practical use. Some possible solutions could be to apply PEI to the lid of the reaction tube beforehand and turn the reaction tube upside down after LAMP amplification or use wax that responds to heat. Furthermore, we should be able to use technology like micro-Total Analysis system or Lab-on-a-Chip, for which much research is being conducted in recent years, to solve this problem.\nWe established an extremely simple method for visually detecting LAMP products in a sequence-specific manner by simply adding a small amount of low-molecularweight PEI to the LAMP reaction solution. The biggest feature of this technique is the ability to visually present sequence information of amplicons without using an expensive source of light or a detector. In contrast, conventional genetic tests require expensive reagents, complex and skillful manipulation, and large devices equipped with an expensive optical system. These are the main reasons why genetic testing is kept within the walls of specific institutions or university laboratories with special equipment and trained engineers. The combination of the LAMP method and the new detection method described here can overcome several factors that have been preventing true practical application of super-simple g-POCT devices for genetic testing. If a simple, inexpensive g-POCT device that is small and light enough to be held in one hand and whose main components are disposable can be developed, the day when parents will be able to genetically test for a pathogen while sitting next to the bed of their child who has a fever will not be far off [16] .\nViral DNA of the hepatitis B virus (GenBank accession number: V00867) digested with BamHI was cloned to pBR322. Reverse transcriptase PCR was conducted for HCV viral RNA (GenBank accession number: AB031663) from patient serum purified using a QIAmp viral RNA Mini kit (Qiagen K.K.) with the PCR primers designed to the sequence of 5' non-coding region and core. The PCR product was cloned to pBR322 according to the established method. Concentrations of the respective plasmids obtained were determined by 260-nm spectrophotometer (Ultrospec 20000, Pharmacia Biotech). The template DNA solutions for LAMP reactions were prepared by serial dilution of the plasmid solutions and purchased lambda DNA solution (Takara Bio, Inc.) with Tris-HCl buffer (10 mM, pH8.0) until it contained 1,000 copies/5 µl.\nThe LAMP reaction was carried out on a scale of 25 µl based on reference 1. Briefly, a forward inner primer (FIP) and a backward inner primer (BIP) at a final concentration of 1. The LAMP primers (Sigma Genosis Japan K.K., HPLC purification grade) were as follows. The hyphens were added between F1c or B1c and F2 or B2. The LAMP reaction for Lambda DNA was used as a LAMP reaction for a basic investigation to clarify the fundamental characteristics of the reaction that occurs between the LAMP product and PEI. A well-established LAMP product [1] for mRNA of prostate-specific antigen (PSA) was used as a LAMP reaction product unrelated to the three LAMP reaction products above. A solution was prepared by serially diluting a LAMP reaction solution with a known concentration (28 µg DNA/25 µl) using LAMP reaction buffer in order to systematically investigate the reaction between the LAMP product and PEI. The concentration of DNA synthesized by the LAMP reaction was determined according to the attached protocol using a PicoGreen dsDNA Quantitation Kit (Molecular Probes, Inc.). The spectrofluorophotometer used was the RF-5000 spectrofluorophotometer (Shimadzu Scientific Instruments, Inc.).\nThe loop segments within LAMP products, i.e., sequences complementary to sequences between either F1 and F2 or B1 and B2, were used as probes for detection, as shown in Fig. 1 . The probes were designed such that the sequences between F1c or B1c and F2c or B2c had a melting temperature (Tm) of 1 to 5°C lower than LAMP reaction temperature (62°C). All fluorescently labeled oligo DNA probes, which were HPLC purification grade, were purchased from Sigma Genosis Japan KK. The sequences of the probe used were as follows. The numbers in parentheses indicate the length and melting temperature (Tm) from supplier's information. \nCommercially available PEI (Wako Pure Chemical Industries, Ltd.) was used without further purification. The average molecular weight of PEI used was 600, 1,800, and 10,000. In this study, the concentration of the PEI aqueous solution is expressed as the concentration of the monomer unit (-C 2 H 5 N-, 46 g/monomer). The PEI stock solution (2.0 mol/l) was prepared by dissolving 4.6 g of PEI in 50 mL of deionized distilled water in a graduated cylinder. At the time of use, the stock solution was diluted with water to the desired concentration.\nWhite precipitate of magnesium pyrophosphate forms in the LAMP reaction solution as a by-product of the amplification reaction [5] . Therefore, after amplification, we first precipitated the magnesium pyrophosphate to the bottom of the reaction tube by centrifugating the LAMP reaction solution for 10 seconds using a small desk-top centrifuge (6,000 rpm). Then, 4 µl of PEI solution adjusted to the desired concentration was added to the supernatant to form an insoluble DNA-cationic polymeric polymer complex at room temperature. A pellet was formed by immediately centrifuging it for 10 seconds using the same centrifuge. A fluorescent image of the pellet was photographed with a fluorescence microscope (VB-G05, Keyence Corporation) equipped with a Handy UV Lamp (wavelength: 365 nm; Vilber Lourmat) and a UV blocking filter (NEO Dynamic L-400, MARUMI Optical Co., Ltd.). Since the magnesium pyrophosphate precipitate was nonfluorescent, it had no effect at all on visualization of pellet fluorescence. To investigate the effect of ionic strength on precipitation titration, aqueous KCl was added to the reaction solution after the LAMP reaction so that it was the prescribed concentration.\nAfter centrifugation, 20 µl of supernatant of the PEI-DNA complex solution was aliquoted to a 384-well assay plate (Corning, Inc.) for fluorometry using a fluorescence plate leader (Polarion; Tecan Japan Co., Ltd.). The percentage of probes captured in the precipitate of the PEI-LAMP product complex was calculated according to the following formula based on the obtained results. " }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: Illicit drug abuse has become an increasing public health and social concern in the past decades worldwide. Drug abuse causes many problems both to individuals and to societies, including loss of productivity, transmission of infectious diseases, crime, family and social disorder, and excessive health care expenditures [1] . Human immuno-deficiency virus (HIV)/AIDS, associated with injection drug use (IDU) and needle sharing to a large extent, has become one of most stunning tragedies in human history. It has caused more than 20 million deaths, and about 40 million people are living with HIV worldwide thus far, with Africa as the most afflicted continent [2] . As the most populous country in the world, China has also observed rapidly increasing drug abuse and HIV/AIDS occurrence in the past 10 to 20 years. China can still shape the course of its epidemic, but it needs to move swiftly and with great resolve [2] . This paper reviews global illicit drug trafficking, drug abuse and its association with HIV/AIDS epidemic in China, and Chinese policies on illicit drug abuse and prevention of HIV/AIDS, and offers additional strategies to governmental crack down on drug smuggling and drug use prohibition to help cope with China's HIV and drug abuse problem. We searched English and Chinese language literature via Medline and the China National Knowledge Infrastructure and reviewed unpublished official data, including national reports on illicit drug control and HIV/AIDS sentinel surveillance data. More than 100 papers and reports were reviewed. Key databases included: \nDrug abuse in China can be traced to the late Qing Dynasty (1644-1911 A. D.), when British colonists forcefully brought Indian opium into China for exchange of silk, tea, and cash. Opium was then locally planned. By the founding of new China in 1949, more than 20 million Chinese people were opium addicts, representing 5% of the total population [3] . After a short nationwide antidrug campaign, drug abuse was reported to be eliminated from the mainland in the early 1950s, and for the next three decades China was believed to be a drug-free nation [3] .\nIllicit drugs reemerged in China in the 1980s as China adopted an open-door policy, and the reemergence was mainly connected with global drug trafficking activities. In Asia, there are two major opiate-producing regions: the \"Golden Triangle,\" comprising three Southeast Asian countries of Myanmar (Burma), Laos and Thailand, and the \"Golden Crescent\" that includes the three Southwest Asian nations of Afghanistan, Iran and Pakistan [4, 5] . The majority of heroin and opium in the current Chinese market is brought from Myanmar into Yunnan Province or from Viet Nam into Guangxi Province, and then it is transshipped along inland trafficking routes to Sichuan, Guizhou, Gansu and Xinjiang or to Guangdong, Shanghai and Beijing [6] [7] [8] . A small potion of heroin/opium is trafficked into Xinjiang from the \"Golden Crescent\" [8] . These drugs further penetrate into other provinces. Since the late 1990s, increasing amounts of amphetamine-typestimulants (ATS) and other chemically related synthetic drugs including amphetamine, methamphetamine and ecstasy have been locally manufactured and consumed in China [4] .\nThe number of drug users documented officially by Chinese public security departments increased from 70,000 in 1990 to 1.14 million by 2004 [9] while the estimated number is 3.5 million [1] . The lifetime prevalence rates of illicit drug use among residents age 15 years or older in high-prevalence Chinese cities increased from 1.1% in 1993 to 1.6% in 1996, and the 1-year prevalence rate increased from 0.9% to 1.2% during this period [10, 11] .\nThe main drug of choice in China is heroin. According to a report of the National Narcotic Control Commission (NNCC), 87.6% of drug users abused heroin in 2002 [12] . The abuse of ATS and MDMA (methylenedioxymethamphetamine or ecstasy) has become popular in city night clubs in recent years [1] . In the 1980s, farmers living in rural bordering areas in Yunnan and Guangxi provinces constituted a large fraction of drug users. Since the early 1990s, more and more urban residents use illicit drugs. The majority of drug abusers are young people with a low education level and limited job skills, although a small proportion of urban users regard using drugs as an indicator of \"high social class.\" NNCC data showed that 74% were aged 17-35 years in 2002 [12] . Experimentation (90%), peer pressure (44%), and relaxation (42%) are commonly cited reasons for beginning to use drugs. Initially, drugs were taken primarily through sniffing/snorting (55%) or smoking cigarettes mixed with drugs (38%) [13] . Injection of drugs became increasingly common among drug users, probably as a result of increasing prices of illicit drugs and greater cost effectiveness of injecting to achieve the desired effect. National behavioral surveillance data showed that the median prevalence of IDU among drug users increased from 35% in April 1995 to 49% in April 2004, and median prevalence of needle sharing among IDUs also increased from 26% to 43% during this period [14, 15] . \"Buying from drug or grocery stores\" and \"buying from hospitals\" were the two most common routes for obtaining injection needles and syringes [16] . Reasons for sharing a needle included: \"do not care about anything else when hooked,\" \"thought it had been cleaned up,\" \"difficult to buy or obtain,\" \"only sharing with selected persons,\" \"no money to buy,\" and \"following other drug users\" [16] . Besides IDU and needle sharing, other risk behaviors including unprotected commercial sex put drug users and ultimately non drug users at high risk of HIV [17, 18] . It is believed that many female drug users sell sex for drugs, and limited published research also provides supporting evidence [18] [19] [20] . However, the impact of the interaction between IDU and commercial sex on HIV risk is not clear. Considering the dramatic increase in the commercial sex industry in China, and the potential bridging role of those with dual risk behaviors in transmitting HIV from high risk groups to the general population, research on the interaction of IDU and commercial sex should be given high priority.\nSince the first AIDS case was detected in 1985 [21] , China has cumulatively reported about 100,000 HIV infections by 2004 [22] . Thus far, the HIV/AIDS epidemic is mainly concentrated in specific geographical areas and sub-group populations [23] . IDU has been the largest contributor to the reported epidemic since 1989, when the first HIV outbreak among IDUs was observed in Ruili City of Yunnan Province bordering with Myanmar [24] . All 31 provinces, autonomous regions and municipalities on mainland China reported HIV infections among IDUs by 2002, and 42% of infections in China were estimated to be due to IDU by 2004 [22] .\nIn 1995, only one out of 8 national sentinel sites for IDU detected HIV infections. The detected prevalence was 0.02% [14] . By 1997, 3 out of 22 sites detected positives, and the average prevalence increased to 6.6%. After that, national average HIV prevalence among IDUs varied between 5.4% and 8.2% [15, 22] . However, dramatic geographic differences in HIV prevalence have been observed. Yunnan and Xinjiang provinces have the most severe HIV infection rates among IDUs. Other provinces along or close to drug trafficking roads, such as Guangxi, Sichuan, Guizhou, Hunan, and Jiangxi, have moderate epidemics [25] . HIV began to spread rapidly in Yunnan province in the late 1980s and early 1990s [26] . High prevalence among IDUs was first detected in 1998 in Urumqi City (28.8%) and in Yining City (82.2%) of Xinjiang Province and later on in selected sentinel sites for IDU in other provinces [27] . These include Guangxi (>10%, 1998 sentinel data), Jiangxi (14.5%, 2000), Sichuan (16-20%, 2002) , Guizhou (17-19%, 2002) , and Hunan (15-20%, 2003) [23] . In 2004, 75% of HIV national sentinel sites for IDU detected HIV infection. More than 20% of HIV prevalence was reported in selected sites in Xinjiang, Sichuan, Guizhou, Guangxi and Hunan [22] . Official HIV prevalence rates in other geographic areas remain <10%; these reports may reflect reality or may be due to failure in detection.\nAt least 7 HIV-1 subtypes (A, B/B', C, D, E, F, and G), and 3 major circulating recombinant forms (CRF01_AE, CRF07_BC, and CRF08_BC) have spread in China [28] [29] [30] [31] . However, the most prevalent HIV-1 strains are subtype B' (44%), C (29%, usually CRF08_BC) and CRF01_AE (13%) [30] . Subtype C and B'/B are the main subtypes among IDUs [30, 32] . Co-circulating of multiple subtypes of HIV-1 in China implies the possibility of interclade recombination. Characterization of genetic variability of HIV-1 may help track the epidemic, generate subtype-specific immunological reagents, develop vaccines, and choose antiretroviral therapy regimens.\nDrug trafficking and abuse are illegal in China. Offenders may be sentenced to prison if smuggling 10 grams or more of heroin and could receive the death penalty for smuggling more than 50 gram of heroin. In 1990, the \"Regulations on Prohibition Against Narcotics\" were enacted with three levels of penalty to be applied to drug users. First-time offenders may be fined and/or allowed to go to voluntary detoxification centers, where they receive 10-day methadone treatment managed by the Ministry of Public Health. The cost of treatment is about 2,000 to 5,000 Chinese yuan -a cost considered expensive for many drug users and their families. If drug users who have gone through a voluntary detoxification program are caught again using drugs, they are sent to compulsory rehabilitation centers (CRC) administered by the Ministry of Public Security for 6 to 12 months. Drug users who relapse users after going through CRC are sent to reeducation-through-labor-centers (RELC) administered by the Justice Department for two to three years [33].\nIn Chinese history, drug abuse and prostitution have been considered \"social evils.\" The Chinese government typically takes \"crackdown\" measures and tries to eradicate these phenomena. China did achieve a success story in the 1950s. Illicit drug abuse and prostitution were eradicated through national anti-drug and anti-prostitution campaigns [3, 34 ]. However, this success has not been repeated in the past two decades. One possible explanation is that China has expanded its market economy and opened further to the outside world. Under this situation, it seems impossible to completely stem manufacture of highly profitable illicit drugs and import of drugs along China's long porous land borders with drug-producing countries.\nCurrently, the Chinese government adopts more pragmatic policies and takes measures targeting both the root and surface of the drug abuse problem. The measures targeting the root include continuously cracking down on drug smuggling activities and discouraging new users through anti-drug education campaigns. The Chinese government actively seeks to collaborate with neighboring countries to prevent drug smuggling across borders and to help Myanmar, for example, to reduce opium poppy cultivation by replacing with crop plantation. Chinese mass media have increased anti-drug education to the general population. Anti-drug education has been included in the curricula for primary and secondary school students [9] . The measures targeting the surface include providing drug detoxification and harm reduction services to drug users. China has about 300 voluntary detoxification clinics, 700 CRC, and 200 RELC. Each can accommodate 100 to 3,000 patients [9] . Both western medications and traditional Chinese medicines are used for detoxification, and community therapy has been provided in some heavily affected areas since the late 1990s [9] . The National Working Group for Community-based Methadone Maintenance Therapy was established collectively by the Ministry of Health, the Ministry of Public Security, and the State \nWhether or not China can shun a generalized epidemic of HIV/AIDS may be largely dependent on how China deals with IDU risk factors and breaks the bridge between IDU and heterosexual transmission. Past experience in China suggests that solely cracking down on drug smuggling and prohibiting drug use can not prevent or solve all illicit drug related problems in the era of globalization. Governmental support, harm reduction programs, voluntary counseling and testing, and utilization of non-governmental organizations are recommended.\nChina has a strong central government. Without government support, it would be not imaginable to achieve success in the campaigns against drug use and the spread of HIV/AIDS. Since the late 1990s, the Chinese central government has stepped up HIV/AIDS control efforts, including setting out national policy framework for responding to HIV/AIDS, increasing funding inputs, expanding collaborations with international organizations. The Chinese National Medium-and-Long-Term Strategic Plan for AIDS Prevention and Control (1998-2010) was formulated in 1998 and set one goal that \"by 2002, health education on preventing HIV/AIDS and STDs should be carried out at all detoxification centers and re-education centers as well as in 80% of jails...\" [37] . The Action Plan (2001-2005) calls for creating \"drug-free communities\" through drug prohibition education and drug detoxification activities, together with active promotion of healthy life styles and behaviors and harm reduction for drug users.\nOn the other hand, there is substantial autonomy at provincial level in some areas. Responses to drug use and HIV/AIDS epidemic vary significantly at provincial and lower administrative levels. For example, Yunnan and Guangxi provinces have done far more than other provinces in supporting, implementing, and advocating for harm reduction interventions for IDUs. Some local governments are not fully motivated to confront drug abuse and HIV/AIDS problems. Some government leaders still ignore or even cover up these problems. They are far more interested in economic growth than HIV/AIDS control and wish for their administrative areas to become \"economy provinces\" or \"economy cities\" rather than \"AIDS provinces\" or \"AIDS cities,\" which is believed to be helpful for their career promotion. Advocacy to and support from government leaders at all administrative levels for harm reduction and community-based prevention are needed.\nHarm reduction includes many strategies, such as methadone maintenance, needle exchange, dispensing other drugs, and outreach services [38] . Harm reduction has been a controversial issue as compared to abstinencebased philosophies [39, 40] . Harm reduction seems to encourage tolerance of social phenomena that are undesirable and hazardous and that may result in social turpitude. Instead, abstinence is considered to be the proper way to address drug problems [39] . In the United States, law and policy restrict the use of federal funds in supporting needle and injection equipment distribution projects. However, many studies refute the concern that access to sterile syringes is an endorsement of IDU and is likely to result in increases in injection and initiation of injection [41, 42] . Harm reduction projects in China are in the pilot phases. Some scholars and health officials still have similar concerns as in some Western countries. They believe that needle exchange services may send a wrong signal of encouraging drug abuse to drug users and the public, and they consider methadone substitution unethical because it uses one drug to replace another drug [43] . As drug use and HIV/AIDS spread rapidly across the country, it is urgent to find supporting domestic evidence that harm reduction will reduce HIV transmission by evaluating harm reduction projects and ultimately scaling up harm reduction efforts.\nIt is easy to access sterile needles and syringes in urban areas of China because they are legally sold and available at pharmacies and medical clinics [16, 44] . However, many drug users live in rural areas; in addition, drug users may share needles and syringes because they can not buy them during night time or do not have money to buy them [16] . Methadone is orally administered, and metha-done substitution can reduce injection and needle sharing of opiate drug addicts. But methadone maintenance therapy is costly and requires drug users to attend clinics on a regular basis. Therefore, methadone substitution and needle exchange services should be made available and affordable at convenient times and in both urban and rural settings, especially in the communities with heavy drug use.\nIn addition to cost and availability, other factors might also affect acceptability of methadone maintenance therapy, such as concerns about the safety and efficacy of the therapy [45] . Greater retention in treatment has been found to result in greater decreases in drug use, criminal activity, and unemployment [46] . The length of drug treatment has a positive association with better post-treatment outcome [46] . However, limited experience with methadone maintenance therapy (MMT) in China shows a high rate of dropouts. International studies have shown that motivational enhancement therapy or motivational interviewing enhances treatment initiation, retention and outcomes in MMT program [47, 48] , and adding behavioral intervention components into MMT programs increases abstinence and reduces HIV risk behaviors [49] . Policy-oriented operational research is needed in China to better understand how to increase the effectiveness of MMT and other harm reduction interventions in the Chinese context.\nThere are still persistent conflicts in the policy and legal landscape. The central government has given explicit support to harm reduction, for example, as stated in the Medium-and Long-Term Strategic Plan and the Action Plan. Some programs have been implemented successfully [36,50,51]. However, in China, as in many other countries, public health and public security authorities frequently approach drug abuse from different perspectives, leading to conflicting approaches at local levels. The crackdown philosophy and detention of drug users in China reflect inconsistent interpretations of \"harm reduction\" and present a challenge to public health officials in implementing methadone substitution and needleexchange programs [52] . Drug users may be reluctant to participate in these programs due to fear of being caught by police officers [50] . It might be impossible to completely solve the dilemma in the near future, but this conflict is expected to gradually reduce for the following reasons. First, Chinese national policies for HIV prevention and control have become much more pragmatic in the past years. MMT and needle exchange programs were almost unimaginable several years ago, but now they are ready to be expanded across the country. We expect that the open policy trend will continue as the Chinese economy is increasingly merged with international markets, and this trend will favor harm reduction programs. Fur-thermore, China's centralized government may achieve an advantage in promoting public health policies if these policies are believed to be correct. Second, inter-agency coordination on public health crisis has been enhanced at both central and local governmental levels since SARS outbreak in 2003, which reduces potential conflict of public health policies. Public health workers should provide policy advocacy to public security authorities and help them change their traditional norms about illicit drug control and obtain their supports for harm reduction. Third, operational research is needed to provide evidence on the benefits of harm reduction programs and convince policy enforcers and lead to revision of unfavorable policy components.\nHIV voluntary counseling and testing (VCT) is often considered the first step for initiating prevention and/or therapy. One of the strategies for addressing the AIDS epidemic is to give people an opportunity to know their HIV status so that they can take precautions to avoid further spread and receive early therapy if they are infected [53, 54] . However, even in developed countries, many atrisk people do not take VCT. A national British survey in 2000 showed that only one-third of IDUs had VCT in the past 5 years [55] . About one-fourth of the 0.8 to 0.9 million infected people in the United States remain unaware that they are HIV positive [54] . In China, there is a large discrepancy between reported (about 100,000) and estimated (about 1 million) cumulative HIV/AIDS cases thus far. Many at-risk individuals do not seek out standard HIV counseling and testing services. The stigma associated with drug use and HIV/AIDS and fear of arrest or knowing a positive result can be major barriers to access to VCT. A survey among 840 pregnant women and 780 health professionals in Yunnan Province -an epicenter of the HIV/ AIDS epidemic in China -found prevalent negative attitudes toward HIV/AIDS. Twenty-three percent of health professionals and 45% of pregnant women thought HIV was a disease of \"low class and illegal\" people; 48% of health professionals and 59% of pregnant women thought that HIV positive individuals should not be allowed to get married; and 30% of the health professionals were not willing to treat an HIV-positive individual [56] . Cost of traditional VCT, low awareness of risk factors for HIV infection, distance, and inconvenience in time also may prevent access to VCT. Possible solutions include development of outreach programs to offer anonymous testing and counseling to those at heightened risk of HIV infection and adoption of new technologies such as rapid saliva testing and counseling strategies to improve the outreach and efficacy of programs.\nNon-governmental organizations (NGO) can play a critical role in the delivery of HIV prevention services and other assistance to persons living with AIDS. The flexibility of NGOs enables them to respond quickly to fill in gaps in health care and social services. NGOs can do what government agencies cannot do or are not willing to dofor example, reaching out without perceived threat to IDUs and other marginalized sub-groups whose behaviors are often stigmatized and also put them at higher risk of HIV/AIDS. A recent survey of 29 NGOs in Central and Eastern Europe showed that most NGOs targeted injection drug users; provided needle exchange and HIV prevention peer education; and delivered AIDS presentations and distributed educational materials [57] . In Africa, where the main transmission occurs via heterosexual activity, NGOs are most likely to direct their attention to the general public and to youth; they provide peer-education or community outreach [58] . In both Thailand and Brazil, where success has been observed in controlling the HIV/AIDS epidemic or reducing AIDS mortality, NGO are believed to play a key role, but their programs lack rigorous and systemic evaluation [59, 60] . NGOs often face several difficulties: lack of financial resources [57, 58] ; lack of communications with governmental organizations [61] ; governmental indifference or opposition; and AIDSrelated stigma [57] .\nChina has large number of government organized NGOs (GONGOs), including Family Planning Associations, Women's Federation, Red Cross, Youth League, trade unions, and diverse academic associations. The members in these GONGOs have formal positions in governmental organizations while they volunteer at GONGOs. The \"true\" NGO that has no relationship to the government is just emerging in China. More and more of existing Chinese GONGOs are getting involved in sexually transmitted disease/AIDS prevention. Since the 1990s, the Chinese government has encouraged them to participate in HIV/ AIDS control. These government-sponsored NGOs support HIV/AIDS education and academic publications and participate in AIDS research and education with foreign governmental and non-governmental partners, and they can serve as a powerful aid to the Chinese government to achieve the goal of stopping further spread of drug use and HIV/AIDS epidemic.\nIn spite of its potential for greatly contributing HIV prevention in China, there is little literature in this area. An exception is a recent study (Chen and Liao, 2005 ) of a Women Federation's HIV prevention program in south China. The study showed that the Women Federation was able to deliver a culturally oriented, multi-level intervention program targeted at female drug users. The data also indicated that the program was successfully in increasing knowledge about HIV/AIDS, increasing condom use, and decreasing needle and syringe sharing among the female drug users in the project. Studies which systematically evaluate the implementation and effectiveness of NGO based intervention programs are greatly needed in the future. \nThe author(s) declare that they have no competing interests.\nHZQ and JES conceived of the study and wrote the first draft of the manuscript, HZQ, HTC and YHR collected the data, and all authors participated in the data interpretation and manuscript revisions." }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: Development of a humanized monoclonal antibody with therapeutic potential against West Nile virus E protein by nickel-affinity chromatography (data not shown). After immunization and screening 2,000 hybridomas, we isolated 46 new monoclonal antibodies that recognized WNV E protein (Supplementary Table 1 online).\nWe evaluated the antibodies for their ability to block WNV infection in BHK21 cells using a standard plaque-reduction assay 23 . Twelve had strong neutralizing activity that greatly exceeded the potency of immune human γ-globulin, with 50% plaque reduction neutralization titers (PRNT 50 ) below 2 µg, whereas immune human γ-globulin had a PRNT 50 value of 500 µg 5 . The inhibitory activity of two neutralizing antibodies, E16 and E24, was reproduced in J774.2 mouse macrophages and SW13 human adrenal carcinoma cells ( Supplementary Fig. 1 online) and thus was not specific to fibroblasts.\nOne of the potent neutralizing monoclonal antibodies, E16, inhibited infection of genetically diverse WNV lineage I strains that were isolated from mosquitoes, birds and horses in New York. E16 neutralized all WNV strains with PRNT 50 values of 4-18 ng and PRNT 90 values of 53-297 ng (Supplementary Table 2 online) . Notably, Fab fragments of E16 inhibited WNV (PRNT 50 , 23 ng), suggesting that neutralization does not require bivalent E protein binding. E16 potently blocked infection with strain 956, the original lineage II strain isolated in 1937 (ref. 24 ), yet was virus specific, as it neither recognized nor neutralized other flaviviruses including distantly related dengue and yellow fever viruses (data not shown) and closely related Japanese and St. Louis encephalitis viruses (Supplementary Table 2 Figure 1 Mapping of monoclonal antibodies to DIII with yeast. (a) Surface display of WNV E protein on yeast. A fusion protein is composed of the ectodomain or DIII of WNV E protein and the yeast Aga2 protein, which becomes attached to the Aga1 protein on the yeast cell wall. Yeast were transformed with the vector alone (pYD1; top row), the entire WNV E ectodomain (amino acids 1-415; middle row), or DIII alone (amino acids 296-415; bottom row). 24 h after induction, yeast were stained with the indicated monoclonal antibodies (negative control, anti-SARS ORF7a) and processed by flow cytometry. Data for a representative neutralizing (E16) and non-neutralizing (E18) antibody are shown. (b) Flow cytometric enrichment for DIII-expressing yeast variants that lose binding of E24. After each round, an increased percentage of DIII expressing yeast are recognized by the polyclonal WNV E-specific antibody but not by E24. After the final round, DIII-expressing variants (boxed region) were harvested. a b Figure 2 Fine epitope mapping of DIII neutralizing and non-neutralizing monoclonal antibodies. (a) Flow cytometry profiles for immunoreactivity by nonneutralizing (E2), weakly neutralizing (E1) and strongly neutralizing (E34) monoclonal antibodies with yeast expressing wild-type and variant DIII. The red, yellow and green arrows point to mutations that abolish yeast surface binding of individual monoclonal antibodies and correspond to distinct regions of DIII (see b). (b) Mapping of neutralizing and non-neutralizing monoclonal antibodies on the surface of WNV DIII. A molecular surface representation is depicted based on the nuclear magnetic resonance structure of WNV DIII 16 . The indicated amino acid residues associated with binding of neutralizing, weakly neutralizing and non-neutralizing monoclonal antibodies are shown in red, yellow and green, respectively.\nTo map our strongly neutralizing monoclonal antibodies, we developed a strategy using yeast surface display 25 . The ectodomain (amino acids 1-415) or DIII (amino acids 296-415) of WNV E protein were expressed as fusion proteins on the yeast cell surface (Fig. 1a) .\nMonoclonal antibodies that recognize DIII alone are considered DIII specific. Monoclonal antibodies that recognized the E ectodomain but not DIII alone may contact residues that map to domain I or II or both, although cooperative contacts with DIII cannot be ruled out. Most of our 46 monoclonal antibodies recognized yeast that displayed the entire ectodomain of E ( Fig. 1a and Supplementary Table 1 online) . Sixteen antibodies recognized yeast that displayed DIII alone, and 10 of 12 strongly neutralizing antibodies localized to DIII. Only two neutralizing antibodies (E53 and E60) recognized the E ectodomain but not DIII alone.\nWe used error-prone PCR mutagenesis of DIII of WNV E protein and yeast surface expression to map antibody contact residues in a highthroughput manner. We performed individual screens to identify DIII mutants that lost binding selectively to strongly neutralizing (E16, E24 and E34), weakly neutralizing (E1), and non-neutralizing (E2 and E22) monoclonal antibodies. To eliminate mutants that abolished surface expression of DIII, yeast were stained sequentially with an Alexa Fluor 647-conjugated individual monoclonal antibodies and an Alexa Fluor 488-conjugated oligoclonal antibody derived from a pool of individual monoclonal antibodies. After cell sorting, we identified yeast that selectively lost expression of an individual monoclonal antibody epitope but retained surface expression of DIII (Fig. 1b) . Multiple independent yeast that lost binding of individual monoclonal antibodies were subjected to plasmid recovery and sequencing. Monoclonal antibodies that localized to DIII and strongly neutralized WNV had reduced binding when residues S306, K307, T330 or T332 were altered ( Fig. 2a and Table 1 ). These are located on adjacent loops and form a contiguous patch 16 on the solvent-exposed surface at the lateral tip of the DIII (Fig. 2b) . Only two other mutations caused considerable loss of binding of any of the ten neutralizing monoclonal antibodies tested: K310E or P315R reduced binding only of E49. No two neutralizing monoclonal antibodies had identical loss-of-binding patterns. For example, S306L reduced binding of E16, E27, E40, E43 and E49 but not E24, E33, E34, E47 and E58. K307R abolished binding of E34, E40, E43, E47, E49 and E58 but affected E16, E24, E27 and E33 less strongly. In contrast, K307E decreased binding of all neutralizing monoclonal antibodies yet did not affect non-neutralizing or poorly neutralizing monoclonal antibodies. Changes in residues T330 and T332 also abolished binding of neutralizing but not non-neutralizing monoclonal antibodies. T330I or T332M strongly reduced binding of all neutralizing monoclonal antibodies with the exception of E27, and T332V or T332A weakened binding of only E24, E27, E33 and E58.\nSix monoclonal antibodies that recognized DIII were either poorly neutralizing or non-neutralizing, and none engaged the dominant neutralizing epitope defined by S306, K307, T330 or T332. E2 and E9 were abolished or reduced by mutation of D381 and H396, and binding of E22 was weakened by a change in P315. D381 and H396 are proximal to one another but physically distinct from the four residues that affect binding of neutralizing monoclonal antibodies (Fig. 2b) . None of the mutations identified by loss-of-binding sorts for E2 or E22 had any effect on two other non-neutralizing monoclonal Individual WNV-specific monoclonal antibodies (25 µg/ml) were mixed with yeast that displayed wild-type or mutant DIII on their surface. After washing, and incubation with an Alexa-647 goat-anti mouse IgG secondary antibody, yeast were analyzed for antibody binding by flow cytometry. The two values represent the percentage of yeast that were positive for DIII expression with a given monoclonal antibody, and in parentheses, the mean linear fluorescence intensity of the positive cells. Yeast were analyzed at 24 h after induction with galactose, which gives a baseline surface expression of DIII of 60-80% positive cells (Fig. 1a) . Bold values indicate an almost complete (>80%) loss of binding, whereas underlined values show a marked (50-79%) reduction in either the percentage or the mean fluorescence intensity of the positive cells. Results are representative of at least two independent experiments for each antibody and DIII variant.\nantibodies, E21 and E23. E1, a monoclonal antibody with weak neutralizing activity, mapped to a site between the non-neutralizing and neutralizing monoclonal antibodies, as mutation of K310 and N394 strongly inhibited binding.\nTo determine whether human antibodies specific for WNV recognize the neutralizing epitope on DIII during infection, plasma was obtained from WNV-positive individuals. Samples from convalescent individuals were negative for WNV RNA but positive for neutralizing WNV-specific antibodies. The individuals reported mild systemic illness, though none progressed to severe disease. To determine whether these samples contained antibodies that localized to the neutralizing epitope on DIII, we tested whether E16 Fab or IgG could compete binding to recombinant, wildtype and mutant N394K and K307N forms of DIII ( Supplementary Fig. 2 online); N394K retains wild-type binding to E16, whereas K307N has markedly reduced binding. E16 equivalently inhibited binding of patient WNV-specific antibodies to wild-type (Fab, 35% ± 8; IgG, 40% ± 12) or N394K DIII (Fab, 32% ± 9; IgG, 34% ± 11), whereas E53 IgG, a WNV-specific monoclonal antibody that recognizes an epitope outside DIII, did not compete binding to wild-type (1% ± 4) or N394K\n(-2% ± 5) DIII. As expected, E16, which only weakly recognizes K307N, poorly competed (Fab 6% ± 3; IgG 9% ± 4) binding to K307N DIII. These data suggest that humans, who clear WNV infection, develop antibodies that recognize an epitope in close proximity to that defined by E16.\nTo evaluate the correlation between neutralization, epitope localization and in vivo protection, we assessed the therapeutic activity of different neutralizing monoclonal antibodies in an established mouse model 5 . Studies were performed with 5-week-old wild-type C57BL/6 mice, which have a ∼10% survival rate 5 . Mice were inoculated subcutaneously with 10 2 plaque-forming units (PFU) of WNV and administered a single dose of monoclonal antibody at day 2 after infection. Notably, 500 µg of the non-neutralizing monoclonal antibody E2 provided no protection (data not shown). In contrast, 100 µg of any of three different neutralizing monoclonal antibodies that map to K307 (E16, E24 or E34) protected greater than 90% of mice from lethal infection ( Fig. 3a-c) . Even a single 4 µg treatment of E16 or E34 on day 2 after infection prevented mortality. Given that humans can present with WNV infection of the CNS, we evaluated the therapeutic efficacy of monoclonal antibodies at later , or (c) E34 monoclonal antibodies. As controls, mice were independently administered saline (PBS) or a negative control monoclonal antibody (anti-SARS ORF7a, 500 µg). The survival curves were constructed using data from two independent experiments. The number of animals for each antibody dose ranged from 20 to 30. The difference in survival curves was statistically significant for all WNV-specific monoclonal antibody doses shown (P < 0.0001). (d) WNV burden in the brain of 5-week-old wild-type mice. At days 4, 5 and 6 after WNV infection, brains were harvested and viral burdens were determined by plaque assay. The following percentage of mice had viral burdens below detection (<20 PFU/g): day 4, 33%; day 5, 22%; day 6, 17%. (e,f) Efficacy of WNV-specific monoclonal antibody therapy at days 4 (e) and 5 (f) after infection. A single dose (0.5 mg at day 4 or 2 mg at day 5) of monoclonal antibody (E16, E24, E34 or anti-SARS ORF7a) was administered either 4 or 5 d after WNV infection. Data reflect approximately 20 mice per condition. The difference in survival curves was statistically significant for all WNV-specific monoclonal antibody doses shown at day 4 (P < 0.0001) and day 5 (E16, P = 0.0009; E24, P = 0.027). (g) Effect of E16 therapy on viral burden. Mice were treated with 2 mg of E16 or PBS on day 5 after WNV infection. On day 9, brains were recovered, homogenized and subjected to plaque assay. For a subset (6) that received PBS treatment, brains were harvested at days 7 and 8 from moribund mice. The data is expressed as PFU/g. Of 16 mice treated with E16 68% (11) had undetectable viral loads in the brain at day 9 whereas all mice (14 of 14) treated with PBS had detectable virus at the time of harvest. The dotted line represents the limit of sensitivity of the assay and the dark bars represent the mean of the log values. The differences were statistically different (P < 0.0001).\ntime points. At days 4, 5 and 6 after WNV infection, we detected virus in the brains of 67%, 78% and 83% of mice, respectively (Fig. 3d) . A single 500 µg dose of E16 or E34 at day 4 resulted in an 80-90% survival rate (Fig. 3e) . A single 2 mg dose of E16 at day 5 resulted in 90% survival (Fig. 3f) and complete clearance of WNV from the brain in 68% of mice by day 9 (Fig. 3g) . Thus, administration of neutralizing monoclonal antibodies to mice with active CNS infection improved survival and induced a virologic cure. As expected, lengthening the interval before treatment was associated with decreased benefit. Administration of E16 at day 6 did not enhance survival (data not shown), although average survival time was increased (9.5 d ± 0.4 to 11.5 d ± 0.4; P = 0.003).\nWe considered humanizing E16 or E34 as a possible therapeutic measure. Humanized monoclonal antibodies have substantially longer half-lives in humans than their mouse counterparts 26, 27 . Sequencing studies indicated that E16 had greater homology to human framework regions, making it simpler to construct a humanized version of E16. We amplified the cDNA encoding the heavy (VH) and light (VL) variable domains from the hybridoma cellular RNA by a 5′ rapid amplification of cDNA ends (RACE) procedure. The VH belongs to mouse heavy chain subgroup II (J558 family) and the VL belongs to mouse κ chain subgroup V. The complementarity-determining regions of E16 were grafted onto the human VH1-18 backbone (Fig. 4a) and human Vκ-B3 backbone (Fig. 4b) to create Hm-E16.1. One (VL-Y49S) and two framework back-mutations (VH-T71A and VL-Y49S) were introduced to create two variants, Hm-E16.1 and Hm-E16.3, respectively. The resulting humanized VH and VL were combined with human γ1 and κ constant regions, fused to an IgG signal sequence and inserted into expression plasmids. To construct the chimerized antibodies, the mouse VH and VL sequences were combined with the human γ1 and κ constant regions.\nWe expressed humanized (Hm-E16) and chimerized (Ch-E16) E16 in HEK-293 cells, and purified them from supernatants by affinity and size-exclusion chromatography (data not shown). The affinity was analyzed by surface plasmon resonance using purified antibody in the solid phase. Mouse E16 binds DIII with an affinity of 3.4 nM and a half-life of 3.9 min. The affinity of the Ch-E16 and Hm-E16 was similar with K D ranging from 7.1 to 21 nM (Fig. 4c) . Hm-E16, Ch-E16, and the parent E16 all had similar PRNT 50 values (Fig. 4d) .\nWe hypothesized that E16 could also control infection in mice through effector functions including antibody-dependent complement fixation and cytotoxicity. To test this, we generated a Ch-E16 N297Q aglycosyl variant that neutralizes WNV (Fig. 4d) but does not efficiently fix complement or bind Fc γ receptors 28 . Mice were administered Ch-E16 or Ch-E16 N297Q at day 2 after WNV infection. Although high doses of Ch-E16 and Ch-E16 N297Q provided virtually complete protection, lower doses of the aglycosyl variant afforded less protection (Fig. 4e) . Administration of 4 µg of Ch-E16 resulted in 84% survival, whereas 4 µg of Ch-E16 N297Q provided only 31% protection.\nTo test which effector function enhanced the activity of E16, we performed studies with 8-week-old C1qa, C4, or Fcgr1 and Fcgr3-deficient C57BL/6 mice (Fig. 5) . These mice all show increased susceptibility to lethal WNV infection compared to wild-type controls. In C1qa or C4-deficient mice, which cannot activate complement by the antibody-dependent classical pathway, E16 had a similar potency compared to wild-type mice. In contrast, in Fcgr1-and Fcgr3-deficient mice, although high doses afforded complete protection, lower doses resulted in higher mortality rates. A dose of 20 µg at day 2, which strongly protected wild-type, C1qa or C4-deficient mice, did not improve the survival rate of Fcgr1-and Fcgr3-deficient mice (P = 0.4). Thus, the Fc region enhances the potency of E16 in mice, by virtue of its ability to bind to Fc γ receptors.\nTo confirm the efficacy of humanized E16, wild-type mice were administered three different versions of purified Hm-E16 at day 2 after infection. Although Hm-E16 variants protected mice against lethal infection (Fig. 4f) , at a dose of 4 µg, the variant (16.3) that showed the highest affinity for DIII (Fig. 4c) was more protective (67% versus 46% survival; mean survival time of 14 ± 1 d versus 11 ± 2 d, P = 0.04).\nWe generated a panel of 46 monoclonal antibodies against WNV E protein, and applied a new high-throughput epitope-mapping strategy to identify a dominant epitope that was recognized by the majority of neutralizing monoclonal antibodies in DIII. This epitope was also detected by convalescent antibodies from individuals who had recovered from WNV infection without clinical consequence. Three neutralizing monoclonal antibodies protected against WNV mortality in a postexposure therapy model. One of these, E16, was humanized and confirmed as therapeutically effective in mice.\nPrevious studies have mapped amino acid contact residues of neutralizing monoclonal antibodies by sequencing in vitro neutralization escape variants, through site-specific substitution of specific charged or polar residues, and by performing binding assays with overlapping peptide libraries. We used error-prone PCR mutagenesis and yeast surface expression to identify contact residues in a high-throughput manner. Although this technique has been used previously 29 , this is the first highthroughput epitope-mapping application. By having a large panel of DIII monoclonal antibodies and selecting only variants that abolished or markedly reduced binding of a few monoclonal antibodies, we minimized the possibility that mutations altered protein folding. We have recently confirmed the recognition sites on DIII for E16 and the validity of the yeast display strategy by solving the crystal structure of the E16 Fab-DIII complex (G.N., T.O., M.D, & D.F., unpublished data).\nOf 12 neutralizing monoclonal antibodies, 10 localized to the distal lateral surface of DIII, results that are consistent with prior studies that mapped three neutralizing monoclonal antibodies against WNV using in vitro escape variants 15, 30 . Our weakly neutralizing and non-neutralizing monoclonal antibodies did not recognize this epitope but localized to distinct regions. E16 recognized the dominant epitope and neutralized all strains that were tested. Sequence analysis of 124 WNV strains in public databases showed almost complete (98.4-100%) conservation of the contact residues S306, K307, T330 and T332. Only two clinically attenuated lineage II isolates had mutations at these residues. Because of the structural homology among flaviviruses 7, 8, 16 , the analogous amino acids that map to the distal lateral surface of DIII can be readily identified. Although this region is highly variable among flaviviruses, most mutations that abolish binding of virus-specific neutralizing antibodies map here 15, 18, 30, 31 , suggesting the existence of an analogous dominant neutralizing epitope for other flaviviruses. We speculate that successful vaccines against WNV or other flaviviruses should induce potent humoral responses against this neutralizing epitope.\nAlthough passive administration of immune human γ-globulin after WNV infection improved survival in mice 4,5 , it may be limited by its low-titer neutralizing activity, variability and risk of transmission of infectious agents. Only two prior studies have shown postexposure therapy of neutralizing monoclonal antibodies with flaviviruses: 6B5A-2 reduced mortality 3-4 d after infection with St. Louis encephalitis virus 6 ; and 503 reduced mortality resulting from infection with Japanese encephalitis virus 5 d 32 . Here, we show that three different neutralizing monoclonal antibodies improved survival even when administered 4 and 5 d after WNV infection. Moreover, therapy with E16 at day 5 completely cleared WNV from the brain at day 9 in 68% of mice. Thus, inhibitory WNV monoclonal antibodies improve clinical and virologic outcome even after viral spread through the CNS, results that agree with studies showing that antibody can mediate viral clearance from infected neurons 33, 34 .\nOur experiments are consistent with a model in which the therapeutic efficacy of monoclonal antibodies is determined by properties in addition to neutralization: (i) the monoclonal antibody (E24) with the strongest neutralizing activity in vitro did not have the greatest efficacy in vivo; (ii) an aglycosyl version of E16 that lacked the ability to fix complement or bind to Fc γ receptors had equivalent neutralizing but reduced therapeutic activity; (iii) E16 was less potent in mice that lacked Fc γ receptors. E16 was humanized as a possible therapeutic measure for humans. Hm-E16 bound DIII with similar affinity and showed efficacy as postexposure therapy. Moreover, it may be possible to improve Hm-E16 by a b c introducing mutations that enhance affinity, creating forms of E16 that more readily cross the blood-brain barrier, and combining monoclonal antibodies that neutralize WNV infection through independent mechanisms. Our results are the first successful demonstration of a humanized monoclonal antibody as postexposure therapy against a viral disease, and suggest that antibody-based therapeutics may have more broad utility than previously appreciated, especially in the treatment of CNS infections in which an effective antibody response is important for limiting virus dissemination and injury to neurons.\nWe cultured BHK-21, Vero and C6/36 Aedes albopictus cells as previously described 35 24, 36) . We also performed neutralization experiments with prototype strains of St. Louis (59268 (Parton)) and Japanese encephalitis (Nakayama) viruses 37 . For in vivo experiments, viruses were diluted and injected into mice as described 23 .\nPurified WNV E protein expression. WNV E protein ectodomain was generated using a baculovirus expression system according to previously described methods for related flaviviruses 38 . The last 45 nucleotides of prM (endogenous signal sequence) and the first 1,290 nucleotides of WNV E protein from the New York 1999 strain 39 were fused downstream of the polyhedrin promoter and upstream of a histidine repeat in a baculovirus shuttle vector (pFastBac, Invitrogen) by PCR using a high-fidelity Taq polymerase (Platinum Taq, Invitrogen). Three days after baculovirus infection of Hi-5 insect cells at a multiplicity of infection (MOI) of 1, supernatants were harvested, filtered, buffer-exchanged and purified by nickelaffinity chromatography according to the manufacturer's instructions. The purified WNV E ectodomain lacks the C-terminal 71 amino acids that are associated with the membrane proximal, transmembrane and cytoplasmic domains.\nPurified WNV DIII. The construction, expression, purification and refolding of DIII of WNV E protein is described in greater detail elsewhere (G.N., T.O., M.D. & D.F., unpublished data). Briefly, wild-type, N394K and K307N DIII were generated from an infectious cDNA clone of the New York 1999 strain of WNV (gift of R. Kinney, Centers for Disease Control and Prevention, Fort Collins, CO) using PCR and Quik-change mutagenesis (Stratagene). After cloning into a PET21 vector (Novagen) and sequence confirmation of the mutations, we transformed plasmids into BL21 Codon Plus E. coli cells (Stratagene). Bacteria were grown in Luria broth, induced with 0.5 mM isopropyl thiogalactoside (IPTG) and pelleted. Subsequently, we lysed bacteria after the addition of lysozyme, sonicated them and recovered DIII as insoluble aggregate from the inclusion bodies. DIII was denatured in the presence of guanidine hydrochloride and β-mercaptoethanol and refolded by slowly diluting out the denaturing reagents in the presence of L-arginine, EDTA, PMSF, reduced glutathione and oxidized glutathione. We separated refolded DIII from aggregates on a Superdex 75 16/60 size-exclusion column (Amersham Bioscience) and concentrated it using a centricon-10 spin column into 20 mM Hepes pH 7.4, 150 mM NaCl and 0.01% NaN 3 . After refolding, wild-type DIII reacted with all domain III-specific monoclonal antibodies including those that recognized conformationally sensitive epitopes.\nGeneration and purification of monoclonal antibodies. BALB/c mice were primed and boosted at 3-week intervals with insect cell-generated, purified, recombinant WNV E protein (25 µg) that was complexed with adjuvant (RIBI Immunochemical). Approximately 1 month after the last boost, we harvested serum and tested it for immunoreactivity against solid-phase purified E. Mice with high titers (>1/10,000) were boosted intravenously with purified E protein (5 µg) in PBS. We harvested splenocytes 3 d later and fused them to P3X63Ag8.653 myeloma cells to generate hybridomas according to published procedures 40 . We purified monoclonal antibodies against WNV or other control antigens by stan-dard protein A or protein G chromatography according to the manufacturer's instructions (Pharmacia).\nFor WNV infection experiments, all wild-type C57BL/6J mice were purchased from a commercial source (Jackson Laboratories). We obtained the congenic C1qa-deficient and C4-deficient mice from G. Stahl (Beth Israel Deaconess Medical Center, Boston, MA) and M. Carroll (The CBR Institute for Biomedical Research, Harvard Medical School, Boston, MA), respectively. We obtained the congenic Fcgr1-and Fcgr3-deficient mice commercially (Taconic). We used mice between 5 and 8 weeks of age depending on the particular experiment and inoculated them subcutaneously with WNV by footpad injection after anesthetization with xylazine and ketamine. Mouse experiments were approved and performed according to the guidelines of the Washington University School of Medicine Animal Safety Committee. For passive-transfer experiments, we administered to mice a single dose of purified monoclonal antibody by intraperitoneal injection at a given time point (day 2, 4 or 5) after infection. To analyze virus production in the brain, infected mice were killed on a given day after inoculation. After cardiac perfusion with PBS, we removed the brains, weighed and homogenized them, and performed plaque assays as previously described 23 .\nExpression of WNV E protein on yeast. The ectodomain or DIII of WNV E protein was expressed on the surface of yeast using a modification of a previously described protocol for surface expression of T cell receptors 29 . Amino acid residues 1-415 (ectodomain) or 296-415 (DIII) of WNV E protein were amplified with BamHI and XhoI sites at their 5´ and 3´ ends, respectively, by PCR from the New York 1999 infectious cDNA clone (R. Kinney, Centers for Disease Control and Prevention, Fort Collins, CO). The resulting products were digested with BamHI and XhoI, and cloned as downstream fusions to the yeast Aga2 and Xpress epitope tag genes in the yeast surface display vector pYD1 (Invitrogen). An upstream GAL1 promoter controls fusion protein expression. These constructs were transfected into the S. cerevesiae yeast strain EBY100 (refs. 25,41) resulting in yeast that expressed the WNV E ectodomain or DIII. Yeast that only expressed the Xpress epitope tag linked to Aga2 were prepared in parallel by transfecting EBY100 cells with the parent vector pYD1. Individual yeast colonies were grown to log phase overnight in tryptophan-free media containing 2% glucose at 30 °C and harvested in log phase. Fusion protein expression was induced on the yeast surface by growing yeast for an additional 24 h in tryptophan-free media containing 2% galactose at 25 °C. We harvested yeast, washed them with PBS supplemented with 1 mg/ml BSA and immunostained them with 50 µl of monoclonal antibody (25 µg/ml) against the Xpress tag or WNV E protein. After 30 min, we washed yeast three times and stained them with a goat anti-mouse secondary antibody conjugated to Alexa Fluor 647 (Molecular Probes). Subsequently, the yeast cells were analyzed on a Becton Dickinson FACSCaliber flow cytometer.\nLibrary construction and screening. We mutated DIII of the WNV E protein using an error-prone PCR protocol 25 that included Mn 2+ and Mg 2+ at concentrations of 0.3 and 2.0 mM respectively. Subsequently, the cDNA library was ligated into pYD1 and transformed into XL2-blue ultracompetent cells (Strategene). The colonies were pooled and the plasmid DNA was recovered using the Qiagen HiSpeed Maxi kit.\nFor each individual antibody, we screened the yeast library of DIII mutants according to the following protocol. To identify yeast that selectively lost binding to a given monoclonal antibody epitope, the library was initially stained with an Alexa Fluor 647-conjugated WNV-specific monoclonal antibody for 30 min at 4 °C. To control for the surface expression of DIII, after washing, yeast were subsequently stained for 30 min at 4 °C with an Alexa Fluor 488-conjugated oligoclonal antibody that was derived from a pool of individual monoclonal antibodies (E1, E2, E9, E16, E24 and E34). After immunostaining, we subjected yeast to flow cytometry and identified the population that was single monoclonal antibody negative but pooled oligoclonal antibody positive. The yeast cells were sorted at an event rate of ∼4,000 cells/s and this population (monoclonal antibody-negative and oligoclonal antibody-positive) was enriched after three rounds of sorting. After the final enrichment sort, we plated yeast and selected individual colonies and tested them for binding to individual monoclonal antibodies. For individual clones that had lost only the desired monoclonal antibody epitope, the DIII-pYD1 plasmid was recovered using the Zymoprep Yeast Miniprep kit (Zymo Research). The plasmid was then transformed into DH5α cells, purified using the Qiaprep Spin Miniprep kit (Qiagen) and sequenced.\nIn some cases, DIII variants with two independent mutations were isolated. To determine which mutation conferred the loss-of-binding phenotype, single independent mutations were engineered by site-directed mutagenesis of DIII-pYD1 using mutant oligonucleotides and the Quik Change II mutagenesis kit (Strategene). All mutations were confirmed by sequencing.\nWe determined the titer of neutralizing antibodies by a standard plaque reduction neutralization titer (PRNT) assay using either BHK21 or SW13 cells 23 . Results were plotted and the titers for 50% (PRNT 50 ) and 90% inhibition (PRNT 90 ) were calculated. The inhibition assay with J774.2 mouse macrophages was performed as follows: we mixed medium and E16 or E24 (2.5 µg of monoclonal antibody) with 5 × 10 2 PFU of WNV, incubated the mixture for 1 h at 4 °C, and then added to 5 × 10 4 J774.2 mouse macrophages in individual wells of a 24-well plate. After 1 h, cells were washed four times with PBS to remove free virus and monoclonal antibody, DMEM with 10% FBS was added, and the cells were incubated for an additional 24 h. We subsequently harvested supernatants for a viral plaque assay on Vero cells.\nCompetition ELISA with human anti-WNV antibodies. After purification and refolding, wild-type, K307N and N394K DIII were diluted (5 µg/ml) in 0.1 M Na carbonate buffer (pH 9.3) and adsorbed to 96-well plates overnight at 4 °C. After blocking with PBS, 2% BSA and 0.05% Tween 20 (PBS-BT), wells were preincubated for 1 h at 23 °C with PBS-BT containing no antibody, E16 IgG (50 µg/ml), E16 Fab (50 µg/ml) or E53 IgG (50 µg/ml). E53 serves as a negative control as it recognizes an epitope in domain I and II of WNV E protein. Subsequently, human plasma (1/40 dilution in PBS-BT, heat-inactivated) was directly added for an additional 1 h at 23 °C. We obtained the human samples with informed consent from seven different WNV-infected patients (gift of M. Busch and L. Tobler, Blood Systems Research Institute, San Francisco, CA). Because the samples were sequentially numbered and not linkable back to the original subjects, they satisfied the criteria for exemption from approval from the Human Studies Committee at Washington University. After six washes with PBS-BT, plates were serially incubated with biotin-conjugated goat anti-human IgG (1 µg/ml), streptavidin-horseradish peroxidase (2 µg/ml) and tetramethylbenzidine developing substrate (DAKO). We determined optical densities at 450 nm with an automatic ELISA plate reader (Tecan) and adjusted them after subtraction of the value obtained from nonimmune human plasma.\nSurface plasmon resonance. Antibody affinity analysis for DIII of WNV was performed by surface plasmon resonance (BIAcore 3000, Biacore, Inc). Binding curves and kinetic parameters were obtained as follows: we captured E16 antibodies by flowing (300 nM, rate of 5 µl/min for 2 min) them over immobilized F(ab)'2 fragment specific for goat anti-human or mouse IgG with Fc region specificity. Subsequently, DIII of the New York 1999 strain of WNV E protein (amino acids 296-415), which was generated in E. coli, was injected (6.25-200 nM, flow rate 70 µl/min for 1.5 min and then allowed to dissociate over 5 min). The F(ab)'2 surface was regenerated by pulse injection of 10 mM glycine (pH 1.5) and 100 mM NaOH before each E16 injection. We analyzed curves with a global fit 1:1 binding algorithm with drifting baseline.\nCloning and humanization of E16. E16 heavy-and light-chain RNA was isolated from hybridoma cells after guanidinium thiocyanate and phenol-chloroform extraction, and converted to cDNA by reverse transcription. The VH and VL segments were amplified by PCR using the 5′ rapid amplification of cDNA ends (RACE) system (Invitrogen). Gene-specific primers (GSP) for VH and VL were as follows: VH-GSP1: 5´-GGTCACTGTCACTGGCTCAGGG-3´; VH-GSP2: 5´-AGGCGGATCCAGGGGCCAGTGGATAGAC-3´; VL-GSP1: 5´-GCACACG ACTGAGGCACCTCCAGATG-3´; and VL-GSP2: 5´ CGGATCCGATGGATAC AGTTGGTGCAGCATC-3´. The PCR products were inserted into the plasmid pCR2.1-TOPO using the TopoTA kit (Invitrogen). We then subjected the resulting plasmids to DNA sequencing to determine the VH and VL sequences for E16. The cDNA sequences were translated and the predicted amino acid sequence determined. From these sequences the framework and CDR regions were identified as previously defined 42 . We joined the mouse VH to a human C-γ1 constant region and an Ig leader sequence, and inserted it into pCI-neo for mammalian expression. We joined the mouse VL to a human Cκ segment and an Ig leader sequence and also cloned it into pCI-neo for mammalian expression of chimeric E16 (Ch-E16). For Ch-E16, site-directed mutagenesis was also performed to change residue 297 from asparagine to glutamine of the heavy chain to eliminate the single glycosylation site on the γ1 Fc.\nHumanized E16 VH consists of the framework segments from the human germline VH1-18 VH segment and JH6 segment 43, 44 , and the CDR regions of the E16 VH, respectively. The humanized E16 VL consists of the framework segments of the human germline VK-B3 VL segment and JK2, (refs. 45-47) segment and the CDR regions of E16 VL. The humanized VH segments were assembled de novo from oligonucleotides and amplified by PCR. The humanized VL segments were assembled by PCR and overlapping PCR. The resulting VH and VL segments were subsequently combined by overlapping PCR with a leader sequence and the appropriate constant region segment and cloned into the expression vector pCI-neo as NheI-EcoRI fragments. We confirmed the DNA sequence of the resulting plasmids by sequence analysis. Site-directed mutagenesis was then performed to substitute mouse for human residues at key framework positions VH-71 (T71A) and VL-49 (Y49S). The resulting plasmids were cotransfected into human 293 cells using lipofectamine-2000 and humanized antibody was recovered from the resulting conditioned medium and purified by protein A and size-exclusion chromatography.\nStatistical analysis. All data were analyzed with Prism software (GraphPad Software). For survival analysis, Kaplan-Meier survival curves were analyzed by the log-rank and Mantel-Haenszel test. For viral burden experiments, we determined statistical significance using the Mann-Whitney test.\nNote: Supplementary information is available on the Nature Medicine website." }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: Local health departments are considered the backbone of public health response plans for any and all infectious disease outbreaks. An influenza pandemic is considered increasingly likely, and is now considered one of the most significant and urgent threats to the nation's public health preparedness infrastructure. It has been argued that of the 12 disaster scenarios recently assessed by the U.S. Department of Homeland Security, pandemic influenza is the most likely and perhaps the most deadly [1] . The United States pandemic influenza plan released in November 2005, lays out a critical role for local and state public health agencies during a pandemic, including: providing regular situational updates for providers; providing guidance on infection control measures for healthcare and non-healthcare settings; conducting or facilitating testing and investigation of pandemic influenza cases; and investigating and reporting special pandemic situations [2] . These specified activities would require an extensive prompt response by local health departments. Current contingency plans account for possible personnel shortages due to influenza morbidity, but previous studies have shown that during extreme scenarios, a varying proportion of healthcare workers may be unable or unwilling to report to duty [3] [4] [5] . This may be even truer for health departments, where unlike more \"traditional\" first responder agencies (such as law enforcement, fire services, and emergency medical services), the capacity and willingness to respond 24/7 to crises is not historically ingrained in the workforces' professional cultures and training. Even in the post-9/11 environment, recent data indicate inconsistent and sometimes slow after-hours response by health departments to urgent events involving communicable disease [6] .\nRisk perception theory provides a revealing framework for better understanding response limitations and needs of the public health workforce. The perceived risk, according to this theory, is a multifactorial phenomenon, involving the summation of actual risk and other peripheral influences independent of the actual risk, such as perceived authority, trust, and situational control; these peripheral influences have been termed \"outrage\" [7] or \"dread.\" [8] .\nBased on these models, it was previously suggested that contributing factors peripheral to the actual risk will have a considerable practical impact on how public health employees would respond in a crisis [9] . Aside from physical and circumstantial barriers such as availability of transportation or dependency of family members, we have identified specific risk perception issues whose impact may be markedly high and of unique importance for the public health workforce's response to a crisis. These factors, or modifiers, stem from a number of features previously suggested to have been associated with elevated risk perception, including manageability of the threat; risk to future generations; direct personal impact; and sense of control over events.\nBased on these modifiers, several major barriers to effective public health workforce emergency response were suggested; these include uncertainty regarding working environment safety, unclear expectations of role-specific emergency response requirements, safety and well being of family members, inadequate emphasis on the critical value of each employee to the agency response efforts, and insufficient emphasis on stress management techniquesall of which may heighten employees' sense of dread due to a lack of personal control [9] .\nIn light of the projected impact of an influenza pandemic, health departments must optimize the response rate of their employees in this crisis scenario. Based on the emer-gency response principle that all disasters are \"local\" [10] , we have set out to assess local public health employees' risk perception and likelihood of reporting to duty during a local outbreak of pandemic influenza, and to uncover the variables that affect these outcomes, thus providing a needed evidence base for health departments' planning and training efforts.\nWe conducted the study in Carroll, Dorchester, and Harford county health departments between March 2005 and July 2005. All three health departments are located in Maryland, and range in size from 132 employees to 225 employees. We selected these health departments because of their location in communities ranging from 30,000 on Maryland's Eastern Shore (Dorchester County) to 235,000 in the greater Baltimore/Towson metropolitan area (Harford County) [11] , thus reflecting the 96% of the nation's local public health agencies serving communities with populations of 500,000 or fewer [12] . Self-administered anonymous surveys were sent to all health department personnel by their respective health departments. Completed surveys were directly mailed to investigators at the Johns Hopkins Center for Public Health Preparedness.\nThe survey included questions on personal characteristics such as job classification, gender, and age. The respondents used a 5-point Likert scale for questions pertaining to a possible flu pandemic: probability of them reporting to work (\"very likely\" to \"not at all likely\"); possibility of being asked by their health department to respond to an emergency (\"very likely\" to \"not at all likely\"); how knowledgeable they thought they were about the potential public health impact of pandemic influenza (\"very knowledgeable\" to \"not at all knowledgeable\"); how confident they were about being safe in their work roles (\"very confident\" to \"not at all confident\"); how likely was their family prepared to function in their absence (\"very likely\" to \"not at all likely\"); how likely they felt their health department would provide them with timely updates (\"very likely\" to \"not at all likely\"); how familiar they were with their role specific response requirements (\"very familiar\" to \"not at all familiar\"); how well they thought they could address the questions of a concerned member of the public (\"very well\" to \"not at all\"); how significant a role they thought they would play in the agency's overall response (\"very significant\" to \"not at all significant\"); how important would be pre-event preparation and training (\"very important\" to \"not at all important\"); how important it was for them to have psychological support available during the event (\"very important\" to \"not at all important\"); and how important it was for them to have psychological support available after the event (\"very important\" to \"not at all important\").\nThe job classification variable was collapsed into technical/support staff (such as computer entry staff, clerical staff (e.g. receptionists), computer specialists, health information systems data analysts etc.), and professional staff. The latter included public health officials, clinical staff (e.g., nurse, dentist, physician), public health communicable disease staff, environmental health staff, public information staff, and other public health professional staff (e.g., health educator, legal professional, financial officer, other).\nWe dichotomized the responses to the job classification question into professional and technical/support categories. Questions about likelihood of reporting to work and pandemic influenza-related attitudes and beliefs were dichotomized into responses with a score two or less, and all other responses. We used logistic regression to compute Odds Ratios to evaluate the association of demographic variables and attitudes and beliefs with selfdescribed likelihood of reporting to work. We used multivariate logistic regression to explore associations between attitudes and beliefs related to pandemic influenza preparedness and self-described likelihood of reporting to work. The model included adjustment for age, gender, and job classification. Similarly, we used bivariate and multivariate (adjusted for age, gender, and job classification) logistic regression models to evaluate the association between the various attitudes and beliefs. In order to assess non-response bias, we compared age, gender, and job classification distributions for the respondents and for all health department personnel.\nWe used TeleForm Version 8 (Cardiff, Vista, CA) and Stata Version 9 (Stata Corporation, College Station, TX) for data capturing and analysis respectively.\nWe received 118 out of 205 (57.6%), 74 out of 128 (57.8%), and 116 out of 198 (58.6%) surveys fromCarroll, Dorchester, and Harford county health departments respectively, resulting in an overall response rate of 58.0% (n = 308). We did not find a statistically significant difference in age and gender distribution between the respondents and all health department personnel. A small yet statistically significant difference in the proportion of technical/support staff (vs. professional staff) was detected (22.4% vs. 32% in the study group and all personnel respectively, p = 0.003), yet no significant difference in the proportions of professional staff subgroups was detected.\nOf the 303 who responded to the question about their likelihood of reporting during a pandemic influenza related emergency, 163 (53.8%) indicated they would likely report to work during such an emergency. Age and gender did not have an association with likelihood of reporting. Clinical staff indicated a higher likelihood of reporting (Multivariate OR: 2.5; CI 1.3-4.7) than technical/support staff (Table 1) .\nOnly 40% of all respondents-45.1% professional staff and 26.1% technical/support staff -felt it was likely they would be asked by their health department to respond to a pandemic influenza related emergency. Perception of Table 2) . Perception of one's existing knowledge about pandemic influenza, and perception of having an important role in the agency's overall response were significantly higher among professional staff compared to technical/support staff (Figure 1 ), In multivariate analysis, increased self-described likelihood of reporting to work during an influenza pandemic emergency was significantly associated with agreement with several constructs, most notably perception of the capacity to communicate risk effectively, perception of the importance of one's role in the agency's overall response, and familiarity with one's role-specific response requirements in a pandemic influenza related emergency. ( Table 2 ).\nThe vast majority (83%) of the respondents felt they would benefit from additional training activities. A lower perceived level of familiarity with one's role was not significantly associated with a higher perceived need for additional training ( (Figure 2 ).\nThe World Health Organization has urged all countries to prepare for the next influenza pandemic, which it termed . The federally adopted U.S. model of all-hazards emergency readiness has presented local health departments with new organizational challenges and learning curves. The all-hazards approach entails an ability and willingness to respond to a broad spectrum of disasters, ranging from the intentional (e.g., chemical, biological, or radiological terror) to the naturally occurring (e.g., weather-related crises or non-bioterrorism related infectious disease) [14] .\nCurrent national contingency plans account for possible personnel shortages within the healthcare and public health settings, mainly due to the expected influenza morbidity among workers. Yet our data suggest that regardless of the expected morbidity among personnel during an influenza pandemic, nearly half of the local health department workers are likely not to report to duty during such an extreme public health crisis. In fact, most of the workers (and nearly three out of four technical/support workers) do not believe they will even be asked to report to work.\nWe have found that the willingness to report to duty during a pandemic varies considerably according to the individual's job classification. Clinical staff state they are significantly more likely to report to duty, compared with all other workers. This difference correlates well with the single most influential construct associated with willingness to report to duty -the perception of the importance of one's role in the agency's overall response. Less than a third of the respondents believed they will have an important role in the agency's response to local outbreaks of pandemic influenza, but within this subgroup, willingness to report to duty was as high as 86.8%. Belief in the importance of one's role was lowest among technical/support staff, environmental health staff, and other non-clinical professional staff (15.1%, 18.4% and 18.8% respectively), groups in which willingness to report was shown to be lowest. We therefore believe further efforts must be directed at ensuring that all local public health workers, but most notably non-clinical professional staff, understand in advance the importance of their role during an influenza pandemic -otherwise they will fail to show up when they are most needed.\nOur findings fit well in the theoretical framework emphasizing risk communication needs of public health workers, who themselves serve as risk communicators [9] . Several factors, previously suggested to be risk perception \"modifiers\" [9] of substantial impact on public health\nProportion of individuals who agreed with each of the attitude and belief constructs by staff type Figure 1 Proportion of individuals who agreed with each of the attitude and belief constructs by staff type. \nTechnical/Support Staff Professional Staff * * workforce's response to a crisis, indeed proved to be important in this context. Lack of knowledge, ambiguity regarding one's exact tasks, and questionable ability in performing one's role as risk communicator were all significantly associated with a higher perceived personal risk and a two-to ten-fold decrease in willingness to report to duty; these factors proved to be more influential even than the perceived level of family preparedness to function in one's absence. It is therefore important to recognize that public health employees, who are intended to serve as purveyors of risk communication for their communities, themselves represent a community with specific perceptions that must be addressed in the context of emergency readiness training.\nThe threat of an impending influenza pandemic is not a new one -pandemics have been taking place once every several decades for over 300 years. Yet it was only in the last couple of years, as highly pathogenic H5N1 strain became increasingly endemic in southeast Asia and as lethal infections with the virus occurred in an alarmingly increasing rate among humans, that the urgency of the situation was openly declared by national and international health authorities. The rapidity of this evolving situation may serve to explain why only one third of the respondents felt they were adequately knowledgeable on pan-demic influenza, and why only one in five respondents felt capable in effectively communicating pandemic risks. This finding is especially noteworthy, in that members of the public health support staff may become frontline telephone risk communicators in a crisis, serving as the first points of interface for concerned callers contacting a health department. Only one of the 35 technical/support staff workers who felt incapable of effective risk communication was willing to report to duty, even though most of them believed the health department will have the ability to provide timely information.\nThe study has some relevant limitations that must be factored into the overall analysis. First, the sample was limited to three non-randomly selected health departments, none of which serves a community larger than 250,000 residents, and all of which have staff sizes under 250. The sample size of 308 survey employees limited this study's power. As the study includes Maryland health departments only, it does not account for potential jurisdictional or regional variations nationwide in response capacity or risk perceptions toward pandemic influenza response. Furthermore, the job classifications -based on those used to develop the CDC-adopted emergency preparedness competencies [15] -do not necessarily map neatly onto functional responsibilities in disaster Odds Ratios of reporting to work in case of a pandemic-influenza-related emergency by staff and attitude or belief construct Figure 2 Odds Ratios of reporting to work in case of a pandemic-influenza-related emergency by staff and attitude or belief construct. \nTechnical/Support Staff Professional Staff response. For example, health educators may play as frontline a role as clinical staff, in terms of their degree of interface with the public in a disaster. Our job categories therefore do not necessarily reflect the relative impacts of job-specific cohorts on disaster response in the event that they do not report to work.\nWe assessed the presence and the direction of nonresponse bias by comparing the distribution of personal characteristics for the respondents and for all health department personnel. The lack of significant difference in age and gender distribution, as well as the lack of significant difference in job classification other than technical/ support staff indicates that the extent of such a bias in the study is probably limited. The small yet statistically significant over-representation of technical/support staff in our study group may potentially have caused a slight underestimation of overall willingness to report. However, as the internal associations between the various variables were also studied separately for the technical/support staff and professional staff (Figure 2) , this over-representation should not impact the general conclusions presented above.\nHaving accounted for these limitations, it is important to note that the findings were internally consistent among the three surveyed health departments. Although none of the health departments served large metropolitan areas and all had fewer than 250 employees, it must also be recognized that only 4% of the nation's local health departments serve populations of 500,000 or more, and that local public health agencies tend to have small staff sizes (with a median of 13 full time employees) [12] .\nInterestingly, our findings show similar patterns to data on the willingness of urban healthcare workers from nonpublic health settings to respond to emergencies: a survey of 6248 employees from 47 healthcare facilities in the New York City area revealed that these workers were least willing (48%) to report to duty during an untreatable naturally-occurring infectious disease outbreak affecting their facility (SARS), compared to other disaster scenarios [5] . In our study we have detected similar rates of likelihood to report to duty, although lower rates could have been expected in our study population since the New York City survey focused on healthcare workers whose organizational cultures are historically much more accustomed than that of local public health workers to emergency response, in a city with a heightened awareness of disaster preparedness in the wake of the World Trade Center attacks and subsequent anthrax attacks [5] .\nIn the face of a pandemic influenza threat, local health department employees' unwillingness to report to duty may pose a threat to the nation's emergency response infrastructure. Addressing the specific factors associated with this unwillingness is necessary to help ensure that existing local health department preparedness competencies [15] will translate into the scope of response described in the nation's pandemic influenza plans [2] . Interventions suggested to enhance the willingness of healthcare workers in non-public health department settings to report to duty in disasters include workforce preparedness education [5] , provision of appropriate personal protective equipment, [4, 14] crisis counseling, family preparedness and social support [5, 16] .\nThese recommendations fit well within the framework of our findings, and we further recommend that such education programs include specialized training emphasizing the specific nature of, and guidelines for, one's role in response to pandemic influenza; the relevance of each worker's role in the effectiveness of an overall public health response; and the workers' ability to provide effective risk communication. Additional research must further focus on best practice models for addressing the above described gaps in local public health response to this urgent public health threat.\nThese data offer a current, evidence-based window into the needs of public health workers who would serve as a backbone of locally-driven emergency response in an influenza pandemic setting. We found that most of these workers feel they will work under significant personal risk, in a scenario they are not adequately knowledgeable about, performing a role they are not sufficiently trained for, and believing this role does not have a significant impact on the agency's overall response. These specific perceptions and needs must be attended, and specific intervention programs must be initiated. In order to reduce the perceived risk associated with the worker's role in an influenza pandemic, each worker must have better understanding of the scenario and importance of his or her personal role within these settings, confidence that the agency will provide adequate protective equipment for its employees, psychological support and timely information, and a belief of being well-trained to cope with emergency responsibilities including the ability to communicate risk to others. In view of what is currently considered to be an impending influenza pandemic, a wide gap between these desired targets and current status exists, that may lead to significant hindrance in the ability of local health departments to function adequately." }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: In 2003, the world witnessed the spread of a novel and deadly virus, namely SARS CoV. The health care workers (HCWs) and hospital systems that bore the brunt of the SARS outbreak continue to struggle with the aftermath of the crisis. Indeed, HCWs -both in clinical care and in public health -were severely tested by SARS. Unprecedented demands were placed on their skills and expertise, and their personal commitment to their profession was severely tried. Many were exposed to serious risk of morbidity and mortality; indeed, approximately 30% of reported cases were among HCWs, some of whom died from the infection [1] .\nAs a number of commentators have noted, SARS exposed the vulnerabilities of our current health care systems and governance structures [2] [3] [4] . The aftermath of SARS and the spectre of pandemic avian influenza make imperative the need to consider the obligations of HCWs for patients with severe infectious diseases, particularly diseases that pose risks to those providing care. It is of pressing importance that organizations representing HCWs -professionals and non-professionals alike -give clear indication of what standard of care is expected of their members in the event of a pandemic.\nMany experts believe that the SARS outbreak was merely a preview of the next flu pandemic that is soon to arrive, possibly from an avian influenza virus [5] . Quite clearly, avian flu threatens to be more widespread than SARS, with the potential to become a truly global pandemic. An honest and critical examination of the role of HCWs during such a crisis is needed in order to provide guidelines regarding professional rights and responsibilities, as well as ethical duties and obligations [6] .\nIn this paper, we address the issue of special obligations of health care professionals (HCPs) during an infectious disease outbreak. We contend that there is a pressing need to clarify the rights and responsibilities of HCPs, especially in the current context of pandemic flu preparedness. Moreover, we argue that these rights and responsibilities would best be codified in professional codes of ethics. Finally, we present a brief historical account of the treatment of the duty to care in professional health care codes of ethics with the intention of opening the social dialogue and advancing the public debate on this increasingly relevant issue.\nGiven that the response by HCPs to the SARS crisis was generally regarded as exemplary, one might ask whether an ethical problem truly exists. There is little doubt that the vast majority of HCPs performed their jobs admirably under considerable stress and significant personal risk. Many HCPs provided exemplary care, and still others behaved in truly heroic fashion. So why, then, formally problematize something that is not a problem?\nAs noted, many HCPs acted in a supererogatory manner during the SARS outbreak [7], none more so than Dr. Carlo Urbani of the World Health Organization, who himself died of SARS after being exposed to the yet unknown virus in the course of carrying out his professional duties. Likewise, scores of nurses, doctors, respiratory technicians, and other professional and nonprofessional health workers laboured extremely long hours at personal risk. This demonstration of going above and beyond the call of duty, which proved necessary to control the disease, was highly morally commendable.\nAt the same time, however, serious concerns did surface during SARS about the extent to which HCPs would tolerate risks of infection [8, 9] . Some baulked at providing care to those infected with the unknown virus. In some circumstances, staffing became an issue in SARS wards and assessment centres; indeed, failure to report for duty during the outbreak resulted in the permanent dismissal of some hospital staff. As a consequence, the risk that was faced during SARS was not distributed equitably, and those HCPs who volunteered to provide care faced the greatest exposure.\nFollowing the outbreak, many of those who treated SARS patients raised concerns about the protections that were provided to safeguard their own health and that of their family members. Conflicting obligations were another significant concern. HCPs are bound by an ethic of care, therefore, obligations to the patient's well-being should be primary. At the same time, however, HCPs have competing obligations to their families and friends, whom they feared infecting, in addition to obligations to themselves and to their own health (particularly those with special vulnerabilities, such as a co-morbid condition). During SARS, some HCPs questioned their choice of career; indeed, some decided to leave their profession and pursue new ventures, indicating an unwillingness or inability to care for patients in the face of risk. Recent survey data from the U.S. indicate that there exists mixed views on the duty to care for patients during infectious disease outbreaks [10] .\nWhat is clear is this: the issue of duty to care has emerged as a matter of paramount concern among health care professionals, hospital administrators, public policy makers, and bioethicists [11] [12] [13] [14] .\nThe ethical foundations of the duty to provide care are grounded in several longstanding ethical principles. Foremost among these is the principle of beneficience, which recognizes and defines the special moral obligation on the part of HCPs to further the welfare of patients and to advance patients' well-being. In modern health care, it is commonly understood and generally accepted that the principle of beneficence constitutes a foundational principle of the patient-provider relationship [15] .\nFor the HCP in general, and for the physician in particular, there are a number of compelling reasons to provide care in the context of an infectious disease outbreak. Clark [12] has recently outlined three such reasons:\n1. The ability of physicians and health care professionals to provide care is greater than that of the public, thus increasing the obligation to provide care Although self-care and self-protection, as well as the care and protection of friends and family members, are acknowledged in pandemic plans, it is evident that the expertise of HCPs is an integral and principal component of the response to a pandemic. There is no other sector of society that can be legitimately expected to fulfil this role and to assume this level of risk.\nArguably, HCPs have consented to greater than average risk by their very choice of profession. While it may be granted that the risk of contracting an infectious disease was likely not a concern for a generation of prospective health care workers, any informed reading of the medical literature in the last 20 years has shown that infectious diseases remain ubiquitous and problematic -notwithstanding overly-optimistic statements regarding the future threat of infectious diseases. It is therefore not unreasonable to argue that HCPs were aware of the greater than average risks posed by their choice of profession.\nIn publicly-funded health care systems, such as those found in many Western societies, there is a strong claim for a social contract between the HCP and society. It is a reasonable and legitimate expectation by the public that HCPs will respond in an infectious disease emergency. Society has granted and permits professions to be self-regulating on the understanding that such a response would occur.\nOne of the characteristics of a self-regulating profession is the development of standards of practice, sometimes referred to as best practice guidelines. These standards are articulated in professional codes of ethics, which are developed on the basis of the fundamental principles and values of the particular profession, as is the case, for instance, with respect to the codes of ethics that were developed long ago in medicine and nursing. Indeed, the code of ethics has a long and respected tradition in the health professions and today most, if not all, the various health and social care professions have codes of ethics in place to provide guidance to their members.\nThe code of ethics is sometimes referred to as an instrument of \"soft law,\" owing to its non-legislative nature [16] . As such, in the health care professions, codes of ethics should be interpreted as guides for ethical reasoning and frameworks for the treatment of individual patients, rather than as substitutes for such reasoning or as an absolute mandate [17] . At the same time, a code that is too vague can render it ineffectual and irrelevant. In an era in which health care and technology are evolving at a rapid pace, efforts are necessary to ensure that codes of ethics remain current, practical, and concordant with public expectations.\nAn informative and comprehensible code of ethics has numerous tangible benefits. Perhaps the greatest benefit would be to dispel confusion and uncertainty for HCPs concerning their professional rights and responsibilities as regards the duty to care. Of course, a detailed treatment of the issue in professional codes of ethics would also serve to increase awareness and comfort levels, perhaps resulting in increased willingness to provide care in uncertain and risky conditions [18] . Additionally, codes guiding professional conduct may effectively serve as norms of standards recognizable and enforceable by law, acting as the foundation of legal obligations and decisions [16] .\nFinally, codes of ethics also serve as potent forms of symbolic communication to the public that is served by the professions. By making explicit the values that health care professions represent, professional codes of ethics can reassure the public that the trust invested in the professions is justified and legitimate, as is properly noted in the following excerpt from the College of Nurses of Ontario Practice Standard on Ethics:\n\"Nurses have a commitment to the nursing profession. Being a member of the profession brings with it the respect and trust of the public. To continue to deserve this respect, nurses have a duty to uphold the standards of the profession.... As members of a self-regulating profession, nurses also have a commitment to help regulate nursing to protect the public's right to quality nursing services. It is in the public's interest that the profession continue to regulate itself by developing and changing the methods of self-regulation to meet the changes in health care and society. Nurses have an obligation to participate in the effective evolution of self-regulation. Self-regulation is a privilege and each nurse is accountable for the responsibilities that accompany this privilege.\" [19] What do current codes of ethics say regarding duty to care during epidemics? It is of no small concern that many current professional codes of ethics fail to provide explicit guidance sufficient to set policy or assure the public in the event of an infectious disease outbreak. The Canadian Medical Association, for instance, released a revised Code of Ethics in 2004, one year after the SARS pandemic in which Canada was particularly affected [20] . Despite the seemingly fortuitous timing of the publication, the revised Code is, quite astoundingly, altogether silent on physicians' duty to care, which might be described as the first among equals of the myriad ethical dilemmas that emerged during the global outbreak.\nThe key revision in the 2004 edition of the CMA Code was the addition of the following item to the 'Fundamental Responsibilities' section: \"Consider the well-being of society in matters affecting health\" [20] . This addition, however, does little to address, in any substantively meaningful way, the duty to care obligations of HCPs in the context of an infectious disease outbreak. Does the addition of this responsibility obligate physicians to provide treatment even when doing so would put their own health in peril? The wording is too vague to be of any significant guidance in clinical practice.\nIn contrast, the American Medical Association (AMA) appears to have recognized the present need to address the issue of duty to care. In the wake of the 9/11 terrorist attack, the AMA has adopted several new ethics policies that focus specifically on the medical profession's obligations and responsibilities in the context of a public health emergency. The following passage is from the AMA policy document \"Physician Obligation in Disaster Preparedness and Response\" that was adopted in June 2004: \"National, regional, and local responses to epidemics, terrorist attacks, and other disasters require extensive involvement of physicians. Because of their commitment to care for the sick and injured, individual physicians have an obligation to provide urgent medical care during disasters. This ethical obligation holds even in the face of greater than usual risks to their own safety, health or life. The physician workforce, however, is not an unlimited resource; therefore, when participating in disaster responses, physicians should balance immediate benefits to individual patients with ability to care for patients in the future.\" [21] While the AMA has taken a step in the right direction by stating the obligations of its members, and it is to its great credit for initiating this process, it remains to be seen whether other national medical associations and other health care professions will follow suit and redress the silence of codes of ethics on the duty to provide care.\nTo some extent, codes of ethics can be seen as reflections of enduring professional values. At the same time, they are also clearly influenced by and are the product of historical circumstances. The CMA, for instance, previously included in its Code of Ethics a strongly worded statement explicitly addressing the obligations of physicians in infectious disease outbreaks. The 1922 version reads as follows: \"When pestilence prevails, it is their [physicians'] duty to face the danger, and to continue their labours for the alleviation of suffering, even at the jeopardy of their own lives\" [22] . This is the only appearance in the CMA Code of this type of strong categorical language regarding the professional duty to care. Interestingly, the specific text cited above appears for the first time in the revision following the 1919 influenza pandemic and then, conspicuously, disappears from the next revision released in 1926.\nThe AMA included the very same provision in its Code of Ethics from 1846 through until the 1970s when it was likewise excised. This marked professional retrenchment from a strong obligation to provide care -as reflected in current codes of ethics -is attracting increased interest of late. A number of explanations have been proposed by academic commentators. For instance, the retrenchment has been linked to the rise of government and corporate intrusions into medical practice [23] . Others, including Clark [12] , have pointed to an increasing general belief originating circa 1950 that infectious diseases had been vanquished. It is most likely the case that both these factors played a significant role in the observed retrenchment over time.\nIrrespective of the reasons underlying the current silence, there can be little doubt that infectious diseases are an increasing clinical reality in the developed world, and have long been a tremendous challenge in the developing world. For this reason alone, the continuing silence of codes of ethics is greatly problematic, both clinically and normatively.\nThere is no current consensus as to how explicitly and stringently the requirements for the duty to care should be stated [14] . In a 2003 survey of 1000 American physicians, respondents reported decidely mixed views on whether they would continue to care for patients in the event of an outbreak. Given that only a narrow majority of the surveyed physicians reported believing in a professional duty to treat patients in epidemics, the authors of the study concluded that there should be a reinforcement of \"the [medical] profession's ethical duty to treat\" in the event of a public health crisis [10] .\nThis call for the reinforcement of the duty to care echoes the 1922 CMA Code of Ethics that clearly stipulates that physicians have a duty to provide care, even at the jeopardy of their own lives. This statement may indeed be considered too strong and too categorical by many today. To require the provision of care even when doing so entails significant risk to the provider would appear to be demanding that all HCPs behave like \"supreme Samaritans\" [12] . Is this reasonable? Is this ethical? These remain open questions, but it is doubtful that all HCPs would adhere to such stringent obligations when faced with a SARS-like crisis. As Emmanuel [24] has instructively noted, the historical record of physicians is decidedly mixed in this regard; indeed, it was in response to the vocal and mounting opposition to treating seropositive patients at the height of the HIV/AIDS epidemic in the 1980s that the medical profession reconsidered the ongoing retrenchment of the duty to care. With the threat of a new epidemic, another round of full and open discourse is required as to whether the acceptable standard of professional engagement should occur at the level of \"supreme\", \"good\", or \"merely decent\" Samaritan [12] .\nThere is presently a need to address the professional duties of HCPs to their patients with the risks to the well-being of society, which may include family, friends, co-workers, and other patients, in addition to the population at large. This is a matter of balance. The content of current professional codes of ethics offers little guidance or reflection of consensus in the health care community. In the wake of SARS and with the current threat of avian influenza, it is clearly time for medicine, nursing, and other self-regulating health care professions to address the issue head on.\nA number of options are open to the professions. One option, which we have already argued is unacceptable, would be to remain silent. On the other extreme, codes of ethics could be revised with a strong emphasis on the professional obligation and duty to provide care during infectious disease emergencies; that is, assume a position leaning towards supererogation, or performing acts that are 'above the call' of duty [7] . Several other options exist between these two extremes. For instance, the codes could reflect a strong but limited duty to care, with the limits clearly specified. Alternatively, there could be a weak emphasis on duty to care -an option more sympathetic to the self-regarding concerns of HCPs [8] -although this may run the risk of dissolution of the generally high regard for the health care professions that exists in society today.\nWe maintain that, with respect to the duty to care, it is not acceptable for codes of ethics to be vague, ambiguous, or otherwise avoid explicit statements of position. This is particularly true in light of calls for additional protections for HCPs during infectious disease outbreaks, including a position statement to that effect issued by the Ontario Medical Association [25] . Such calls for danger pay and/or enhanced disability insurance could be justified if the professions expressed a strong commitment to the ethical duty to provide care during public health emergencies. In the absence of such commitment, however, any additional measures to protect and safeguard the well-being of HCPs would appear self-serving and misplaced.\nIn the current context of pandemic influenza planning, as with other public health emergencies, there is an acute need for strategies to encourage greater discussion and dialogue among all interested parties and stakeholders [26] . A first step would be for the professional colleges to create a forum to engage their memberships and encourage the exchange of views on the issue. Such an exchange could then inform the development of formal position statements on the duty to care during communicable disease outbreaks, as well as the development of clear and unambiguous guidelines regarding the professional rights and responsibilities and the ethical duties and obligations of HCPs during such outbreaks. Such statements ought to be made publicly available (e.g., prominently posted on the websites of professional health care colleges and associations) in order to encourage sustained dialogue on the issues raised by professional colleges. A next step would be to foster public debate and dialogue on the positions taken by the various health care professions. To promote this public debate, a working group at the University of Toronto Joint Centre for Bioethics has produced an ethical framework to guide preparedness planning for pandemic influenza, based in part on experiences and study of the SARS crisis [27] . The framework presents a 15-point, value-based ethical guide for pandemic planning, including the value of duty to care. This report has been made publicly available via the internet and the use of webcasting and electronic town hall meetings are being planned to facilitate an open exchange. It is of utmost importance to promote a public discourse on these issues and, most importantly, to give a voice to all those who would be directly affected by a communicable disease outbreak. Together, health care professionals and the general public should participate in discussions to determine whether and when it is legitimate for HCPs to eschew the duty to care in the face of personal risk.\nIn light of the recent experience of Canadian physicians, nurses, and other HCWs on the frontlines of the SARS outbreak, we submit that the Canadian health care community should lead the charge to address issues of duty to care and ethical obligations in times of public health emergencies. In place of open and honest discussion, we currently have vagueness and ambiguity. In our view, health care codes of ethics should speak specifically to this issue in order to guide professional behaviour during infectious disease outbreaks. Indeed, the time to address the ethical duty to provide care is at hand -before the arrival of the next public health emergency." }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: There is accruing evidence that green tea may have anticancer activity (1) , but the mechanisms for this action are poorly understood. Green tea is produced from the shrub Camellia sinensis (Fig. 1) ; leaves are dried but not fermented so that the green coloration attributed to polyphenols is retained. Commercially prepared green tea extracts contain $60% polyphenols (1) . These polyphenols are the source of bioflavonoids, which have strong antioxidant activity.\nThe major bioflavonoids in green tea are epicatechins. Like all bioflavonoids, the tea catechins have three hydrocarbon rings; hydroxyl molecules are found at the 3, 5, and 7 positions (Fig. 2) . The four major tea catechins are epicatechin (EC), EC 3-gallate (ECG), epigallocatechin (EGC) and EGC 3-gallate (EGCG). The relative proportions of EC, ECG, EGC and EGCG in non-decaffeinated green tea are 792 ± 3, 1702 ± 16, 1695 ± 1 and 8295 ± 92 mg 100 g À1 dry wt, respectively; corresponding proportions in non-decaffeinated black tea are 240 ± 1, 761 ± 4, 1116 ± 24 and 1199 ± 0.12 mg 100 g À1 dry wt (1) .\nEpicatechins have apparent activity against human cancer: they reportedly may promote apoptosis (2) (3) (4) (5) (6) , arrest metastasis by inhibiting metalloproteinases (7, 8) , impair angiogenesis (9, 10) and reverse multidrug resistance (11, 12) . Although all epicatechins except EC can potentially suppress cell proliferation (13) (14) (15) (16) (17) (18) , EGCG appears the most promising and is therefore under clinical investigation in chemoprevention trials (19) . However, given the wide range in physiologic potency of the different catechins, an exclusive focus on EGCG is probably short-sighted. EGCG is reportedly more effective than EGC in decreasing the intestinal absorption of cholesterol (20) and it is the most potent catechin inhibitor of HIV-1 reverse transcriptase (21) , but ECG has the strongest collagenase inhibitory effect (22) and the highest antioxidant potential (23) . By contrast, only EGC is a potent mediator of oxidative modification and an inhibitor of xanthine oxidase during hepatic catabolism of purines (24) .\nWe hypothesized that the in vitro anticancer action of the various catechins varies with the type and stage of malignancy. We tested this hypothesis by examining proliferation of catechin-treated cell lines derived from organ-confined or metastatic prostate cancer (CaP) and from moderately or poorly differentiated epithelial ovarian cancer (EOC). The goal was to obtain data that would be useful for developing chemopreventive and therapeutic clinical trials in patients with gender-specific and non-specific solid tumors.\nFour gender-specific human cancer cell lines were used. The HH870 androgen-receptor-negative CaP cell line was developed at Hoag Cancer Center, Newport Beach, CA, from an organ-confined primary tumor that had been resected from a 56-year-old, previously untreated Caucasian (25) . This tumor was Gleason Grade 3/4, with no evidence of vascular or perineural invasion or extracapsular extension (stage T2b). The DU145 metastatic CaP cell line (American Type Culture Collection line HTB-81) was derived from a brain lesion of 69-year-old male Caucasian. It is androgen insensitive and does not express prostate-specific antigen. Two EOC cell lines developed at Hoag Cancer Center were also used: HH639 was from a poorly differentiated clear cell, Grade 3 carcinoma in the omentum and left ovary of a 56year-old Caucasian female; HH450 was from moderately differentiated metastatic cells recovered from the abdominal fluid of a 52-year-old Asian female.\nAll four cell lines were cryopreserved in liquid nitrogen freezer at À70 o C. For recovery of cryopreserved cells, the vials were transferred to a 37 o C water bath for 15-30 s, further thawed at room temperature and then transferred to a 15 ml polypropylene tube with a Pasteur pipette. An aliquot of 9 ml of RPMI-9% fetal bovine serum (FBS) was added in drops. The cells were allowed to settle for 5 min and then centrifuged at 4 o C for 10 min at 300 g. Supernatant was removed, and cells were suspended in fresh RPMI, gently tapped and vortexed. Cell viability was monitored by 0.2% trypan blue dye exclusion, and cell count was determined using a hemocytometer. Cells recovered from cryovials were grown in RPMI-1640 with glutamine (Invitrogen, Carlsbad, CA) supplemented with 9% FBS, HEPES buffer, gentamycin (5 mg%) and fungizone (0.5 mg%), at 37 C in a humidified atmosphere of 5% CO 2 . Upon confluency, cells were detached with sterile EDTA-dextrose (137 mM sodium chloride, 5.4 mM potassium chloride, 5.6 mM dextrose, 0.54 mM ethylene diamine tetra acetate (EDTA), 7.1 mM sodium bicarbonate) at 37 C for 5-15 min (or $45 min for HH639), recovered with cold RPMI-1640-9% FBS and resuspended in the same medium. Use of trypsin was avoided for harvesting the cells. Cell viability and cell count were reassessed before cells were seeded in culture flasks.\nAll epicatechins used in this study (Fig. 2) with 50, 60 or 100 mM of each epicatechin or with no epicatechin (control) in RPMI-1640 with glutamine (Invitrogen), 9% FBS, 0.54% HEPES buffer, gentamycin (5 mg%) and fungizone (0.5 mg%).\nAll experiments used 25 ml sterile polystyrene tissue culture flasks with a vented blue plug seal cap (Beckton Dickinson, Franklin Lakes, NJ, Cat. No. 353107). Each flask contained stock solution with or without epicatechin in concentrations of 50 mM (five flasks for each epicatechin and five flasks for control) and 25, 75 and 100 mM (three flasks for each epicatechin and three flasks for control). Cells (0.25 · 10 6 ) suspended in 10 ml of the RPMI-1640-FBS solution described above were transferred to each flask and allowed to grow until control cells reached confluency. The cells were detached with sterile EDTA-dextrose at 37 C for 5 min, recovered with cold RPMI-1640-FBS medium and resuspended in the same medium.\nCells were counted using a hemocytometer; trypan blue dye exclusion was used to determine the number of viable versus dead cells. The interval between seeding and confluent growth of control cells was used to calculate the doubling time and the number of cell cycles. The 50% inhibitory concentration (IC50) of each catechin in each cell line was calculated using a software program (Microcal Origin Corp, OriginLab Corporation, Northampton, MA). The cells were photographed directly from the flask using light microscopy (Olympus IX-70, Japan).\nAnalyses of variance and Fisher's least significant difference (LSD) method were used for pairwise comparisons of values significant at the 0.05 level. \nOrgan-confined prostate cancer cell line HH870 and primary and metastatic epithelial ovarian cancer cell lines (HH450 and HH639) seeded (2.5 · 10 5 cells) in flasks with or without various concentrations (25, 50, 75 or 100 mM) of ECG or EGCG were photographed under a light microscope after the untreated control cells reached confluency (Fig. 3 ). Both ECG and EGCG significantly affected the density of each cell line at or above 75 mM. The decrease in cell density at higher concentrations is much pronounced for ECG than for EGCG, a finding significant considering recommendations of clinical trials with EGCG (19) .\nThe mean density or viable cell number (in millions) (n ¼5 per treatment) of different cell lines was examined with or without catechins (50 mM) (Fig. 4) . The cell density was measured when growth of untreated cells reached confluency. Statistical analysis by ANOVA as well as by pairwise comparison showed that both ECG and EGCG significantly affected the cell density. ECG decreased the cell density of prostate cancer cells DU145, HH870 and ovarian cancer cell line HH639 more potently than EGCG. But EGCG inhibited the growth of ovarian cancer cell line HH450 better than ECG, suggesting the need to determine relative efficacy of ECG and EGCG in clinical trials for different cancers.\nTumor Cell Doubling Time: ECG versus EGCG Figure 5 shows the influence of the four epicatechins on cell doubling time. ECG and/or EGCG prolonged the doubling (14) EGC and ECG inhibited the growth of a human lung cancer cell line, PC-9 cells as potently as did EGCG, but EC did not show significant growth inhibition. The mechanism of growth inhibition by EGCG was studied in relation to cell-cycle regulation. EGCG (50 and 100 mM) increased the percentages of cells in the G \nThe relative cytotoxicity (CTX) of ECG to carcinoma HSC-2 cells and normal HGF-2 fibroblasts cells from the human oral cavity, as compared with other polyphenols in tea, was evaluated. For the HSC-2 carcinoma cells, ECG, CG and EGCG grouped as highly toxic, EGC as moderately toxic, and C and EC as least toxic. For the HGF-2 fibroblasts, ECG and CG grouped as highly toxic, EGCG as moderately toxic, and EGC, C and EC as least toxic. The CTX effects of the polyphenols were more pronounced to the carcinoma, than to the normal, cells. Table 1 summarizes the effects of EC, ECG, EGC and EGCG on viability, doubling time and cycling of the four cell lines. Untreated cells from each line reached confluency in about 2.5 cell cycles. EC did not affect the proliferation of DU145, HH870 or HH450 cells but it reduced the proliferation of HH639 cells by half (P < 0.05) and prevented their confluent growth (Table 1) . EGC did not affect the proliferation of any cell line (Table 1) , whereas EGCG arrested proliferation of all four lines. ECG, followed by EGCG, was the most potent inhibitor of cell growth and cycling.\nProliferation of each cell line (n ¼ 3 per treatment) was monitored with or without ECG or EGCG at concentrations of 0, 25, 50, 75 and 100 mM. The dosimetric results plotted in Fig. 6 shows concentration-dependent suppression of cell growth by ECG and EGCG. The suppressive effect on cell density was striking at higher concentrations of ECG and EGCG. ECG was a more potent inhibitor of cell growth than EGCG. At 25 mM of EGCG, cell numbers for HH870 and DU145 were significantly higher than control values. Based on the results plotted in Fig. 6 , IC50 values were calculated. The IC50 values are 24-30 mM for ECG, versus 42-89 mM for EGCG (Table 2 ). ECG suppressed growth at all higher concentrations tested (Fig. 6) , whereas EGCG significantly (P < 0.05) enhanced proliferation of CaP cells at 25 mM, a finding relevant to chemoprevention trials with EGCG only.\nGreen tea is widely consumed in Japan and China and its polyphenolic components have a chemopreventive effect against cancer in vitro and in vivo (39) . A cup of green tea contains 100-150 mg catechins, of which 8% are EC, 15% are EGC, 15% are ECG and 50% are EGCG (40) . Although numerous investigations have shown the role of EGCG in cancer chemoprevention, only a few studies have attempted to compare the relative antitumor efficacy of all four catechins (Table 3) . When we used a systematic approach to assess the effect of various catechins on cell lines derived from gender-based cancers, we found that each catechin's antitumor activity depended on the type of tumor. EGCG was not always the most potent chemopreventive agent.\nMost of the earlier literature (Table 3) indicates that EGCG is the most potent growth inhibitor of cell lines from glioblastoma, melanoma and cancers of the breast, colon, lung, prostate (androgen-receptor-positive), pancreas, liver and mouth. EGCG prevents proliferation of DU145 cells by arresting the cell cycle at G 0 /G 1 -phase (19) . Gupta and others (26) have documented that G 0 /G 1 -phase arrest is independent of p53 mutation, and EGCG treatment of DU145 induces the cyclin kinase inhibitor WAF1/p21. These observations suggest that EGCG imposes a cell-cycle checkpoint (19) . However, our results showed that ECG may be more potent than EGCG for inhibition of primary and metastatic CaP and EOC cells (Fig. 4, Tables 1 and 2) . ECG significantly reduced cell proliferation (Table 1, Figs 2 and 3) and increased mean doubling time (Table 1, Fig. 4) .\nThe in vitro effect of chemopreventive agents can be studied when tumor cells are in a matrix (1, 4, 27) or in a suspension (28, 29) . We used the suspension method because it exposes the entire cell surface to the chemotherapeutic agent. Our findings confirm an earlier report that used the matrix method to show that ECG is more potent than EGCG in suppressing the proliferation of DU145 CaP cells (4) . Thus reported differences in the relative efficacy of different catechins may not be due to differences in methodology.\nNot all tumor cells are killed by catechins. In our study, ECG (50 mM) induced death of most but not all HH639 cells. Doubling ECG's IC50 concentration might increase the tumor kill rate if ECG does not epimerize to CG. Our in vitro dose of 100 mM is equivalent to 29 mg (EC/EGC) to 45 mg (EGCG/ ECG), far less than the 100-150 mg (50% of which is EGCG) in one cup of green tea. However, Lee et al. (41) reported that plasma levels of EGCG and EGC in healthy volunteers increased to 78 and 223 ng ml À1 , respectively, 20 min after drinking brewed green tea (1.2 g of tea solids in 200 ml hot water). This suggests that drinking more than 10 cups of green tea may be necessary to maintain a plasma concentration of EGCG equivalent to that used in vitro by a dose of 50 mM or 22.5 mg. Kaegi (42) suggested a daily intake of 13 cups of green tea as a chemopreventive measure. Because this level of tea consumption is impractically high, chemoprevention of cancer with catechins may require administration of the appropriate catechin in a purified form.\nIn conclusion it may be stated that both green and black tea polyphenols are important components of antitumor aspect of complementary and alternative medicine (CAM), which play a significant role in the American health care system and in patients who suffer from chronic problems (43) . While green tea catechin gallates such as EGCG and ECG possess potent antitumor activities, their epimers, commonly found in black tea, act as potent inhibitor of proteases involved in replication of viruses, including coronoviruses (44) . There is a need to understand preventive and therapeutic potential of catechin gallates from both green and black teas. We are currently designing a phase I chemopreventive study to examine the effects of purified EGCG and ECG in patients who have been chosen observational management of organ-confined prostate cancer." }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: Chronic obstructive pulmonary disease (COPD) is a respiratory disease characterized by an airflow limitation and inflammation of the lower airways [1] . As the disease worsens, some patients experience 'exacerbations' of their principal symptoms of dyspnoea, cough and sputum. These exacerbations frequently result in a visit to a general practitioner's office or to a local hospital for treatment. Exacerbations occur in COPD patients at a median of three times a year with half of them being unreported [2] [3] [4] . The heterogeneity of COPD exacerbations make them difficult to define, classify and manage due to their range of symptoms, varied treatment requirements, seasonal occurrence, and ambiguous aetiology [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] .\nTo address this problem, attempts have been made to develop a consensus definition for COPD exacerbations [15] . Recently, the American Thoracic Society (ATS) and European Respiratory Society (ERS) adopted the following definition: 'an event in the natural course of the disease that is characterised by a change in the patient's baseline dyspnea, cough and sputum beyond day-to-day variability sufficient to warrant a change in management' [1] .\nThe severity of an exacerbation has been also difficult to classify despite the various schemes that have been proposed to deal with this issue [4, [15] [16] [17] . The ATS and ERS have also jointly suggested a classification based upon severity and the type of medical management used, i.e., Exacerbation Level I is home treatment, Level II is hospitalization, and Level III is specialised care [1] . The aim of this scheme is to improve the existing management of exacerbations and to serve as an aid in the assessment of treatment efficacy. Different operational definitions for COPD exacerbations have been proposed in the past and these have helped determine their relative importance, in particular their relationship to COPD progression [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] . However, these definitions have relied primarily on symptoms, and this along with the absence of a standard classification for the degree of symptom severity, has delayed the development of new therapies for this condition. The current therapies for exacerbations have been evaluated based on their ability to reduce symptoms, and to improve a patient's forced expiratory volume in one second (FEV 1 ) since the latter is strongly correlated with COPD mortality. However, FEV 1 does not discriminate well between the stable and exacerbative states of COPD, particularly during the later stages of this disease. Hence, the development of biological markers, or biomarkers that are more sensitive and specific to the severity of COPD exacerbations would provide investigators with new insights and directions for further research.\nAt this time, only a few clinical variables or inflammatory mediators have been shown to be associated with COPD exacerbations and their related morbidity and mortality. Some of those include: age [18] [19] [20] ; FEV 1 , forced vital capacity and peak expired flow [19, 21, 22] ; body mass index [20] ; albumin [20, 22, 23] ; sodium [23] ; pH [24, 25] ; eosinophils [26] [27] [28] [29] ; interleukins 6 and 8 [29] [30] [31] [32] ; fibrinogen [31] ; and C-reactive protein [33] . Significant clinical events such as the number of exacerbations per year, the number of hospital admissions per year, time to relapses, and days in hospital have been regarded as useful measures in clinical studies designed to assess drug efficacy and cost-effectiveness as well as to standardize existing hospital support programs for COPD [34] [35] [36] [37] [38] [39] . However, it is not known how these measures change with increasing severity of COPD exacerbations. Therefore, we have surveyed the medical literature to identify which of the commonly accepted variables and suggested markers for COPD exacerbations change according to the ATS/ERS' levels of exacerbation severity. The long-term aim of our work is to assess the sensitivity and specificity of potential markers for use in future COPD studies as well as to determine how such markers can be further studied and fully integrated into the development of new drugs for COPD.\nWe searched standard databases since 1966 using medical search headings and related terms as obtained from major consensus documents related to COPD exacerbations. The major keywords were 'exacerbation', 'unstable', 'acute', 'bronchitis', and variants of the term 'COPD'. This phase of our search retrieved a total of 843 citations. For these citations, we read the title and abstract of each citation so as to exclude citations that concerned exacerbations of coronary artery disease, myocardial infarction, cystic fibrosis, asthma, pulmonary emboli, and community pneumonia. Citations for case studies, letters, reviews, meta-analyses, and animal studies were also excluded.\nAfter this initial screening, we identified 387 citations to papers that were of possible interest. We retrieved the original articles in electronic and hard copy forms, and then critically read each article. As a result of this step, we arrived at a total of 268 studies in our final review and analyses. We selected these studies based on the availability of demographics, spirometry, clear study eligibility criteria, and the potential markers being used to assess exacerbations.\nThe objectives of this literature review and data analyses were to determine which of the baseline measures com-monly used in COPD exacerbation studies change with the extent of the exacerbation and disease severity, and to determine whether COPD exacerbations can be modelled as 'events' or 'time-to-event' in future investigations.\nInitially, we considered various exacerbation definitions and classification schemes, in particular, those suggested by Rodriguez-Roisin [15] as well as those described by Pauwels and colleagues [17] . However, we determined that the ATS/ERS' operational classification of exacerbation severity [1] was the most sensible and feasible system for systematically assessing the patient baseline characteristics and biomarker information from the majority of published studies. We therefore used this classification scheme and the related clinical history, physical findings and diagnostic procedures for managing exacerbations to perform our data abstraction. From each study, we retrieved the reported demographics, spirometry, smoking status, clinical, cytological and biochemical variables as well as suggested markers of the severity of the exacerbation at baseline conditions, i.e., immediately prior to, or during the exacerbation event but before the time in which the intervention of interest was investigated (Table 1 ). Whenever such variables were measured in stable conditions, we also abstracted this information. For each study, we noted the type of definition used to define an exacerbation such as symptom-or event-based as well as the research question asked, the experimental design used, any sponsorship, and the presence or absence of data from individual study patients. Data was then further organized according to sample size and smoking status when available. Cytological and biochemical data were also classified according to their collection methods. These included sputum induction, bronchial biopsy, bronchoalveolar lavage (BAL), exhaled breath sampling, and blood sampling.\nWe were also aware of the possibility that for some study groups in severity Levels II and III (as per the ATS/ERS criteria) included in this review may have experienced a similar quality of care or medical management that was not reported adequately in the original publication. In attempt to correct for this problem, we combined the exacerbation data from Levels II and III into an 'in-patient' category and then compared it to Level I that we regarded as the 'out-patient' category.\nWe collected and calculated study means, medians, standard errors, standard deviations, 95% confidence interval, and inter-quartile ranges using the statistical algorithms in Microsoft Excel 2002. We then conducted fixed effect meta-analyses to obtain mean point estimates, 95% confidence intervals, and two standard deviations for each exacerbation level [40] . Exacerbation Severity Levels I and II, II and III, and I and III were each compared using a twotailed Z-test. The alpha level of p < 0.05 was adjusted for multiple testing according to the Bonferroni correction procedure [41] . In the event that a specific exacerbation severity level had a large number of studies in which only median data were available, the data were considered to be normally distributed and medians were treated as means. Since many studies did not publish data for individual patients, we were limited in addressing non-normality in the data by using a log 10 -transformation.\nWe again performed a fixed effect meta-analysis to obtain mean point estimates, 95% confidence intervals, and two standard deviations for in-patient and out-patient categories of each measure. We then compared each category using a two-tailed Z-test and a p-value of 0.05.\nOur search strategy yielded 268 suitable studies that met our selection criteria. These studies were published between 1979 and July 2004 -Week 2. (The references for these studies can be found at the LACDR Division of Pharmacology website [42] ). The total number of study subjects included in this review was 142,407. Of this group, 18% fell in Exacerbation Severity Level I, 78% in Level II, and 4% in Level III. When we re-analysed the data according to out-or in-patient settings, 18% were out-patients and 82% in-patients.\nMeta-analyses of typical study demographics showed that there was significant overlap in 95% confidence intervals and study data distributions for the three exacerbation severity levels except for age where study patients in Level II had a mean age of 64.2 years (95% confidence interval (CI): 62.9 to 65.5 years) compared to 68.0 years (95% CI: 65.9 to 70.1 years) for patients in Level III (p = 0.002) ( Table 2 ). When the demographics were re-analyzed according to patient settings, we determined that only body mass index was statistically different between the out-patient setting (mean point estimate: 26.2 kg/m 2 ; 95% CI: 23.8 to 28.7 kg/m 2 ) and the in-patient setting (mean point estimate: 23.4 kg/m 2 ; 95% CI: 22.5 to 24.3 kg/m 2 ) (p = 0.038) ( Table 3) .\nThe spirometry measures Forced Expired Volume in 1 Second (FEV 1 ) and Forced Vital Capacity (FVC), both in percent predicted, decreased from Exacerbation Levels I to II (p < 0.017) but remained unchanged from Levels II to III ( Figure 1A and 1C, respectively). However, when Levels II and III were combined to create an 'in-patient' category for each of these variables, there was a statistically signifi-Respiratory Research 2006, 7:74 http://respiratory-research.com/content/7/1/74 cant decrease for the in-patients versus the out-patients (p < 0.05) ( Figure 1B and 1D, respectively). We also observed the same trend for FEV 1 /FVC (Figure 2A and 2B ). For all other spirometry measures, there were too few studies available in Level III for meta-analysis.\nWe found for smoking that pack years increased with exacerbation severity, but only Levels I and II were statistically different (p = 0.015) ( Figure 2C ). When we compared pack years between patient settings, it was statistically higher for the in-patients than the out-patients (p = 0.010) ( Figure 2D ).\nIn terms of the hemodynamic measures, only heart rate showed a statistically significant difference being higher in Level II than Level I (p = 0.014) with no difference Many study variables were measured at or around the time of the exacerbation. If these variables were measured in the stable condition of these COPD patients, i.e., measurements were taken weeks or months prior to the exacerbation, then these were also obtained.\nbetween Levels II and III ( Figure 3A ). Heart rates were also higher for in-patients than out-patients (p = 0.011) (Figure 3B ).\nThe clinical measures of dyspnoea, i.e., the breathing rate ( Figure 3C ) and Borg dyspnoea score, tended to increase from Levels I to II and then decrease from Levels II to III. However, only breathing rate demonstrated clear statistical differences between the three levels (p < 0.017). Only Levels II and III of the Borg Dyspnoea Score were statistically different (p < 0.001); a statistical comparison of these levels with Level I was not possible due to lack of data. When patient settings were compared, only breathing rate showed a clear statistical difference being statistically lower for in-patients than out-patients (p = 0.003) ( Figure 3D ).\nExacerbation Levels II and III were statistically different with respect to pH (p = 0.003) and bicarbonate (p = 0.002) in that pH decreased from Level II to III whereas bicarbonate increased. However, there was insufficient Level I data for each variable to allow for statistical comparisons with the other Levels. There was also insufficient data available to compare out-patients with in-patients.\nIn terms of blood gas measures studied, only arterial carbon dioxide tension (PaCO 2 ) showed a statistically significant increase with increasing exacerbation severity (p < 0.017) ( Figure 4A ) as well as out-versus in-patients (p < 0.05) ( Figure 4B ). In the case of oxygen saturation, it gradually decreased with increasing exacerbation severity with statistically significant differences between Levels I and II (p < 0.001) as well as Levels I and III (p = 0.011) ( Figure 4C ). It also decreased going from an out-patient to an inpatient setting (P < 0.001) ( Figure 4D ).\nThe six minute walking distance challenge test seemed to show a decreasing trend with increasing exacerbation severity but such changes did not reach statistical significance. This was also the case when the out-and in-patients were compared. Many other variables related to spirometry, respiratory status, exacerbation and hospital event categories also did not change significantly with exacerbation severity or out-and in-patients (See additional file 1). There was not enough data in the bacteriology and virology categories to permit any meta-analyses. Of the 268 studies sampled, only half contained data about the biochemical variables.\nWe conducted this review of the COPD exacerbation literature to determine which commonly-accepted baseline variables and suggested markers changed in a consistent manner with the severity of COPD exacerbations. As our index of COPD severity, we used the recently published ATS/ERS operational classification of exacerbation severity for medical management. This is because most of the published literature rarely provides sufficient details to characterise the severity of a patient's exacerbation. In addition, we also analyzed the same data according to out-and in-patient settings so as to account for possible overlaps in medical management between Levels II and III but were not reported in the original publication.\nThe long-term aim of our work is to improve the quality and applicability of exacerbation management through the identification of sensitive and specific markers that can be used for the assessment of treatment effects. This review identified a few potential markers of exacerbation severity.\nWhen we assessed the spirometry measures FEV 1 and FVC in % predicted, as well as FEV 1 /FVC, we observed statistically significant differences with exacerbation severity, and between out-and in-patients ( Figures 1A-D and 2A -B). One draw-back was the paucity of such information in Level III studies. This confirms the clinical situation that as exacerbations worsen and more specialised care is required, spirometry measurements are less likely under baseline conditions or during an exacerbation [14] . Thus, such data is rare in many published studies.\nThe number of smoking-related pack years increased with exacerbation severity and showed a clear difference between out-and in-patient settings ( Figures 2C and 2D) , a finding that is consistent with the idea that the more a COPD patient smokes, and for longer, the higher the likelihood that COPD exacerbations will be more severe.\nAccording to the mean point estimates obtained in this In-patient (N=81;n=5768) PaCO2 (mmHg) * B study, COPD patients with 40 to 60 pack-years of smoking will experience an increase in the severity of COPD exacerbations. However, our conclusion regarding this finding is limited by there being data from only two studies at Level III.\nAlthough heart rate varied little between Exacerbation Levels II to III, it is important to note that it was substantially elevated in patients ( Figure 3A ) with the clearest difference being between in-and out-patients. This is possibly associated with the anxiety and dyspnea that experienced when an exacerbation occurs. The increase in heart rate of course increases the oxygen requirements of the heart. The increased heart rate may also be the result of underlying cardiovascular disease that is more prominent in severe COPD patients [43] .\nThe relationship of pH and bicarbonate to exacerbation severity are consistent with the signs of respiratory acidosis evident in COPD patients with exacerbations [1, 24, 25] . However, due to the shortage of data in Level I, proper statistical conclusions about each of these variables are difficult to make. In relation to this, breathing rate significantly increased from Levels I to II and then decreased from Levels II to III ( Figure 3C ). The first observation may reflect components of the exacerbation episode (i.e., anxiety and dyspnea) as well as the physiological need to breathe more to maintain adequate blood gas levels. The reduction at Level III possibly reflects the results of the specialized care where patients are given ventilatory support so as to return the breathing rate to normal. The Borg Dyspnea Score showed the same trend as breathing rate, although insufficient data in Level I did not allow for further comparisons. When out-and inpatient data were compared for each of these variables, only breathing rate demonstrated a clear statistical difference ( Figure 3D ). The Borg Dyspnoea Score on the other hand did not have enough studies in the out-patient category to perform any statistical test. Overall, the observed trends were consistent with the fact that management of dyspnoea is one of the main factors generating the high hospital costs associated with COPD exacerbations [44] . In keeping with the direct measures of dyspnoea, arterial carbon dioxide tension showed a clear relationship with exacerbation severity and patient management settings ( Figures 4A and 4B ) that is consistent with the conclusions reported in the medical literature [20, [45] [46] [47] . Arterial oxygen tension in contrast did not change with exacerbation severity or patient setting. Possibly this lack of correlation reflects the immediate administration of supplemental oxygen given to hypoxaemic patients in a hospital setting. There was however a decreasing trend in oxygen saturation with increasing exacerbation severity and clear differences between out-and in-patient settings ( Figures 4C and 4D ) that are consistent with the present thinking on blood gas changes.\nMost of the other commonly accepted measures and suggested biomarkers poorly reflected exacerbation severity, or the fact that there was not sufficient data to undertake a meta-analysis (See additional file 1). This finding recalls a 2001 US Department of Health and Human Services report on exacerbation treatment outcomes from over 200 randomised controlled trials [14] . The aim of that study was to create new guidelines to improve the management of COPD exacerbations. That study also concluded that the current literature was limited in terms of the number of studies and the amount of detail available as well as the reliability and accuracy of the clinical assessments used to discriminate between COPD exacerbations and other causes of worsening respiratory status. Thus, our observations agree with previous observations regarding the assessment of the unstable COPD literature.\nAs previously discussed, most of the studies used for this review were predominately with hospitalized patients (Level II). However, most COPD occurs in an out-patient setting (Level I) [48] [49] [50] [51] [52] [53] . This has implications for our study since the latter population was poorly represented.\nOur basic categorisation was according to the ATS/ERS' operational scheme for classifying the severity of COPD exacerbations as well as to out-and in-patient categories. To our knowledge, we are the first to undertake this type of literature review and thus we were faced with a lack of consistency in the definition of exacerbations as used in the various studies. We tried to overcome this difficulty by selecting and ranking clinical studies so as to improve the comparability of subjects between studies.\nWe were also aware that the clinical studies we analysed differed with respect to which comorbidities or identifiable causes for exacerbations were reported. Most patients were elderly and therefore were more likely to be suffering from one or more co-existing diseases such as asthma or cardiovascular disease. Such co-morbidity makes interpretation of our findings more difficult with respect to the true causes of exacerbations. If their aetiology could be determined, then susceptible patients such as those in Level I could be identified and new treatments developed to help prevent their onset and related hospital costs.\nFinally, the compatibility between the studies of COPD exacerbation that we analysed may have been limited by substantial variations in the time and location of studies. Exacerbations are more likely in summer [5] but many studies failed to report the time of year or the time period for study implementation. Thus, seasonal effects, combined with the low incidence of exacerbations per patient, could represent an inherent bias. In addition, different institutions probably had different standards with respect to diagnosis and management of COPD exacerbations when these studies were performed. Such variations may also explain any observed inconsistencies in our findings. However, we attempted to overcome this possible bias in Exacerbation Levels II and III by the subsequent re-analysis of this data on the basis of out-patient and in-patient settings.\nAs observed in The additional online file, there was a scarcity of information particularly for biomarkers at different exacerbation levels. It is also unclear to us whether any of the variables that changed with exacerbation severity are causally-related. Hence, longitudinal studies and/or less restrictive eligibility criteria would be needed to address all these questions. One difficulty in tackling such problems is the enormous amount of time and expense involved in implementing such studies. In addition, the current methods for data analysis in clinical studies have limitations imposed by the assessment of the reduction in frequency or total suppression of exacerbation episodes (i.e. rare event or \"non-event\").\nTo overcome these drawbacks and obtain more accurate evaluation of treatment effect on COPD exacerbations, alternative analytical methods based, for example, on predictive mathematical models such as hidden Markov chains or Bayesian forecasting should be tried. Such models can characterise and predict rare events without undertaking a full-scale, long-term longitudinal study. This approach to predicting rare events has been used previously in studies of migraine, epilepsy and various cardiovascular diseases where the size of treatment effect is measured in terms of a reduction in the frequency of the repetition of an event within a given probability or within a given time period [54, 55] . One example of a mathematical model development includes the use of a Markov model to predict COPD exacerbation rates in a clinical trial of the inhaled anticholinergic bronchodilator tiotropium [56] . In this example, the model was developed on the basis of prior knowledge of the exacerbation rate as estimated from meta-analyses of randomised controlled trial data. This gave the probabilities for COPD exacerbations for different stages of COPD. In another study, a proportional hazards model was used to identify risk factors for COPD patients hospitalised due to an exacerbation [44] . The current ATS/ERS guidelines for exacerbations do not consider the implications of using probabilistic models as a means of assessing the severity of COPD exacerbations or the effect of treatment [1] . A modelling approach may offer new insights into which variables related to COPD exacerbations should be investigated.\nFrom a research planning perspective, our study findings have generated some hypotheses and related considerations that could be evaluated in future clinical trials. One hypothesis is that the combination of variables that we observed to change in our study (i.e., FEV 1 , FVC, FEV 1 / FVC, arterial carbon dioxide, breathing rate, heart rate, pack years, and oxygen saturation) could represent a new definition for a 'severe' exacerbation event. Most definitions in the literature, including the recent ATS/ERS definition, do not indicate any assessment of (patho)physiological variables as signs of an exacerbation. They simply regard the exacerbation as a worsening of the normal day-to-day symptoms and/or an adjustment in medical management [17] . A definition that encompasses a clear set of objective measures would be useful to medical practitioners who predominantly rely on clinical judgement or past experiences for diagnosing an exacerbation and its severity as well as for assessing treatment effect.\nAnother important consideration for future clinical trials is the assessment of treatment effect based on predictions of exacerbation frequency and intensity. In other words, the collection of data such as the rate of onset and resolution of an exacerbation from longitudinal studies could be used to determine probabilities of second, third, fourth, etc., exacerbation events in individual patients [54] . The alteration of such probabilities with an experimental treatment could be a more sensitive and reliable approach for assessing treatment effect in clinical trials than recording daily changes in symptoms or medical management.\nLastly, our findings were obtained from COPD patients that had experienced at least one exacerbation during the study assessment period. In the same studies, there were also patients who did not experience an exacerbation. This indicates that a fraction of COPD patients may be regarded as being susceptible to an exacerbation whereas another fraction is 'exacerbation-free'. It would be interesting to determine how the variables we identified in our study change in the latter patient group according to FEV 1 . Some published studies have stratified COPD patients on the basis of exacerbation frequency; this is generally done by categorising patients as having either 'infrequent' or 'frequent' exacerbations if they had less than or greater than a mean of three exacerbations per year, respectively [57] . In our study, we were unable to make this distinction between COPD patients since many of the published studies did not provide individual patient data on exacerbation frequency. We are currently investigating a commercial database of clinical trials that will enable us to look at patients with 'infrequent' or 'frequent' exacerbations. The results of this work could help us better select patients as well as identify potential markers for future longitudinal studies.\nThe current management and treatment of COPD exacerbations is primarily dependent on the evaluation of the symptoms rather than the signs related to the exacerbation event. We found that arterial carbon dioxide tension and breathing rate consistently varied with the severity of COPD exacerbations and with in-versus out-patients. Other commonly-accepted measures and suggested biomarkers for exacerbations failed to show consistent trends or lacked sufficient data to permit any meta-analysis. We recommend the design of longitudinal studies looking at the frequency of exacerbations as well as the use of more advanced modelling techniques to improve the selection of potential markers for the categorization of the severity of COPD exacerbations and the assessment of treatment effect in future studies.\nCOPD -Chronic Obstructive Pulmonary Disease " }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

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[ { "from": "human", "value": "Full Text: LEARNING OBJECTIVES: Recognize that the presenting features of amyloidosis are protean and depend on the organ systems involved. Recognize that cardiac involvement is common in primary amyloidosis and that symptomatic heart involvement carries a poor prognosis. CASE INFORMATION: 66 year old man without PMH presented 8 months of progressive exertional dyspnea and fatigue. Evaluation revealed volume overload with anasarca and signs of biventricular heart failure. Laboratory studies revealed renal insufficiency with a creatinine of 1.8 and 2+ proteinuria. serum protein electrophoresis with immunofixation showed a monoclonal IgA kappa paraprotein; urine protein electrophoresis was unremarkable. Echocardiogram revealed an ejection fraction of 20%. Right ventricular function was moderately reduced and right and left ventricular hypertrophy was noted. ECG showed atrial fibrillation with a ventricular response of 105 BPM and low voltage. Pt. underwent right heart catheterization with endomyocardial biopsy. Tissue stained positive with crystal violet and Congo red with characteristic green birefringence under polarization microscopy. This result together with the paraprotein established the diagnosis of AL amyloidosis. IMPLICATIONS/DISCUSSION: Amyloidosis is a disease characterized by extracellular accumulation of insoluble protein fibrils in various organs and tissues. Classification of amyloidosis is based on the nature of the precursor proteins that form the fibril deposits. AL amyloidosis (primary) is a plasma cell dyscrasia in which clonal plasma cells in the bone marrow produce monoclonal immunoglobulin light chains. Clinical manifestations reflect the organ systems involved with kidney and heart involvement being most common. Clinically evident cardiac involvement occurs in up to 50% of patients with AL amyloid and often leads to significant cardiac dysfunction. Diastolic dysfunction is caused by diffuse infiltration of the left ventricular myocardium with resultant stiffening and impaired relaxation and usually precedes systolic dysfunction. Systolic dysfunction results from replacement of functional myocardial tissue with amyloid protein. The conduction system can be affected by amyloid infiltration as well, leading to conduction disturbances, arrhythmias or sudden death. ECG findings include low voltage, conduction abnormalities such as AV block, atrial fibrillation and pseudoinfarct patterns. This case demonstrates a classic presentation of AL amyloidosis with significant cardiac involvement. AL amyloidosis has a median survival of one to two years but patients with symptomatic heart involvement have a median survival of only six months. LEARNING OBJECTIVES: Recognize the importance of counseling patients who have received previous valve replacements that continued intravenous drug use may disqualify them from lifesaving interventions. CASE INFORMATION: A 44 year-old presented with progressive dyspnea, fevers and substernal tightness. He had a endocarditis requiring porcein valve replacement in 1993. He started using intravenous cocaine and heroin weekly one month prior. He had a fever of 40 C and a holosystolic murmur. Blood cultures grew steptococcus viridans. Transesophageal echocardiography showed aortic valve vegetations on all leaflets and a ring abscess. Toxicology screen was positive for cocaine. Ampicillin and gentamicin were initiated. After six weeks of therapy, the aortic vegetations and blood cultures persisted. Cardiothoracic surgery was consulted, but declined intervention, citing the likely recidivistic drug use and futility of care. IMPLICATIONS/DISCUSSION: Surgical intervention in active intravenous drugs users is an issue of ethical debate. Ninety percent of heroin users will reuse even after successful rehabilitation. Continued intravenous use represents a hundred-fold increased risk of recurrent endocarditis. The apparent futility of surgical intervention in this setting has prompted surgical services to adopt a``one strike and you are out policy.'' This is almost a certain death sentence for patients meeting surgical criteria. The ethics committee was involved but could not establish ethical grounds for challenging surgery's decision. The precedent of withholding organ transplantation from continuing drug users was applied. Physicians should counsel patients who have received previous valve replacements that continued intravenous drug use may disqualify them from lifesaving interventions. Added emphasis on rehabilitation following valve replacement may be the only sure method to avoid this dilemma. \nA 53 year-old woman presented with four months of weakness and a rash. The weakness was progressive, symmetric, and proximal. The muscles of both her upper and lower extremities were affected. The erythematous rash was located on the inner thighs and arms bilaterally. She could not raise her arms and had difficulty holding her head erect; her leg weakness prohibited standing independently. Sensation and reflexes were not affected. She had normal serum electrolytes and complete blood count. Her AST was 200 U/L, ALT 78 U/ L, ALKP 52 U/L, CK 7021U/L, and ESR 29 mm/hr. Her TSH, hepatitis panel, ANA, rheumatoid factor, anti-DS DNA, c-ANCA, and p-ANCA were normal. A muscle biopsy of left thigh showed vasculitis and perifascicular atrophy and necrosis. Methylprednisolone and methotrexate were initiated with resolution of her symptoms. An age-appropriate screen for cancer was negative. IMPLICATIONS/DISCUSSION: The diagnosis of dermatomyositis/polymyositis (DM/PM) requires a high degree of clinical suspicion, as it is established based on the combination of clinical, laboratory, and muscle biopsy findings. This patient demonstrated the proximal muscle weakness characteristic of DM/PM. Other causes of proximal muscle weakness, including electrolyte abnormalities, hypothyroidism, inclusion body myositis or drug induced (HMG CoA reductase inhibitors or steroids) were excluded by history and laboratory data. Perifasicular inflammation suggests dermatomyositis; perimysial inflammation is characteristic of drug-induced myositis. Diagnostic criteria for dermatomyositis include symmetric proximal muscle weakness, elevated serum muscle enzymes, perifasicular inflammation on muscle biopsy, and rash. The classic dermatologic findings of PM/DM include mechanic's hands (synthetase AB subset), heliotrope lids, Gottron's sign and shawl sign . Physicians should be aware that most cases of DM/PM present without the classic findings of DM/PM. Systemic corticosteroids are used for treatment. Immunosuppressive agents are used in cases refractory to corticosteroid treatment. Due to an association with underlying malignancy, an age and gender appropriate cancer screen should be performed in all patients diagnosed with dermatomyositis.\nA CASE OF PREGNANCY, HYPOXIA, AND TOO MUCH PROTEIN. R.A. Andrews 1 , E.L. Salerno 2 ; 1 University of Connecticut, Farmington, CT; 2 University of Connecticut, Columbia, CT (Tracking ID #76994) LEARNING OBJECTIVES: 1) Discuss diagnosis, eitiology, and management of pulmonary alveolar proteinosis. CASE INFORMATION: A.G. is a 29 year old female 15 weeks into her fourth pregnancy who presents for diagnosis and treatment of severe hypoxia. Ten weeks prior to admission, she begins having progressive dyspnea on exertion, non-productive cough, weakness, fatigue, nausea, and diarrhea, but no fevers and occassional chills. Two days prior to admission she is referred to the emergency room for severe hypoxia. Patient denies problems with previous pregnancies and is HIV negative. Past medical history and social history are essentially unremarkable. At presentation, she is afebrile with a blood gas on room air that shows a PaO2 of 64. Cardiovascular exam is benign, but diffuse course rales are heard on lung exam. The patient has perioral and acral cyanosis and clubbing. Chest xray shows a diffuse patchy alveolar infiltrate. HCT is 49.3 with normal chemistries and negative D-dimer. Patient is placed on a non-rebreather mask at 100% and started on broad spectrum antibiotics. On day two, she is transferred to intensive care with a PaO2 of 54 on 100% oxygen. Video-assisted thorascopic biopsy is obtained providing the diagnosis of pulmonary alveolar proteinosis. Patient then undergoes whole-lung lavage with improvement in chest xray and PaO2 saturation. IMPLICATIONS/DISCUSSION: Pulmonary alveolar proteinosis (PAP) is a non-inflammatory disease of the lungs affecting men more than women (2:1) typically between 30 and 50 years of age. The primary defect is either a failure to degrade, or increased production, of surfactant causing an accumulation of amorphous proteinaceous material within the alveoli. The exact eitiology remains unclear. Patients generally present with dyspnea on exertion, 30% are asymptomatic. Common symptoms are dry cough, weight loss, malaise and fatigue. Rales are present in 50% of cases. Typical xray findings reveal perihilar alveolar infiltrates with typical``bat wing'' appearance. Diagnosis is made from BAL (bronchioalveolar lavage) or lung biopsy. Lavage produces a milky effluent composed of amorphous granular material that stains PAS (periodic acid-Schiff) positive. On biopsy, inflammatory cells are rare and there is no fibrosis. Alveoli are congested with proteinaceous material and dysfunctional macrophages. Standard of care is whole-lung lavage with 30±40% of patients resolving with only one lavage while the majority of patients require repeat lavage at 6±12 month intervals. Newer treatments include high doses of GM-CSF (granulocyte macrophage-colony stimulating factor) and possibly bone marrow transplant. Prognosis of PAP has been shown to be a 74% 5-year survival. LEARNING OBJECTIVES: 1. Recognize the long term complications after heart surgery for congenital defect in childhood. 2. Appreciate that patients living in precarious conditions need careful management that goes beyond an initial impression, although the risk of somatizing exists. CASE INFORMATION: A 25 year old male asylum-seeker, living in Switzerland for a month, with a history of surgical repair of a pentalogy of Fallot 20 years before with no subsequent complications, presented with breathing-related chest pain responsive to paracetamol, exertional dyspnoea and orthopnoea ± on the same day that five of his compatriots living in the same asylum-seeker centre located in a public bomb shelter with the same symptoms! He was in good general health, his blood pressure was 93/66 mmHg, and he had a regular heart rate of 100 beats/min and a respiratory rate of 25/min. His chest was slightly asymmetrical, with slight arching of the left hemithorax, the sternotomy scar appeared normal, the apical impulse was widened and pulsations were apparent across the whole sternal and left parasternal area (Harzer's sign). Auscultation revealed a holosystolic, non-radiating crescendo murmur of 3/6 in the left parasternal area. Diastole was free and peripheral arteries were normal. Laboratory tests were within normal limits, excluding acute conditions such as pulmonary embolism or cardiac ischaemia. The ECG revealed sinus tachycardia, a vertical frontal axis of the QRS complex and complete right bundle branch block. A transthoracic echocardiogram revealed a voluminous mass with an echo-dense border and an echo-poor centre adjacent to the free wall of the right ventricle, no vascularized (free of internal blood flow), exerting an obvious compression on the right ventricle, the intraventricular pressure being estimated at 80 mmHg based on the high-velocity severe tricuspid regurgitation. Magnetic resonance imaging and computed transverse tomography confirmed the presence of a hypodense mass, in close contact with the pericardium (Fig. 1 & 2) . The severe compression of the right ventricle prompted the surgical removal of this mass. The mass was adhering to the rib cage and the free wall of the right ventricle. Bacteriological investigation revealed the presence of an abscess with coagulase-negative staphylococci. Postoperative progress was uneventful, and the medium-term outcome favourable. No abnormal cavity was visualized during transthoracic echocardiography at the 3-month postoperative control. IMPLICATIONS/DISCUSSION: While infections are known to occur after sternotomy, usually involving coagulase-negative staphylococci, the formation of an abscess in the anterior mediastinum several years after the intervention appears to be exceptional. This diagnosis came to mind only after the more common postoperative complications, e.g. pseudoaneurysm or pericardial haematoma, and the wide range of possible diagnoses covering tumours, infections and malformations of the anterior mediastinum had been considered. Although the signs and symptoms were not very specific and potentially psychogenic in a patient living in precarious conditions, we were inclined to consider a wide range of differential diagnoses, given the patient's history.\ncare, her pulmonary status gradually returned to baseline and she was tapered off phenytoin without subsequent seizure activity. IMPLICATIONS/DISCUSSION: There are very few reports of a pulmonary embolus (PE) presenting as seizure. Given the acuity of onset of this patient's symptoms and the severity of her initial respiratory acidosis, as well as the lack of neurologic findings, we presume that her seizure represents the onset of her pulmonary embolus, though we can not definitively establish causal relationship. Clinical suspicion of PE may be compromised by the attribution of symptoms to the seizure, such as tachypnea, tachycardia, and hypoxia. The initial evaluation of seizure patients is primarily neurologic, delaying the diagnosis of a cardiopulmonary syndrome. In this particular case, the patient's medical history played a key role in the delayed diagnosis of PE, as her pulmonary symptomatology was attributed to asthma, despite the fact that she had been well controlled on minimal medication prior to admission, and that her clinical signs did not completely resolve with the treatment of her exacerbation. Furthermore, this young, active woman had low risk for thromboembolic disease, especially in the setting of a negative evaluation for hypercoaguability. She did use oral contraception, but this particular medication includes only 0.10mg levonorgestrel and 0.2mg ethinyl estradiol and the patient was a nonsmoker. Thromboembolism should therefore be considered in any patients with acute dyspnea, evidence of cerebral hypoperfusion, hypoxia, or unexplained seizure, even in the setting of pulmonary pathophysiology. year-old healthy female who presented to the emergency department after having her first seizure. She was found by her son in bed with jerking of her left upper extremity. When she regained consciousness she found that she bit her tongue and had urinary incontinence. She was well up until three days before when she developed a constant low-grade headache. Her last menstrual cycle was three months before presentation although she reports some light vaginal bleeding one week prior. Per the patient she had no sexual activity for six months. Physical exam on admission was significant for blood pressure of 145/82 and 1+ ankle edema. The neurological exam was completely normal. The patient did not know she was pregnant on admission but an initial beta-HCG was 12,700. Her urinalysis showed 3+ proteinuria. An ultrasound confirmed the presence of a 30 week gestation fetus. A head CT showed low attenuation lesions in both subcortical parietal lobes and in the left frontal lobe. The patient was transferred to Labor and Delivery with the presumptive diagnosis of eclampsia. She received intravenous magnesium sulfate and steroids to enhance fetal lung maturity. The next day after some fetal distress she delivered a viable male infant via cesarean section. Her vaginal bleeding one week prior to admission was due to placental abruption. IMPLICATIONS/DISCUSSION: Eclampsia is not commonly encountered in the practice of internal medicine but it should be considered in the evaluation of a new-onset seizure disorder in women of reproductive age. It has an incidence of approximately 5 cases per 100,000 live births. It is characterized by generalized seizures or coma in the presence of preeclampsia (hypertension, edema, proteinuria) . It is most common in nulliparous women in the teenage years and early twenties and in women over 35. More than 20% of cases occur before 30 weeks gestation, and 75% of cases occur at term or within 48 hours of delivery. It remains the second most common cause of maternal death in the United States. Treatment consists of oxygen, intravenous magnesium and antihypertensive medications. Anticonvulsant medications may be given for recurrent seizures. The fetus should be delivered promptly. Unfortunately this woman did not recognize that she was pregnant therefore she did not receive any prenatal care. This case underscores the importance of pregnancy testing in all women of reproductive age with a new-onset seizure disorder to expedite the diagnosis. Eclampsia should be part of the differential diagnosis in the evaluation of a new seizure disorder in all women of reproductive age unless she proves not to be pregnant. Early recognition and treatment of this disorder can prevent some potentially devastating consequences.\nWHAT IS THE MEANING OF TRISMUS? H.A. Batal 1 , R.H. Harris 1 ; 1 Denver Health Medical Center, Denver, CO (Tracking ID #74878) LEARNING OBJECTIVES: 1) Identify the differential for trismus. 2) Recognize the acute presentation of tetanus. CASE INFORMATION: A 20 yo previously healthy Mexican native presented with 4 days of left cheek spasm associated with pain. He denied any trauma, abrasions or lacerations, thought that he had received his vaccines as a child, and denied any systemic symptoms such as fever or myalgias. He also denied sore throat or dental pain and did not have a personal or family history of neurologic problems, including tics. He had bitten his tongue a number of times due to his left cheek spasm and was finding it difficult to eat solid food, although his intake of liquids was adequate. On examination he had normal vital signs and did not appear to be in any distress. He spoke through slightly clenched lips, but was handling his secretions without difficulty. He had no apparent spasms or tics involving other muscle groups. His neurologic exam was normal, although he refused to protrude his tongue, as he was afraid that he would bite it. His exam showed poor dentition but no localizing dental infection and no evidence of pharyngeal abscess. Submandibular and parotid glands were non-tender. He was noted to have spasm of his left masseter muscle, and broke a tongue blade when an attempt was made to keep his mouth open for examination. He was only able to open his jaw approximately 2 cm. Laboratory examination showed a normal CBC, calcium, and electrolytes. A dental consultant was unable to find any underlying odontogenic etiology for the patient's trismus. Dental radiographs and head MRI were unrevealing. Because the diagnosis of acute tetanus was entertained, the patient was admitted for 48 hours of observation during which time his symptoms did not change. He was discharged with a diagnosis of trismus of unknown etiology and therapy with tegretol at 200 mg bid was initiated. IMPLICATIONS/DISCUSSION: Trimus is defined as spasm of the masticatory muscles, with difficulty in mouth opening, and is a characteristic early symptom of tetanus commonly referred to as lockjaw. The differential diagnosis of trismus is limited and besides acute tetanus includes odontogenic infections, peritonsilar abscess, strychnine poisoning, and drug-induced dystonic reaction. In addition, trismus can be confused with the jaw claudication seen in temporal arteritis as well as the pain of trigeminal neuralgia. Widsom tooth (3rd molar) infections are the most common odontogenic cause of trismus and notably can present without evidence facial swelling. Acute tetanus is important to recognize because it can progress to death as a result of respiratory failure, but the prognosis can be favorable if supportive treatment is initiated promptly. Predictors of inadequate immunity are older age, birth outside the US, low educational status, and poverty. LEARNING OBJECTIVES: To recognize the presentation and therapy of thyrotoxic periodic paralysis. CASE INFORMATION: A 26 yo Latino male was brought to the ER complaining of 12 hours of progressive upper and lower extremity weakness to the point that he could no longer get out of bed. The symptoms began with leg cramps shortly after a family picnic. He had no complaints of abnormal sensation, difficulty breathing or swallowing. He denied recent illness or vaccinations, or ingestion of canned food. He also denied past medical problems, medications, or illicit drug use. His mother and sister had hypothyroidism. The examination showed an alert, well developed male with a mildly enlarged thyroid, but no tremor or lid lag. Strength in the upper extremities was 3/5, and 2/5 in the lower extremities. Deep tendon reflexes were 1 plus bilaterally, and the cranial nerve exam was normal. Labs were remarkable for Na 142, K 1.8, Cl 111, CO2 20. A TSH was drawn. The EKG showed SR with flattened T waves and prominent U waves. After a total of 120 meq of combined IV and PO KCL, the serum K was 5.1 and the patient's symptoms and EKG findings had completely resolved. The following day, the TSH and FT4 returned at <0.03 and 2.48 respectively, consistent with hyperthyroidism. The patient was diagnosed with thyrotoxic periodic paralysis. Propranolol was initiated and a thyroid scan scheduled as an outpatient. Despite compliance with medications, the patient returned to the ER with a recurrence of symptoms after a large Christmas dinner. The iodine uptake scan showed a mildly enlarged gland with homogeneously increased uptake: 20 % (5±17%) at 4h, and 48 % (8±30%) at 24 hours, consistent with Graves disease. Therefore, the patient was treated with radioactive iodine ablation. Four months into followup, the patient was doing well, but had become hypothyroid. IMPLICATIONS/DISCUSSION: Thyrotoxic periodic paralysis is a well described disease in up to 2 percent of all Asian men afflicted with hyperthyroidism. However, it is being increasingly recognized in patients of other racial backgrounds including Native Americans, Latinos and rarely Caucasians or those of African descent. This disorder is caused by overactivity of the Na-K-ATPase channel in the presence of increased thyroid hormone, which shifts potassium intracellularly. The inital treatment involves modest potassium supplementation and non-selective beta blockers. The more common familial periodic paralysis is related to the Na-Ca channel, and presents in a similar fashion, frequently after a carbohydrate rich meal or exercise. It is important for clinicians to understand that thyrotoxicosis of any etiology is an underlying cause of hypokalemic periodic paralysis because treatment of the thyroid disorder will also prevent further paralytic episodes.\nAN ATYPICAL CASE OF POLYARTERITIS NODOSA. B.N. Batson 1 , J.W. Blackston 1 , C. Subramony 1 ; 1 University of Mississippi Medical Center, Jackson, MS (Tracking ID #75156) LEARNING OBJECTIVES: Recognize the clinical and lab findings of polyarteritis nodosa. Perform an appropriate workup when polyarteritis nodosa is suspected. CASE INFORMATION: A 46 y/o BM was transferred complaining of abdominal pain for 4 days that had not improved with conservative treatment. Outside x-rays demonstrated dilated loops of bowel without obstruction. He denied fever, chills, nausea, and vomiting but reported fatigue, anorexia, and a 40-pound weight loss over the past 6 months. He also reported progressive weakness and numbness of both legs which limited ambulation. Physical was significant for BP of 178/114 and a diffusely tender abdomen without guarding, rebound or mass. He had faint bowel sounds and was heme positive on rectal. He had 2/5 strength in bilateral hip, knee, and ankle flexors and extensors. Sensation was diminished in both feet up to mid-shin. Reflexes and Babinski's were normal. CXR was negatvie except for blebs, but KUB showed multiple dilated loops of bowel with air-fluid levels. Lab included WBC 14.1, Hct 27, and plts 417. BUN was 23 and creatinine 1.2. Total protein was 6.7 with albumin 2.3. Hep BsAg was positive; p-ANCA was negative. On HD#2, CT abdomen showed a 9x5cm right lower quadrant mass, moderate ascites, and patchy kidney enhancement with wedged-shaped hypodensities. Renal ultrasound showed normal kidney size with inhomogeneous architecture. On HD#3, sural nerve biopsy was suspicious for mononeuritis multiplex. Later on HD#3, a mesenteric arteriogram was normal. By HD#4, the patient had decreased abdominal pain with stable hematocrit, but creatinine had risen to 2.7. On HD#5 colonoscopy was normal. He then had an MRI-guided biopsy of the RLQ mass which yielded only red blood cells. On HD#8 surgical exploration found bloody ascites and an 8x10cm mass of the ileocolonic junction requiring hemicolectomy. Ascitic cytology was negative for malignancy. Pathology of the mass showed extensive polyarteritis nodosa involving the small-and medium-sized submucosal and mesenteric arteries. IMPLICATIONS/DISCUSSION: While this patient exhibited many classic symptoms of polyarteritis nodosa, his workup of that suspected diagnosis was confounded by an abdominal mass and false-negative mesenteric arteriogram. One must remember that the diagnosis of PAN is best made with tissue biopsy of an involved organ system. While a positive p-ANCA is suggestive of PAN, and a mesenteric arteriogram showing pseudo-aneurysms is sufficient for diagnosis, both of these tests are positive in less than 50% of patients with known polyarteritis nodosa.\nnormalize. Magnetic resonance angiography of the abdomen revealed severe stenosis of the distal left renal artery consistent with FMD. The patient underwent contrast-enhanced renal artery angiography that confirmed the diagnosis. Balloon angioplasty of the stenotic left renal artery was performed. The day after angioplasty, the patient's plasma renin decreased to 50 microU/ml and BP to 130/70. IMPLICATIONS/DISCUSSION: Renovascular hypertension should be suspected in patients with age of onset before 20 or after 50 years, severe or refractory hypertension, acute rise in BP over previously controlled hypertension, rise in plasma creatinine concentration especially after starting an angiotensin-converting enzyme (ACE) inhibitor, abdominal bruit, absence of family history of hypertension, a unilateral small kidney, and recurrent acute pulmonary edema or unexplained heart failure. RAS may result from FMD or atherosclerotic disease. FMD most commonly affects women between the ages of 15 and 50 and accounts for less than 10% of cases of RAS. Atherosclerosis is responsible for the other 90% of cases. Gadolinium-enhanced magnetic resonance angiography is the best noninvasive imaging modality to diagnose RAS with 100% sensitivity and 95% specificity. Renal angiography is the gold standard to confirm the diagnosis. Percutaneous angioplasty is the treatment of choice for renovascular hypertension secondary to FMD. Patients with hypertension secondary to atherosclerotic RAS should be treated with aspirin, cholesterol-lowering agents, and smoking cessation. ACE inhibitors and diuretics can be used to control hypertension but should be avoided in the presence of bilateral stenosis. Balloon angioplasty is less successful in atherosclerotic RAS but placement of stents improves the success rates. Surgical revascularization should be considered in patients who fail angioplasty. LEARNING OBJECTIVES: 1. Identify a likely cause of recurrent painful blistering diseases in young adults 2. Identify the classical presentation, manifestations and prognosis of Epidermolysis Bullosa 3. Recognize the main management issues in caring for Epidermolysis Bullosa patients CASE INFORMATION: A 19 y/o latina woman presented for evaluation of painful blisters over her left hand and both feet after recent initiation of jogging. She reported a history of recurrent painful blisters affecting both her hands and feet since childhood. More severe blistering occured in areas where her skin was rubbed. Steroid creams provided no improvement. Lesions in the past healed over about a month, leaving hyper-pigmented scars. This current flare involved both her feet and left hand, but no other area. Systems review was negative. Other family members were not affected. She took Tylenol with hydrocodone for pain. On examination the patient had multiple blisters, 2±3 cm in diameter, mostly on the plantar surface of both feet. The soles of her feet were thickened and pigmented in circular patch-like forms. The blisters were exquisitely tender to touch. Initial differential diagnosis of this patient's condition included bullous pemphigoid, dermatitis herpetiformis, porphyria cutanea tarda and bullous SLE. A skin biopsy performed by a dermatologist demonstrated histologic separation of the basal layer. After immunohistochemistry, a diagnosis of epidermolysis bullosa, simplex form, was made. IMPLICATIONS/DISCUSSION: Epidermolysis Bullosa (EB) is the term given to three major groups of approximately 16 variants of rare dominant and recessive genetic diseases in which minor trauma causes non-inflammatory blistering (mechanobullous disease). There are as many as 150,000 Americans afflicted, mostly children, but variants present up to third decade of life. Those with medium to more severe kinds of EB can live into their thirties. Mortality is predominantly related to suprainfections. Management consists of all efforts to avoid trauma, particularly to the hands and feet via mittens and soft slippers. Adequate long-term pain control is a crucial aspect of taking care of EB patients, as is prevention and early treatment of supra-infections of the blistering lesions. Clasically all EB variants are resistant to all therapy, though literature reports exist of rare responders to treatment with Phenytoin, a known collagenase inhibitor, as well as IVIg. Genetic counseling is essential and fetal skin biopsy techniques have been developed for prenatal diagnosis. The diagnosis of glucagonoma was confirmed by pathologic examination. Upon removal of the tumor, her rash subsequently cleared without any complications. IMPLICATIONS/DISCUSSION: Glucagonoma syndrome is a rare syndrome with an estimated incidence of about 1 in 20 million people. It occurs with equal frequency in men and women and is usually detected between 40 to 60 years of age. The syndrome is caused by excessive production of glucagon by the alpha-secreting cells of the pancreas. Patients may have a characteristic rash, called``necrolytic migratory erythema'', that involves the perioral and perigenital regions. There may also be accompanying glossitis, angular chelitis, and blepharitis. The rash usually responds poorly to topical treatment. The only curative treatment for the syndrome is surgery. Glucagonoma syndrome should be considered in any patient with: 1) newly diagnosed diabetes and 2) a rash that does not resolve with proper topical treatment and is consistent with necrolytic migratory erythema. 10% of all strokes. There is a much broader differencial of disorders that cause infarction in younger people. Full evaluation is required to determine an underlying cause to treat patients accordingly and prevent recurrence. As opposed to stroke in the elderly which are often due to atherosclerosis and embolism, only 40% of strokes in the young are accounted for by these two etiologies. Other important causes include dissection, vasculitis, blood disorders, pregnancy, mitral valve prolapse, migraine, and patent foramen ovale. Given this patient had simulanteous anterior and posterior strokes, some of these etiologies are more likely, including: proximal emboli or a disseminated process such as vasculitis, blood disorder or drug use. This young person's stroke was a result of cocaine use. Proposed mechanisms by which cocaine causes stroke is that it induces vasoconstriction, vasospasm, hypertension and/or vasculitis. Case reports have documented stroke secondary to cocaine use in patients who had infarction in multiple distributions. Several of these cases were confirmed to have cocaine-induced vasculitis on autopsy despite having had negative MRA. LEARNING OBJECTIVES: To distinguish the toxicity of``crystal meth'' (methamphetamine) from other syndromes in a patient presenting with psychomotor agitation and tachycardia. CASE INFORMATION: A 22 year-old male with unremarkable PMH presented to the ED via ambulance with the acute onset of diaphoresis, tachycardia, and restlessness. He had been in police custody for approximately 48 hours, and charged with public intoxication. After his symptoms developed, he admitted to ingesting a``2 gram rock of crystal meth'' to avoid charges of illegal drug possession. Physical exam revealed an anxious but alert WM, BP 119/72 mm Hg, HR regular at 179 bpm, unlabored RR 16 per min., rectal temp T 99.3 degrees F, and O2 sat. 97% on RA. Auscultation of his chest was clear and his neurologic exam was nonfocal; but he was tremulous, pale, and his skin cold and clammy. He required substantial benzodiazepine doses to achieve sedation: diazepam 5mg IV en route to the ED, and a total of 8mg IV lorazepam and 14mg IV midazolam in the ED. The patient was electively intubated for``airway protection.'' His hospital course was uneventful; he was extubated within the first day of admission and transferred back to incarceration in 2 days. CPK levels were normal in the ED, but subsequently increased to ten times the ULN. A serum toxicology screen was positive for amphetamines and cannabinoids, and negative for salicylates, acetaminophen, methanol, ethanol, and ethylene glycol. Confirmatory testing revealed a serum methamphetamine level of 80,000 ng/dL. IMPLICATIONS/DISCUSSION: Delayed methamphetamine toxicity, defined as symptom onset 48 hours after ingestion, has not been previously reported in the medical literature. The crystalline form of methamphetamine, also known colloquially as``crank,'' is a stimulant drug of abuse that is usually nasally inhaled, snorted, or smoked. It is rapidly absorbed from any mucous membrane and produces almost immediate metabolic and psychotropic effects. This patient reported orally ingesting the drug in a plastic container, thus with gastrointestinal transit time and even partial digestion of the container, the drug may have been easily absorbed later in the distal GI tract. Alcohol withdrawal with incipient delirium tremens may also explain his presenting symptoms, but his markedly elevated CPK, a usual laboratory finding with acute methamphetamine overdose, and strongly positive serum methamphetamine level at the time of maximal symptomology, suggest methamphetamine toxicity as the etiology of his distress. This case stresses the need for medical personnel to obtain complete drug exposure histories from agitated patients, including explicit questioning with regard to drug ingestion stemming from attempts to conceal illegal activities.\nCARDIAC TAMPONADE DUE TO SEVERE MYXEDEMA. J. Blonsky 1 , V. Shivaswamy 1 , K. Hoskison 1 , R. Sekhar 1 , D. Perry 1 ; 1 University of Oklahoma, Tulsa, OK (Tracking ID #75122) LEARNING OBJECTIVES: Recognize hypothyroidism as cause for cardiac tamponade. Diagnosis of cardiac tamponade secondary to hypothyroidism. Treatment of cardiac tamponade caused by severe tamponade. CASE INFORMATION: We report a 53-year-old male with an enlarging right testicle, progressive dyspnea, severe dependent edema, and cold intolerance. Physical examination revealed faint heart sounds, ascites, ankle jerks with delayed relaxation, and an enlargement of the right scrotum consistent with a hydrocele, and a clinical diagnosis of cardiac effusion with tamponade was entertained. Further evaluations revealed low voltage complexes on the EKG, and marked cardiomegaly on a Chest Roentgenogram with bilateral pleural effusions. An echocardiogram showed a large pericardial effusion with signs of early tamponade, and a pericardial paracentesis drained 1400ml of exudate. Pericardiocentesis led to significant clinical improvement. To confirm a unifying diagnosis thyroid functions were diagnosed of profound hypothyroidism with a TSH of 52.5 iu/ml. The patient responded well to thyroid hormone replacement therapy, and demonstrated continued recovery at clinic follow-up visits. The scrotal swelling was confirmed as a hydrocele by ultrasound. Hypothyroid effusions involving the pleural, pericardial, and peritoneal cavities have been described, but uncommon clinically. Pericardial effusions tend to be large, and asymptomatic, as the slow temporal progression permits compensation due to pericardial stretching. The clinical presentation suggests chronic cardiac failure and constrictive pericarditis with pleural effusion, ascites, and anasarca. EKG findings include bradycardia and microvoltage complexes. Treatment with thyroid hormone replacement is nearly always followed by steady regression of the effusion. Pericardiocentesis is indicated for tamponade, after a confirmatory echocardiogram. This presentation illustrates the importance of detecting hypothyroidism as cause for cardiac tamponade. Prompt diagnosis is vital to prevent cardiogenic shock, and includes early suspicion on the basis of a detailed physical examination, and rapid diagnosis by thyroid functions and echocardiography. Evacuation of the pericardial fluid, and thyroid replacement therapy leads to complete regression and resolution of the condition. IMPLICATIONS/DISCUSSION: Hypothyroidism may lead to cardiac tamponade. Prompt diagnosis is vital to prevent cardiogenic shock. Complete resolution of pericardial fluid following evacuation and thyroid replacement therapy.\nA HISPANIC MAN WITH LYMPHADENOPATHY AND EOSINOPHILIA. D. Bolger 1 ; 1 Johns Hopkins University, Baltimore, MD (Tracking ID #76421)\nLEARNING OBJECTIVES: 1. Recognize the clinical features of Kimura's disease. 2. Recognize that a constellation of nonspecific clinical signs can represent a specific entity when pursued. 3. Develop a differential diagnosis for lymphadenopathy and differentiate accordingly. CASE INFORMATION: A 63 year old man born in the Dominican Republic but residing in New York City for 30 years presented to a medical clinic with 2 years of right neck swelling. The gentleman initially noticed a small, painless bump in his neck. The bump enlarged to an eventual size of 8cm by 14cm. Similar bumps also arose in the right neck, left neck, and right axilla. Eventually the predominant lesion became painful and itchy. The patient felt healthy otherwise, denying fevers, fatigue, and weight loss. The patient had a history of hypertension, PPD positivity with negative CXRs, and remote microscopic hematuria. He was taking hydrochlorothiazide and prazosin for his hypertension. He was married with 2 children. A He traveled to the Dominican Republic every year for holiday. No other travel was noted. His physical exam was remarkable for a mobile nodular mass 8cm  14cm in the right anterior cervical neck. There were discrete subcutaneous nodules on both sides of the neck. There was lymphadenopathy in the left neck and right axilla. The nodules and lymph nodes were mobile and firm but without fluctuance. There was no hepatosplenomegaly or other lymphadenopathy present. Pertinent labs included a wbc count of 8100/mm3 with 38% eosinophils (absolute eos count 3078) IgE levels were high at >1800kIU/L. HIV test was negative. Excisional biopsy of lymph node revealed follicular hyperplasia with distinct lymphoid follicles with surrounding fibrosis, abundant eosinophils, post-capillary venules, no granulomas or necrosis, and preserved architecture. Cultures and smears were negative for AFB, fungi, bacteria, and Reed-Sternberg cells. Flow cytometry was negative for lymphoma. In addition, skin snips from the patient were negative for microfilariae. The patient was started on prednisone with rapid resolution of lymphadenopathy, however this recurred when prednisone was tapered. The patient was restarted on full dose prednisone and scheduled for curative radiotherapy. IMPLICATIONS/DISCUSSION: Kimura's disease is a rare disease usually seen in Asian men. They present with lymphadenopathy of the head and neck and eosilophilia. Pathology reveals preserved lymph node architecture with necrosis, predominant eosinophils, endothelial venules, and capsular fibrosis. Often lymphadenopathy is attributed to an infectious agent or malignancy. As this case illustrates, there are other causes to consider. A thorough evaluation and exclusion of important entities such as Hodgkin's disesase, extra-pulmonary TB, and Cat Scratch Disease is critical. Histological evaluation often provides a definitive diagnosis, but not always. The pathology along with the clinical signs (features) were required for the diagnosis in this case. osteomyelitis and an epidural phlegmon. Magnetic resonance imaging (MRI) showed cord compression. Needle biopsy of the abscess was performed, yielding prurulent material. In consultation with Infectious Disease and Pulmonary services, empiric treatment for TB was held as it could mask a pyogenic process. By the eleventh hospital day, needle biopsy failed to grow organisms. Polymerase chain reaction (PCR) results of the biopsy aspirate was positive for mycobacterium tuberculosis (M.Tb) and the patient began therapy with isoniazid, rifampin, pyrazinamide and ethambutol. He subsequently underwent surgery for spinal stabilization and was discharged on an 18 month course of antibiotics. IMPLICATIONS/DISCUSSION: Low back pain is the fifth leading reason for medical visits in the United States. A careful history identifies``danger'' signals of infection or malignancy, such as constitutional symptoms, immunosuppression, age and unremitting nocturnal pain unrelieved by recumbency. In Pott's disease, systemic symptoms are often absent, and patients do not undergo testing until the process is advanced. CXR is not useful in diagnosis since more than 50% of patients do not have lung disease. The primary focus starts in the anteroinferior aspect of the vertebral body of the thoraco-lumbar spine. Local destruction leads to collapse, disc herniation and cord compression. Likewise, a tuberculous abscess, seen in more than 50%, can impinge and compress the spinal cord. CT, myelography, and MRI are useful in diagnosis. While biopsy and culture are sensitive and specific, serology and PCR are useful for timely diagnostic confirmation. Literature reports culture sensitivity for mycobacterial detection is improved by culture media supplemented with oleic acid, albumin, dextrose, and catalase, however detection is not specific to M. Tb. PCR of fixed, paraffin-embedded tissue samples, was shown to have sensitivity and specificity of 94% and 83% respectively. In summary, TB remains a major public health problem. Before the disease can be treated it must be recognized. TB should be considered in cases of spinal osteomyelitis. Early institution of therapy reduces morbidity and mortality.\nASTHMA OR ARE WE MISSING SOMETHING. C. Borrego 1 , E. Flenaugh 1 , D. Norman 1 , J. Henao 1 ; 1 Morehouse School of Medicine, Buford, GA (Tracking ID #74416)\nLEARNING OBJECTIVES: Diagnosis and Treatment of Pulmonary Blastoma. CASE INFORMATION: This patient is a twenty-seven year old black male who has a past medical history of asthma since eight years old. He is a non-smoker. He states that eight months ago he started to work at a police impound. Where he states he was exposed to a lot of dust. Six months later he started having shortness of breath, cough with whitish to yellowish sputum, and wheezing with associated left lateral chest pain. He then went to the hospital were he was treated for an asthma exacerbation with improvement and subsequently discharged. He then returned with chest pain, left sided rated 8/10 and admitted. On physical exam blood pressure was 90/43, pulse 97, respirations 20, and temperature 97.3 degrees Fahrenheit. Room air pulse oximetry was 97%. He has decreased breath sounds on the left hemithorax. Otherwise physical exam was normal. Chest x-ray showed left lung to be completely opacified by tumor mass or pleural effusion, with some cystic components. CT scan off chest showed a large soft tissue mass completely replacing the left lung of mixed density most likely representing a neoplastic process such as sarcoma, invading and obstructing the left main bronchus. The bronchoscopy showed the right lung to be patent and 3 cm below the carina going to the left main stem bronchus, the airway was completely occluded by a tumor mass in the lumen. Endo bronchial biopsy was performed at that time, which revealed pulmonary blastoma, monomorphic epithelial type (well differentiated fetal adenocarcinoma). IMPLICATIONS/DISCUSSION: Pulmonary blastoma is hard to be diagnosed before operation. The main symptoms can be cough, expectoration. This tumor is extremely rare and manifest's a mixture of epithelial and mesenchymal neoplastic cells. This is a low grade tumor that consists of immature embryonic epithelial elements forming tubules and glands and primitive lung structures, surrounded by a malignant-appearing mesenchymal stroma. Other tumors with better differentiated background mesenchymal elements or neuroendocrine cells in the stroma have also been reported and may represent part of this tumors spectrum. These tumors presents in children and adults and most commonly appear as parenchymal masses. Pulmonary blastoma is more common in women, and is usually diagnosed by surgical resection. This is usually curative, although if late relapses and metastasis occur they are usually not curable. For palliative treatment radiation therapy may be used, and a role for chemotherapy is not defined. LEARNING OBJECTIVES: Acute Myeloid Leukemia (AML) is an exceedingly rare diagnosis to find in association with Acute Intermittent Porphyria (AIP). Only one case report documents such an occurrence. CASE INFORMATION: We present the case of a 61-year-old man with a long-standing history of known Acute Intermittent Porphyria, (AIP) diagnosed in 1996, who was admitted to the internal medicine service for treatment of pharyngitis and severe dehydration. He had never undergone any chemotherapy and his AIP was fairly well controlled, with only four exacerbations noted including the sentinel episode. At presentation for his current admission the patient described fatigue and increasing retropharyngeal pain associated with deglutition, which had steadily progressed during the preceding three days. His CBC was significant for hemoglobin of 10.6 g/dl, WBC of 170.4/mm3, and a platelet count of 102,000/mm3. The differential was 24% segmented neutrophils, 0% bands, 3% lymphocytes, 57% monocytes, and 7% blasts. The patient had hepatosplenomegaly and a global petechial skin rash. Bone marrow studies were done to characterize the underlying etiology. Cellularity to fat ratio was 100:0 with normal bony spicules. Myelofibrosis was completely absent, and granulopoiesis was increased with an abnormal appearance. The blast estimate was greater than 20%. Over the subsequent week the patient experienced a rapid clinical decline with worsening coagulopathy. In accord with decisions made by the patient, his family, and consulting hematologists, chemotherapy was not instituted. AIP is the most common and most severe of the porphyrias.\nIMPLICATIONS/DISCUSSION: Although there are case reports of AML occurring with other hepatic porphyrias, there is only one known prior case report of AIP and AML occurring in tandem. This represents the second known incidence of these two disorders occurring simultaneously.\nSARCOIDOSIS PRESENTING AS CERVICAL CORD SYNDROME. D. Bradley 1 , E. Lower 1 , R. Baughman 1 , L. Coberly 1 ; 1 University of Cincinnati, Cincinnati, OH (Tracking ID #73947) LEARNING OBJECTIVES: To recognize that sarcoidosis is a rare but treatable cause of spinal cord lesions that can mimic malignant tumors of the spine clinically and radiologically. CASE INFORMATION: A 53 year-old African American male presented with a six month history of worsening neck pain, progressive upper and lower extremity weakness, and difficulties with bowel and bladder function. He also noted severe fatigue and a 30 pound weight loss over the last eight months. Physical examination was remarkable for flaccid paralysis of his right upper extremity, 3/5 strength of his left upper extremity, and 3/5 strength of his lower extremities. He had decreased rectal tone. Significant laboratory findings included a normal CBC, chemistry panel, and liver function studies. Calcium was elevated at 11.5 mg/dL (nl 8.4±10.2), an ACE level was normal. Cerebral spinal fluid studies were unremarkable. MRI of the spine showed extensive abnormal signal involving the entire cervical cord extending through T3 with diffuse, patchy nodular enhancement and edema from C3±C7 suspicious for malignancy. CT of the chest revealed mediastinal adenopathy. Biopsy of subcarinal lymph nodes showed noncaseating granulomas consistent with sarcoidosis. The patient was treated with high dose corticosteroids and IV cyclophosphamide (off label use as a steroid sparing agent) with marked improvement. Repeat MRI six months later was normal. IMPLICATIONS/DISCUSSION: The spinal cord is involved in less than 1% of patients with sarcoidosis. It is rarely the first manifestation of disease. Sarcoidosis of the spinal cord can be can be clinically and radiologically indistinguishable from a malignant tumor of the spine. Definitive diagnosis requires pathologic examination of the spinal cord lesions or, as in our patient, other associated lesions such as hilar adenopathy. Prompt treatment with high dose corticosteroids is essential to a favorable outcome. Steroids may need to be continued indefinately. There has been some success using steroid-sparing agents such as cyclophosphamide to decrease the dose and duration of steroid therapy. Sarcoidosis is a treatable condition that should be included in the differential diagnosis of spinal cord lesions.\nZOSTER SINE HERPETE: A CASE OF MASKED DIAGNOSIS. J. Bratcher 1 , S. Patel 1 , K. Cynamon 1 , J. Andrieni 1 ; 1 Lenox Hill Hospital, New York, NY (Tracking ID #75529)\nLEARNING OBJECTIVES: 1) Expand the differential for abdominal pain; 2) Diagnose an atypical presentation of herpes zoster in the absence of skin lesions; 3) Recognize that zoster sine herpete is a clinical diagnosis. CASE INFORMATION: A 58 year-old male with history of nephrolithiasis presented with abdominal pain, fever, and chills. Physical exam was significant for low-grade fever and left upper quadrant tenderness. Radiographic studies revealed a 5.3  4.6 cm dissecting aortic aneurysm at the level of the diaphragm, and a separate dissection of the right common iliac artery. The patient was admitted with a diagnosis of an infected aortic aneurysm and evaluated by vascular surgery. The patient continued to complain of left upper quadrant pain, described in a definite dermatomal distribution along the left lower costal margin with radiation to the left flank. Despite absence of any physical evidence of rash, the description was suggestive of herpes zoster. Varicella titers were then ordered and positive IgM titers were revealed. Zoster sine herpete was diagnosed, and the pain spontaneously resolved without medical intervention. On follow-up, the patient remains asymptomatic, never having developed a rash, and repeat CT scans reveal stable aneurysms. IMPLICATIONS/DISCUSSION: Herpes Zoster (Shingles) is a reactivation of varicellazoster virus, which lies dormant in the dorsal or cranial nerve ganglia. Upon recurrence, patients typically have a prodrome of fever, along with pain or tingling in the affected dermatome (50% thoracic, 20% cervical, and 10% lumbosacral). In most cases, the virus creates vesicles along the dermatome after three days, which then crust and resolve within two to three weeks. A small percentage of patients, however, develop acute segmental neuralgia, with a concomitant rise in varicalla IgM antibodies in the absence of skin lesions. This is zoster sine herpete, and as our case illustrates, patients can be initially misdiagnosed with a host of other illnesses, including myocardial infarction, cholecystitis, appendicitis, or diverticulitis. Very little has been published concerning zoster sine herpete since many cases spontaneously resolve without ever having reached the diagnosis. In this particular case, a CT of the abdomen revealed a dissecting aortic aneurysm, which was unrelated to the patient's chief complaint. By relying more on physical exam, the patient's description of the pain, and prior radiographic studies, the diagnosis might have been made more efficiently. This case stresses the importance of history and physical exam in the clinical diagnosis of zoster sine herpete. Otherwise, the diagnosis can be masked by confounding information. ventricular response. CXR (photo to be included) disclosed severe tension pneumothorax with complete collapse of the left lung and slight mediastinal shift to right. Emergent placement of a chest tube resulted in re-expansion of the lung, and stabilization. The patient did well and was discharged from the hospital 2 days later. IMPLICATIONS/DISCUSSION: Use of complementary/alternative medicine, often in concert with``mainstream'' medical treatment, is common and widely practiced in the United States. Acupuncture is effective as sole or adjunctive therapy for management of many types of pain, and is generally regarded as safe. The current vignette describes a case of tension pneumothorax directly attributable to acupuncture treatment. Traumatic injury from acupuncture is rare, but review of the medical literature discloses numerous reports of adverse events resulting from acupuncture treatments. Pneumothorax is the most commonly reported traumatic injury, especially among patients predisposed by presence of COPD; other risks include cardiac tamponade, peripheral nerve injury, and injury to blood vessels including pseudoaneurysm formation. Non-traumatic adverse effects of acupuncture include infections (hepatitis and other blood-borne diseases, as well as reports of septicemia and endocarditis.) The overall safety profile of acupuncture is positive, but patients and physicians should be aware of potential risks of treatment. State laws vary widely regarding requirement for certification; referral to licensed or certified medical acupuncturists is recommended. year-old woman presented with one month of fevers, evanescent rash, myalgias and arthralgias, severe fatigue, and anorexia with 20 pound weight loss. Examination disclosed T 39.48C, tender posterior cervical lymphadenopathy, no obvious synovitis, and a salmon-colored macular exanthem on the lower extremities. CBC showed normocytic anemia (Hgb 9.8,) slight leukocytosis (WBC 11.1 with 82.3% granulocytes,) normal LFTs except for slight elevation of AST, normal BUN/Cr, UA, and CXR. Further tests revealed ESR 90, negative ANA and RhF, and ferritin >2000. Further diagnostic workup included negative PPD, negative blood cultures, normal echocardiogram, normal CT scan except borderline mediastinal lymphadenopathy and mild hepatosplenomegaly, and negative titers for HIV, EBV, CMV, and Lyme disease. Lymph node biopsy disclosed reactive node with no evidence of a clonal population of cells. The patient was diagnosed with AOSD and had minimal response to aspirin. She was treated with steroids and is presently minimally symptomatic on hydroxychloroquine and NSAID. IMPLICATIONS/DISCUSSION: AOSD is a rare disease (incidence 1.6 cases/million persons/yr) that highlights the importance of clinical history and physical examination, since there is no definitive test to make the diagnosis. Diagnostic criteria have been proposed (will be delineated.) ASOD usually follows one of three patterns: self-limited (monophasic) disease with complete resolution of symptoms within one year, intermittent (polyphasic) course with complete remission between flares, or persistent active disease, with chronic destructive arthritis. First line treatment is aspirin or other nonsteroidal anti-inflammatory drugs (NSAIDs;) patients with more severe disease may respond to steroids. There are no controlled trials of immune modulating drugs in ASOD. Reports exist of success using methotrexate to control systemic symptoms; other agents used (all off-label) include gold salts, azathioprine, cyclophosphamide, cyclosporine, sulfasalazine, and intravenous immune globulin. Recent reports suggest etanercept and infliximab may have benefit in patients not responding to other therapies. A 74 year old white female presented with a 3 day history of`d ifficulty getting out of a chair'', dyspnea on exertion and``tea colored'' urine. Her past medical history was significant for depression and hypercholesterolemia. Her past medications included simvastatin (40 mgs po qd) and nefazodone (150 mgs po bid). She had been started on the nefazodone 3 months ago for treatment of depression and shortly thereafter developed a fever, nausea/vomiting and muscle spasms. Physical examination revealed significant weakness of the proximal musculature: 3/5 strength in the shoulder abductors and 2/5 strength in the hip flexors. Her laboratory data revealed: CK 37,050, AST 1,744, ALT 1,029, BUN 17, Creatinine 0.8 , Urinalysis: SpGr 1.020, pH 6.0, protein 100, large blood, granular casts. The nefazodone and simvastatin were discontinued and a forced bicarbonate diuresis was initiated. Polymyositis was suspected and steroids were started empirically pending the outcome of testing. An EMG showed no evidence of an underlying myopathy). ANA and an anti-Jo-1 antibody tests were negative. A muscle biopsy showed age-appropriate atrophy but no necrosis of myocytes and no evidence of lymphocytic infiltrate, thus ruling out the diagnosis of polymyositis. Her renal function remained normal throughout the hospitalization. Her CPK was 1,776 eight days after admission and she gradually regained strength in her proximal muscle groups. She was thought to have developed rhabdomyalyis secondary to a drug interaction between simvastatin and nefazodone. IMPLICATIONS/DISCUSSION: To our knowledge, this is the second case report of an adverse event associated with the concomitant use of simvastatin and nefazodone. Simvastatin is metabolized via the CYP3A4 system and nefazodone is a direct inhibitor of CYP3A4 enzymes. While myositis and rhabdomyolysis are adverse events associated with simvastatin monotherapy, we speculate that the introduction of nefazodone into this patient's regimen sufficiently inhibited the CYP3A4 system to cause an increase in serum simvastatin concentration that was adequate to cause rhabdomyolysis. Therefore, we would recommend avoidance of CYP3A4 inhibitors such as nefazodone in those receiving simvastatin therapy.\nCOLON CANCER, WITH ANGIOGRAPHIC EVIDENCE OF VASCULITIS. I.D. Bucaloiu 1 , S. Dubagunta 1 , K.K. Pachipala 1 , N.R. Kamal 1 , F. Fata 1 ; 1 Geisinger Medical Center, Danville, PA (Tracking ID #76029) LEARNING OBJECTIVES: 5-Fluorouracil (5-FU) is one of the most commonly used chemotherapeutic agents in clinical oncology practice, alone or in different combinations. In stage III colorectal cancer, 5-FU based therapy has been shown to be associated with a survival benefit. We report a case of ischemic small bowel injury in a patient who received adjuvant chemotherapy with Oxaliplatin, 5-FU and LV and was found to have angiographic evidence of vasculitis in the superior mesenteric artery (SMA) territory. CASE INFORMATION: A 73 year-old male underwent a left hemicolectomy for Stage III colon cancer. He was enrolled in an adjuvant chemotherapy research protocol consisting of 5-FU 500-mg/m2 intravenous (IV) weekly boluses each over 1 hour, LV 500-mg/m2 IV weekly infusions each over 2 hours and Oxaliplatin (OP) 85 mg/m2 IV infusions every 2 weeks for 6 weeks followed by two weeks of rest. After the third weekly dose of cycle one of 5 FU/LV he presented with weakness, diarrhea, crampy abdominal pain and poor appetite. His abdomen was distended and he had heme positive stools. Stool cultures and clostridium difficile toxin antigen were negative. He was not neutropenic. Colonoscopy revealed dusky edematous changes in the ascending colon and focal ulceration with patchy submucosal hemorrhages in the distal ileum. Biopsies of the colon mucosa showed densely neutrophilic infiltrates of the lining epithelium and associated mild focal cryptitis. A SMA angiogram was performed and it revealed beading in ileal branches, consistent with the ischemic territory. Vasculitis serologies were negative. The patient was treated conservatively with hydration and IV antibiotics and later he was rechallenged with a 20% reduction of 5-FU dose with no recurrence of his symptoms in 12-month follow-up. IMPLICATIONS/DISCUSSION: Our patient had superficial erosions and ulcerations in the ileum as well as an ischemic appearance of the colon. The characteristic findings of``beading'' on the angiogram suggest an underlying ischemic mechanism related to 5 FU based therapy. This case will add to the series of patients reported with 5-FU induced vascular injury to the small bowel.\nTHROMBOTIC THROMBOCYTOPENIC PURPURA AND EXTENSIVE BONE MARROW NECROSIS SECONDARY TO METASTATIC SIGNET RING CELL ADENOCARCINOMA. I.D. Bucaloiu 1 , J. Brady 1 , S. Walker 1 , K.K. Pachipala 1 ; 1 Geisinger Medical Center, Danville, PA (Tracking ID #76352) LEARNING OBJECTIVES: Although rare, the association between Thrombotic Thrombcytopenic Purpura and extensive Bone Marrow Necrosis can be the presenting picture of metastatic adenocarcinoma. CASE INFORMATION: A previously healthy 49 years old male was transferred from another hospital with a two-week history of severe, intractable back pain, short-term memory loss fatigue and shortness of breath. On exam he was afebrile, icteric and had multiple ecchymoses on the trunk. Complete blood count revealed severe anemia and trombocytopenia, with serum hemoglobin of 4.6 g/dl, a platelet count of 16  109/L and white blood bell count of 3.22 x 109/L. There was evidence of severe hemolysis with serum lactate dehydrogenase of 2659 U/L and Serum Haptoglobin of 7 mg/dL. Direct Coombs test was negative. The absolute reticulocyte count was 42.6 K/uL. The peripheral smear revealed schystocytes and thrombocytopenia. A CT scan of chest, abdomen and pelvis showed splenomegaly, and was otherwise unremarkable. The initial bone marrow aspirate and biopsy revealed extensive necrosis of bone marrow with marked reduction of normal hematopoietic elements. Flow cytometry was negative for a clonal lymphoid proliferation. The repeat contralateral iliac crest biopsy revealed among the extensive necrosis, small aggregates of highly atypical cells with pleomorphic hyperchromatic nuclei. Some cells had signet ring morphology and they stained positive with mucicarmin and cytokeratin stains, which indicate a carcinomatous origin. The patient received multiple blood transfusions, high dose steroids and plasmapheresis, but none of these interventions had a significant impact on the clinical course. His delirium worsened, developed respiratory failure and expired on the 7th day of hospitalization. IMPLICATIONS/DISCUSSION: Massive bone marrow necrosis is known to occur in the context of malignancy, usually hematopoietic, lymphomas, leukemias, but also solid tumors. At the same time TTP has been reported to occur in the setting of neoplasms, especially adenocarcinomas, usually gastric, pancreatic and breast. The association of TTP and massive bone marrow necrosis, although rarely reported in the literature may represent as illustrated in our case report the heralding picture of metastatic cancer. It is important for physicians to be aware of this association as it forces one to search for an unapparent neoplasm. and pelvis with subsequent abdominal MRI confirmed cavitary lesions in the left upper lobe and revealed a 12  10  7.5 cm adrenal mass. A serum ACTH was low at 3 pg/mL (normal 9± 52 pg/mL) and a serum free morning cortisol of greater than 50 mcg/dL (normal 6±20 pg/mL) confiming a diagnosis of Cushing syndrome. Urine catacholamines were normal. The adrenal mass was later surgically removed with pathology confirming a diagnosis of adrenocortical carcinoma. Sputum cultures obtained by bronchoscopy grew significant colonies of Nocardia Farcinica and Nocardia Nova. The patient was started on intravenous trimethoprimsulfamethoxazole for six weeks followed by oral therapy. IMPLICATIONS/DISCUSSION: Nocardiosis is a rare finding in immunocompromised patients with an estimate of 500±1000 new cases per year. Most cases involve the lung with inhalation as the mode of entry. Although an immunocompromised state predisposes patients to Nocardia infection, steroid use is considered to be an independent risk factor. Our patient, while not taking exogenous steroids, did have very high levels of endogenous cortisol. Definitive diagnosis is typically made by routine culture of an isolated specimen. The most effective treatment for Nocardiosis remains speculative and is based mostly on small retrospective studies and case reports. Trimethoprim-sulfamethoxazole demonstrates synergy against Nocardia and general consensus supports high doses of this drug initially followed by long-term treatment with lower doses for up to one year due to the high degree of recurrence of disease. As demonstrated in this case, one should consider Cushing syndrome as a risk factor for the development of Nocardia infections. year-old man presented with one day of abdominal and back pain. He was a daily heroin user and his last injection was two hours prior to presentation. During his evaluation, he became uncooperative and combative, subsequently leaving against medical advice. Two hours later he was found disoriented on the street. His temperature was 39 C; he had nuchal rigidity and tenderness to palpation over the lumbar spine. There were no murmurs, purpura, or petechiae. He rapidly developed respiratory failure and was intubated. Blood, urine, and CSF cultures grew methicillin-resistant Staphylococcus aureus. He received seven days of vancomycin, and rifampin, After stopping a seven day course of antibiotics, his fever returned. A repeat lumbar puncture revealed persistently positive MRSA. Vancomycin and rifampin were restarted , and his fever resolved. An MRI of the lumbar spine demonstrated pyomyosits of the paraspinal muscles. He received 42 days of vancomycin and rifampin with clinical improvement. IMPLICATIONS/DISCUSSION: Back pain in the intravenous drug user, especially when associated with fever, must prompt high suspicion for meningitis, discitis, oteomyelitis of pyomyositis. Although meningitis from Staphyloccocus aureus is usually secondary to neurosurgical procedures, intravenous drug users are susceptible to staph CNS infections due to their frequent innoculation and impaired immunity. With the increased availability of antibiotics on the street, methicillin-resistant Staphyloccocus aureus is increasingly becoming a community acquired pathogen. As this case illustrates, serious CNS infections in an intravenous drug user may warrant empiric vancomycin until definitive cultures return. Staphyloccocus aureus bacteremia requires at least two weeks of intravenous antibiotics; longer courses are required for deep-seated infections. A secondary focus should always be sought, as Staph is a rare primary infection. This case illustrates the importance of maintaining a high degree of suspicion for Methicillin-resistant Staphyloccocus aureus in intravenous drug users presenting with back pain and fever.\nMITOCHONDRIAL TOXICITY AND HIV THERAPY. S. Burridge 1 , L. Coberly 1 ; 1 University of Cincinnati, Cincinnati, OH (Tracking ID #75159) LEARNING OBJECTIVES: Recognize the clinical presentation of mitochondrial toxicity secondary to nucleoside reverse transcriptase inhibitors. CASE INFORMATION: A 43-year-old white male with AIDS presented with altered mental status, nausea, vomiting, right upper quadrant pain and myalgias. Medications included didanosine, efavirenz, lopinavir/ritonavir and stavudine. His temperature was 101.28F, HR 120, BP 135/85 and RR of 26. Physical examination was significant for bitemporal wasting, oral thrush, dry oral mucosa, ketotic breath, pain to palpation in the right upper quadrant and a liver span of 20cm. There were no identified sources of infection. Laboratory examination was significant for sodium 126mmol/L, potassium 5.1mmol/L, chloride 88mmol/L, and bicarbonate 13mmol/L. Lactate was markedly elevated at 9.9mmol/L. Transaminases were 111 IU/L and 128 IU/L, alkaline phosphatase was 209 IU/L and total bilirubin was 4.10 mg/ dL. Triglycerides were greater than 1,000 mg/dL. Abdominal CT revealed hepatomegaly with fatty infiltrate. Medication toxicity was suspected and HAART therapy was discontinued. Riboflavin supplementation was initiated. IMPLICATIONS/DISCUSSION: Mitochondrial toxicity is a rare syndrome that has been reported in patients treated with nucleoside reverse transcriptase inhibitors, particularly zalcitabine, didanosine, and stavudine. The diagnosis is clinical. Patients present with nausea/ vomiting, abdominal pain, weight loss, malaise, fatigue, dyspnea and sometimes fever. Laboratory evaluation reveals lactic acidosis, hypertriglyceridemia and elevated liver enzymes. Imaging studies demonstrate hepatomegaly with fatty infiltration. The mechanism of mitochondrial injury is postulated to be inhibition of the mitochondrial gamma polymerase, the enzyme that directs the replication of mitochondrial DNA. The mitochondria are unable to replicate, the citric acid cycle is blocked and lactate production increases. Fatty acid oxidation is inhibited leading to accumulation of lipid droplets. Treatment involves the administration of mitochondrial supportive therapy such as riboflavin, thiamine, ubiquinone and acetyl-carnitine in addition to the cessation of antiretroviral therapy. Unrecognized, this syndrome carries a 60% mortality attributed to worsening acidosis with resulting cardiovascular collapse or progressive hepatic failure. (Tracking ID #76589) LEARNING OBJECTIVES: To recognize the dilemma posed by an inconclusive MRI in cases of intracerebral mucormycosis and to emphasize the need for brain biopsy. CASE INFORMATION: A 42 year-old male with HIV presented with a five-day history of headache, fever, left sided weakness and multiple falls. Physical exam showed a left sided, central, facial nerve palsy, weakness and left sided hyperreflexia. The patient's CD4 count was 56. Cryptococcus, toxopolasmosis, tuberculous meningitis, atypical infectious agents and lymphoma were considered in the working differential diagnosis. CT scanning and CSF analysis were negative. MRI revealed a mass in the right basal ganglia with associated edema and a midline shift. An MRI guided brain biopsy showed mucormycosis. In spite of amphotericin B therapy he expired ten days later. IMPLICATIONS/DISCUSSION: Mucormycosis in an immunocompromised host with HIV has rarely been described in the literature. Notably, in such cases neither CT nor MRI scanning is sufficient to make the diagnosis. In these instances, surgical exploration is mandatory and can provide crucial diagnostic information.\nDOWN BUT NOT OUT. S. Agresta 1 , J. Canterbury 1 , J. Wiese 1 ; 1 Tulane University, New Orleans, LA (Tracking ID #77016) LEARNING OBJECTIVES: 1. Recognize an under-diagnosed cause of rhabdomyolysis. CASE INFORMATION: A 55 year-old obese man presented with severe shortness of breath after being found at his nursing home. His morbid obesity (550 pounds) confined him to a wheelchair. On the evening prior to admission, he fell from his wheelchair without a loss of consciousness or traumatic injury. A nursing aid found him twelve hours later. At presentation, he complained of diffuse pain; he noted associated shortness of breath but no chest pain or palpitations. His heart and breath sounds were normal; pitting edema extended to the knees. His vital signs were normal. His urine was scant (less than 10 cc/hr) and dark brown in color. His CPK was 35,000 U/L, BUN 75 mg/dL and creatinine 2.1 mg/dL. IMPLICATIONS/DISCUSSION: Although rhabdomyolysis is usually seen with crush injuries or toxic myopathies, prolonged stasis and extended isometric contraction as occurs with seizures should also prompt immediate evaluation for the syndrome. A timely diagnosis is essential, as the degree of renal tubular necrosis is proportional to the time and duration of tubular exposure to the myoglobin pigment. Aggressive diuresis decreases this exposure time; this is the main focus of treatment. The color of the urine (brown as opposed to yellow or clear) predicts the pigment concentration in the tubules, and is a better measure of severity and treatment efficacy than the CPK level. Early diuresis, with or without alkalinization, should be maintained until the urine is consistently clear. Acute renal failure is often attributed to the primary disease (sepsis, MI) in patients found down. Rhabdomyolysis should be excluded as an independent cause of renal failure in all patients found down for an undetermined or prolonged period of time.\nLISTEN TO ME, NOT MY THYROID. E. Chan 1 , J. Wiese 1 ; 1 Tulane University, New Orleans, LA (Tracking ID #77077) LEARNING OBJECTIVES: 1. Listen to the patient's previous history to establish appropriate management decisions. 2. Use the history of the patient's symptoms to distinguish Graves disease from propylthiouracil allergy. CASE INFORMATION: A 23 year-old woman presented with recurrent palpitations, muscle weakness and dyspnea. She had a past history of Graves disease but had refused propylthiouracil therapy. She had a heart rate of 130, and a temperature of 104 F. There was right eye exophthalmos, a diffusely enlarged thyroid. A venous hum was heard over the thyroid; she had proximal muscle weakness and hyperreflexia. The thyrotropin levelwas 0.06U/ ml and the FT4 was 17.01 g/dl. She was treated with with potassium iodide, propylthiouracil and propranolol with resoluton of her symptoms. Three days later, she developed severe generalized arthralgias and myalgias with progressively deteriorating muscle strength and swelling of her fingers. Her anti-nuclear antibody, anti-histone antibody, and acetylcholine receptor antibody were negative. The anti-striated muscle antibody, rheumatoid factor and p-ANCA were also negative. The propylthiouracil was discontinued based upon the hypothesis that the new symptoms were due to an allergic response, Her symptoms resolved the following day. Upon further questioning, she noted a similar reaction to a previous treatement with propythiouracil as the cause of her non-compliance with this therap. A subtotal thyroidectomy, resulted in complete resolution of her symptoms. IMPLICATIONS/DISCUSSION: The appropriate management decision is often obtained by listening to the patient. In the thrill of treating Graves's disease, the reason for the patient's previous non-compliance with propylthiouracil was ignored. Although an allergic reaction to propylthiouracil is rare, it is characterized by arthralgias, myalgias, anxiety and tachycardia when it occurs. Distinguishing this reaction from continued symptoms of Graves's disease requires a meticulous history. Thyroidectomy is the treatment of choice in patients who are unable to tolerate propylthiouracil therapy. Carefully listening to why patients have declined previous therapy is important in preventing recurrence of previous adverse effects.\nincluded rolling and lifting weights on an exercise ball. After returning home he became nauseated, diaphoretic, weak, and experienced excruciating left flank pain followed by an episode of watery diarrhea. His symptoms worsened and he presented to the emergency room the following morning. Physical examination at that time revealed a well-nourished man in marked distress secondary to intractable pain. There was significant tenderness to minimal palpation and percussion in his lower left quadrant and over his left costovertebral angle region. An initial computerized tomography (CT) of the abdomen showed an insignificant left renal cyst. Laboratory evaluation was significant for leukocytosis with a left shift, mild normocytic anemia, slightly elevated creatinine and CPK, and urine which showed pyuria but was sterile. Overnight he experienced fever and worsening pain with apparent peritoneal signs. The next morning a chest x-ray was done which showed no evidence of pneumonia. CT of the abdomen was repeated. In view of the patient's declining condition, this subsequent CT was read as a possible abscess. Aspiration of the renal abscess grew Streptococcus pneumoniae, as did all blood cultures. Predisposing factors for pneumococcal bacteremia were excluded. He recovered fully after a two-week course of ceftriaxone and four weeks of levofloxacin. IMPLICATIONS/DISCUSSION: Streptococcus pneumoniae is a very uncommon pathogen in renal abscesses. Extrapulmonary pneumococcal infection is most often metastatic from the lung. No respiratory disease was noted in our patient. Only two published cases of Streptococcus pneumoniae renal abscesses have been reported. These will be discussed, one in full detail. The etiology of our patient's Streptococcus pneumoniae continues to be a mystery. Two hypotheses will be presented. LEARNING OBJECTIVES: Often uncommon diseases present initially in the primary care setting, and appropriate referral and diagnosis are essential. The following case illustrates an unusual skin condition seen in a primary care setting in order to expand the generalist physician's differential diagnosis of dermatologic conditions associated with renal failure. CASE INFORMATION: The patient is a 36-year-old woman with a history of type I diabetes complicated by renal failure for which she underwent a kidney-pancreas transplant ten years ago. Her transplanted kidney remained functional until three months prior to presentation, at which time hemodialysis (HD) was initiated. Within one month of starting HD, she developed thickening and hardening of the skin on her left hand, which rapidly expanded to involve the entire left arm, and the right hand and arm. Within two months, the feet, lower legs, buttocks, and areas of the back had areas of skin hardening. The face, abdomen, and chest were spared. These areas of skin hardening were associated with minimal overlying skin changes, but she developed burning paresthesias during this time for which she was treated with oxcarbazepine. A skin biopsy confirmed the diagnosis of nephrogenic sclerosing dermopathy (NSD), with the findings of increased numbers of dermal spindle cells and thick collagen bundles in the reticular dermis. IMPLICATIONS/DISCUSSION: NSD was first described in September 2000 in a case series of 14 patients with a fibrotic skin condition resembling scleromyxedema. Each patient had a history of renal failure and 12 of the patients were receiving HD at the time of diagnosis. Patients displayed skin thickening and hardening with overlying brawny skin discoloration, which was localized to the torso and limbs with sparing of the face. Several patients complained of painful paresthesias beginning with the onset of skin hardening. In each case, the skin condition has been progressive with no known effective therapy. The etiology of NSD is unclear, but infectious agents or toxic agents (toxins in the dialysate, endogenous toxins not cleared by dialysis) have been proposed. Resolution of the skin lesions occurred in one case when HD was stopped following resolution of acute tubular necrosis. Through accurate referral and diagnosis of patients, more can be learned about the etiology, prevention, and treatment of NSD. Early recognition of NSD may allow early referral for transplantation to prevent progression of disease on HD, although there is not definitive evidence to date that NSD regresses following transplantation. A 58 year old Mexican man presented with progressive shortness of breath and a dry cough for over 1 month. Review of systems was positive for dyspnea on exertion, three pillow orthopnea, and an eight pound weight loss. His past history is noncontributory, and he uses no medication, tobacco or alcohol. On physical exam breath sounds were decreased with bilateral rales 1/2 way up. He was tachycardic with a loud P2, a jugular venous pressure of 10cm and 2+ edema to the shins. Chest x-ray showed bilateral pleural effusions; right greater than left. Echocardiogram revealed an ejection fraction of 70% with pulmonary hypertension. Laboratories were significant for a normocytic anemia with low iron, TIBC and reticulocyte count; ESR 116; urinalysis with trace protein and 20 red cells; and negative serologies for hepatitis B/C, HIV, and coccidiodes. CT chest was negative for pulmonary embolism and infiltrate. Diagnostic thoracentesis removed 200cc of hazy fluid with pH 7.48, 1250 white cells (81% lymphocytes) and cultures negative for bacteria, fungi and acid fast bacilli (AFB). Cytology was negative for malignant cells. Rapid reaccumulation later prompted removal of another 1L of fluid. A tuberculin skin test was 8 mm but sputum AFBs were negative. Further work-up revealed a positive ANA (1:2560 titer) and strongly positive anti-Smith antibody with negative anti-dsDNA and ANCA. He was diagnosed with SLE and started on prednisone 40 mg daily with resolution of the effusion and improvement in pulmonary hypertension. IMPLICATIONS/DISCUSSION: Pleural effusion is the initial manifestation in 1%, and pleurisy in 3%, of SLE patients, more often in those with late-onset (after age 50). Lupus pleuritis usually presents as pleuritic chest pain with fever, cough and dyspnea but rarely with massive effusions. Pleural symptoms may precede other manifestations of SLE by months or years, particularly in men. To diagnose pleural effusions caused by lupus pleuritis, one must rule out other causes including pulmonary embolism, congestive heart failure, infections (i.e. parapneumonic or tuberculous), and malignancy. Effusion ANA titer detection is a very sensitive but nonspecific test while anti-DNA antibodies or LE cells in the pleural fluid are diagnostic but not very sensitive. Treatment includes NSAIDs for mild cases and systemic corticosteroids for more severe cases. Early diagnosis and prompt treatment can greatly decrease morbidity from this disease. year old man presents with complaints of progressive leg swelling, erythema, 30 lb weight loss, and severe bilateral hip pain over 3 months. He initially noted several fluid filled blisters and pedal edema that spontaneously resolved. Over the next several weeks he described progressive bilateral hip pain requiring the use of crutches for ambulation. The month prior to admission the patient was bed bound secondary to pain. He denied other constitutional symptoms. Of note, he had eliminated fruits and vegetables from his diet 3 years ago. The patient had emigrated from Ethiopia 35 years ago. Physical exam revealed poor dentition, a perifollicular petechial rash of his lower extremities, hyperpigmentation of his upper thighs and bilateral lower extremitity edema. MRI was significant for lower extremity soft tissue edema and osteoporotic femoral heads with abnormal marrow signal. Laboratory values were notable for a normocytic anemia, normal coagulation panel, and electrolyte panel and a Vitamin C level of <0.12 mg/dL. Punch biopsy of the petechial lesions demonstrated perifollicular hemorrhage and corkscrew hair follicles. Vitamin C supplemenation 1000mg per day was initiated with resolution of symptoms over several weeks. Repeat MRI of his LE noted resolution of soft tissue edema and improving osteoporosis. IMPLICATIONS/DISCUSSION: Symptoms of scurvy have been described as early as 1500 BC by the ancient Egyptians. In contemporary history, epidemics have been described in the Renaissance era, when sailors were frequently affected, and during the 19th century, by those who experienced the Great Potato Famine, American Civil War armies, and the California Gold Rush communities. A cofactor and strong reducing agent, vitamin C is crucial to numerous biochemical pathways including collagen synthesis, norepinephrine synthesis, prostaglandin synthesis, and antioxidant function. Collagen synthesis disruption in particular leads to the clinical syndrome of scurvy including ecchymoses, bleeding gums, petechiae, and arthralgias. Other signs and symptoms include muscle pain, microcytic anemia, decreased wound healing, mood disturbances, and anorexia. While scurvy occurs rarely in the US, typically it is seen in elderly or alcoholic patients and those with diets devoid of fruits and vegetables. Sudden cardiac death has been reported in these patients however, morbidity and mortality largely occurs as a result of hemorrhage which can occur in any organ. Skin biopsy findings of perifollicular hemorrhage and follicle corkscrewing are pathopneumonic for the disorder. Symptoms typically improve over days to weeks with the addition of Vitamin C to the diet. Prognosis is excellent with timely diagnosis and treatment.\nNOT JUST ANOTHER DIARRHEA: A CASE OF SECONDARY AMYLOIDOSIS ASSOCIATED WITH LYMPHOMA. J. Chowdhury 1 , A. Gomez 1 ; 1 UCLA San Fernando Valley, Sylmar, CA (Tracking ID #76168) LEARNING OBJECTIVES: 1) Recognize lymphoma as a chronic inflammatory state that can be associated with secondary amyloidosis 2) Distinguish between primary and secondary amyloidosis. CASE INFORMATION: A 44 year old Hispanic male with non-Hodgkin's lymphoma, diagnosed and treated in Mexico with chemotherapy and radiation five years ago presents with five months of large volume, watery diarrhea. He had four to six episodes of blood-tinged diarrhea per day, with abdominal cramps, nausea and vomiting. The patient had anorexia with a twenty pound weight loss, orthopnea and bilateral lower extremity edema. His blood pressure was 60/palp, pulse 88, and he was afebrile. He had dullness to percussion and decreased breath sounds over the left lower lobe as well as 1+ pitting edema in BUE and 2+ pitting edema in the BLE. A CBC was normal, BUN 38, creatinine 1.3, PT 29.6, INR 2.47, PTT 47, albumin 1.4, AST 10, ALT 13, alkaline phosphatase 153, total bili <0.1, LDH 762. Urinalysis showed small blood, 3+ proteinuria, 2 rbc's and 3 wbc's. CXR showed a left-sided pleural effusion. The patient was admitted to the ICU and started on IV steroids and pressors. Thoracentesis revealed a transudate with many lymphocytes, but no malignant cells. CT scans revealed a mass in the left upper lobe of the lung and left hemithorax volume loss, colon dilation to 6 cm and small bowel dilation to 3 cm. Biopsy of the lung mass revealed Hodgkin's lymphoma. Bone marrow biopsy was positive for both crystal violet and Congo Red, suggesting amyloidosis. Rectal biopsy showed friable mucosa and revealed apple green birefringency when viewed with Congo Red stain, confirming the diagnosis of gastrointestinal amyloidosis. Unfortunately he succumbed to the disease a week later while on steroids and chemotherapy. IMPLICATIONS/DISCUSSION: Both primary and secondary amyloidosis present in a variety of ways, including heavy proteinuria, edema, hepatosplenomegaly, hematochezia, congestive heart failure, and carpal tunnel syndrome. Secondary, or AA, amyloidosis occurs in chronic inflammatory states, such as rheumatoid arthritis, inflammatory bowel disease, or osteomyelitis. However, reports of an association with lymphoma are rare. Obtaining a good past history for chronic inflammation is nevertheless important when diagnosing AA amyloidosis. The only disease linked to primary, or AL, amyloidosis is multiple myeloma. Once the diagnosis of amyloidosis is made from tissue biopsy, the distinction between primary and secondary amyloidosis is made based on the presence of a chronic inflammatory state in secondary amyloidosis or the presence of certain paraproteins, found in about 90% of primary amyloidosis. We diagnosed this patient as secondary amyloidosis based on his history of lymphoma.\n``MY TOOTH IS KILLING ME!'': OVER THE COUNTER ANALGESIC RISKS AND HEPATITIS C. E. Coffey 1 , E. Schneider 1 ; 1 Hennepin County Medical Center, Minneapolis, MN (Tracking ID #76561) LEARNING OBJECTIVES: 1. Recognize increased risks of acetaminophen and nonsteroidal antiinflammatory drugs (NSAIDs) for patients with Hepatitis C. 2. Recognize importance of adequate medical and medication history, even for common complaints. CASE INFORMATION: 50 year old male presented to a walk-in clinic seeking medication for a two week old toothache. Nonprescription pills at home didn't help. He also reported 2 days of nausea, vomiting, loose bowel movements, diffuse myalgias, generalized weakness. He denied alcohol, infectious contacts. PMH included hepatitis C, alcohol and cocaine use. Physical exam remarkable only for an ill appearing male with an abscessed tooth and mild abdominal tenderness. Labs included K 3.8, BUN 32, creatinine 2.9, ALT 3460, AST 2575, bilirubin 5.1, alk p'tase 135, lipase 111, INR 1.4. Acetaminophen level 52. Salicylate level was not elevated. He later admitted to taking up to 20 tablets of naproxen and acetaminophen a day over the last few days. He was admitted to the medicine service, and improved with acetylcysteine and supportive care. He was counselled about acetaminophen use. A dentist extracted the abscessed tooth before discharge. IMPLICATIONS/DISCUSSION: Acetaminophen and NSAIDs are associated with increased transaminases in patients with hepatitis C. Renal failure has been associated with NSAIDs and with paracetamol poisoning.This case illustrates that the presenting problem isn't always the most serious. The non-pain symptoms, past history and specific information on his self medication were keys to diagnosis. Given potential toxicities of analgesics, patients with hepatitis C are well advised to seek advice for pain issues rather than self medicating.\nDIAGNOSIS AND TREATMENT OF POST PRANDIAL NAUSEA, ALLERGY AND EOSINOPHILIA. D. Coleman 1 , J.S. Scolapio 1 , J.R. Cangemi 1 , J.C. Guarderas 1 ; 1 Mayo Clinic Jacksonville, Jacksonville, FL (Tracking ID #76262) LEARNING OBJECTIVES: Eosinophilic gastroenteritis, is a disorder characterized by infiltration of eosinophils into the mucosa of the gastrointestinal tract. As a clinical entity with recognizable symptoms it is a rare disorder. Here we discuss the extensive treatment course of a refractory case of eosinophilic gastroenteritis. CASE INFORMATION: A 16-year-old male patient who presented with post-prandial fullness and nausea. He had a history of seasonal allergies, asthma, and peripheral eosinophilia. Endoscopy of the stomach with mucosal biopsies revealed predominate eosinophils. A diagnosis of eosinophilic gastroenteritis was made. He was treated with a proton pump inhibitor (Prilosec) an oral cromolyn (for gastrointestinal symptoms), initially. After no response, he began on high dose prednisone with a gradual taper, with clinical response, to every other day prednisone indefinitely. Several years later, he again experienced symptoms of dysphagia, early satiety, nausea and occasional vomiting. He had been compliant with his medications. The oral steroids (20 mg every other day) had not been tapered because of recurrent symptoms upon dose reduction. As a result of his prolonged predisone use, bone mineral analysis was performed and showed signs of early osteoporosis. He was treated with Fosamax 5 mg a day, vitmain D 50,000 IU twice a week, and Calcium 1000 mg a day. A trial of an elemental nutrition formula was attempted in hopes of reducing his gastrointestinal eosinophilia. Although his peripheral eosinophil count significantly decreased on this elemental diet (Vivonex) he was unable to continue with it because of its poor taste. Given his refractory symptoms and continued risk of complications from chronic steroid use he was treated with Interferon Alpha, starting at a low dose and gradually increased to a dose of 1 million units per day. His peripheral blood eosinophils initially dropped from 5410 to 1020 but then slowly began to increase. In early 2002 his gastrointestinal symptoms recurred and his peripheral blood eosinophils began to rise. He had been compliant with medications. Because the majority of his symptoms were gastroesophageal, we elected to try oral flonase. Since this newest therapy was begun, he has experienced a marked drop in peripheral blood eosinophils (the lowest to date) and his gastrointestinal symptoms have markedly improved. IMPLICATIONS/DISCUSSION: This case is unique because it illustrates an unusual approach to a refractory case of eosinophilic gastroenteritis. Most known and reported therapies had failed in our patient with the exception of high dose steroids and, more recently, oral Flonase. Unfortunately his dependence upon corticosteroids resulted in the serious sequelae of osteoporosis. Although a systemic illness with marked peripheral eosinophils, the symptomatology of this patient was predominantly gastrointestinal. RAPIDLY PROGRESSIVE HEPATOSPLENOMEGALY. K. Cooke 1 , S. Rusch 1 ; 1 University of Illinois, Peoria, IL (Tracking ID #76040) LEARNING OBJECTIVES: Discuss the differential diagnosis of rapidly progressive hepatosplenomegaly while highlighting a typical presentation of an uncommon lymphoma. CASE INFORMATION: A previously healthy 32-year-old woman initially presented to her primary care physician with pleuritic left shoulder pain and a normal physical exam. Laboratory results then included platelets of 104,000/L; alkaline phosphatase of 125 U/L; alanine aminotransferase of 58 U/L; and aspartate aminotransferase of 65 U/L. Two weeks later, the patient returned with acute onset of fevers, fatigue and nausea. Physical examination was benign except for marked hepatosplenomegaly. The liver was palpable 11cm below the right costal margin and the spleen 5cm below the left costal margin. Laboratory results included platelets of 42,000/L; alkaline phosphatase of 1,045 U/L; alanine aminotransferase of 285 U/L; and aspartate aminotransferase of 610 U/L. Admission computed tomography showed diffuse homogenous enlargement of the liver, spleen and kidney with a few mildly enlarged retroperitoneal lymph nodes. Serologic evaluation was negative for hepatitis A, B and C viruses, cytomegalovirus, Epstein Barr virus, herpes simplex virus, toxoplasmosis and human immunodeficiency virus. Bone marrow biopsy on day two of admission was negative for malignancy, infection and amyloid. Screening for antinuclear antibody and extractable nuclear antigen antibodies was negative. The patient's history did not reveal risk factors for unusual infections or toxins. Noninvasive doppler ultrasound was not consistent with Budd Chiari. Blood cultures were negative and alpha-fetoprotein level was within normal limits. During the ensuing four days, the patient rapidly deteriorated with additional liver enlargement of 4cm, falling platelet count and metabolic acidosis. On day five of admission, a transjugular liver biopsy demonstrated alphabeta-hepatosplenic T-cell lymphoma. IMPLICATIONS/DISCUSSION: Rapidly progressive hepatosplenomegaly is not widely discussed in the literature. However, reviewing MEDLINE and EMBASE databases, one can conclude that it is most common in hematologic malignancies and infection. The above typical presentation of an uncommon peripheral T-cell lymphoma illustrates that lymphoma can present without lymphadenopathy or bone marrow involvement. Hepatosplenic T-cell lymphoma is an aggressive cancer with a poor prognosis. Despite aggressive treatment, our patient died on the 28th hospital day.\nA PAIR OF PAINFUL HANDS. A. Cooper 1 ; 1 University of Pennsylvania, Philadelphia, PA (Tracking ID #73721) LEARNING OBJECTIVES: 1. To review the American College of Rheumatology diagnostic criteria for rheumatoid arthritis (RA). 2. To discuss initial treatment of newly diagnosed RA. CASE INFORMATION: A 39 y/o homeless woman with a past medical history significant for hypertension, diabetes, hepatitis C, and asthma on no medications was admitted to the inpatient psychiatric unit for depression with suicidal intent. The medicine service was consulted for evaluation of hand pain. The patient had been having 2 months of progressively worse pain in both her hands, as well as 1 month of swelling and morning stiffness lasting 90 minutes before improvement. The symptoms made it difficult for her to make a fist or hold her cigarette. Over the last 1 week, all of her symptoms markedly increased. Additionally, she recently noted a sensation of numbness and tingling in her first three fingertips of the right hand that wakes her from sleep. The patient denied any fevers, chills, night sweats or other joint involvement. She denied dry eyes or mouth, as well as urethritis/vaginitis. Review of systems in detail was otherwise negative. Social history was significant for a 20 pack year smoking history. Family history was noncontributory. Physical exam was significant for bilateral symmetric swelling, mild erythema and boggy tenderness of 8 proximal interphalangeal joints. There was no involvement of the distal interphalangeal joints or thumbs and minimal tenderness of the MCP joints. There was no wrist tenderness. Remainder of the joint exam was unremarkable. Remainder of the physical exam was significant for a blood pressure of 130/90, mild expiratory wheezes bilaterally at the lung bases, and a positive Phalen's sign. Pertinant negatives included a temperature of 97.9, moist mucous membranes, no cardiac murmurs, no rashes or nodules and a negative Tinel's sign. Laboratory results were significant for normal electrolytes, renal function, TSH, WBC and hemoglobin levels. The ESR was normal at 9. Anti-Scl 70, dsDNA, and complement levels were normal. The ANA was weakly positive at 1:160. The patient had a positive RF of 104 IU/mL (normal 0±29 IU/mL). Parvovirus IgM was negative, as were Hep B serologies and HIV. IMPLICATIONS/DISCUSSION: Discussion: This patient was diagnosed with early moderately severe RA. The American College of Rheumatology criteria for diagnosis of RA were initially developed to help classify patients for clinical studies. However, they have proved useful in diagnosis as well. The guidelines are as follows: A patient has RA if at least 4 of these criteria are met (the 1st 4 of which must have been present at least 6 weeks): 1) Morning stiffness, usually lasting at least 1 hour before maximal improvement. 2) Arthritis of at least 3 or more joint areas. 3) Arthritis of hand joints. 4) Symmetric arthritis. 5) Rheumatoid nodules. 6) Serum rheumatoid factor. 7) Radiographic changes±erosions, unequivocal bony decalcification adjacent to affected joints. Radiographic changes are present in 15% of patients in the 1st year of disease, in >90% of patients after the 1st 2 years. The initial diagnosis of RA may be difficult because early RA may satisfy few of the diagnostic criteria. Additionally, systemic lupus erythematosis, Sjogren's syndrome, and mixed connective tissue disease may also commonly present with a positive rheumatoid factor, and may be difficult to differentiate from RA early in the course of illness. Finally, seronegative RA, or RA presenting in a single large joint such as the knee, poses it's own obvious diagnostic dilemma. The diagnosis of RA is supported by findings of carpal tunnel syndrome, as seen in this patient, as well as weight loss, fever, anemia, serositis, pulmonary nodules and peripheral neuropathy. Therapy for RA is usually initiated with non-steroidal antiinflammatory drugs (NSAIDs). Prednisone is added for rapid improvement when severe joint symptoms are present, and is usually tapered after inflammation is controlled. Disease modifying anti-rheumatic agents (DMARDs) are added if symptoms are not controlled with NSAIDs, if symptoms are moderate or severe in nature, or if radiographic changes are present. DMARDS are medications that have been proven to reduce the rate of bony destruction in RA. The most common DMARD used for initial treatment is methotrexate, started at a dose of 7.5 mg/week. The maximum dose of methotrexate is 25 mg subcutaneously/week. It is important to monitor patients for methotrexate toxiticy including liver function tests (cirrhosis), pulmonary symptoms (interstitial lung disease), and bone marrow toxicity. LEARNING OBJECTIVES: 1) Apply epidemiologic factors to decisions of whether to screen for hepatocellular carcinoma (HCC) in chronically Hepatitis B-infected individuals. 2) Recognize the limitations of current screening modalities for HCC. CASE INFORMATION: An asymptomatic 23 year-old male student presents to clinic after being told by a former sexual partner that he had``given her an infection.'' He has no significant past medical or surgical history. His review of systems is negative. He was born in China and immigrated to the United States at two years of age. He uses a half-pack-per-day of tobacco, alcohol to intoxication 1±2 times per week, no recreational drug use. His physical examination is normal. His laboratory values are significant for being hepatitis B (HBV) surface antigen positive, surface antibody negative, core antibody positive, e antigen negative, and HBV viral load undetectable. Hepatitis C and Human Immunodeficiency Virus (HIV) are negative. His liver function tests are all within normal limits.\nObservational studies show that among perinatally-infected individuals, incidence of HCC begins rising in young adulthood (20±30 years of age). Among those who are infected later in life the incidence of HCC begins rising at 40 to 50 years of age. Other major risk factors for HCC include presence of cirrhosis, e-antigenemia, exposure to other hepatotoxins including alcohol and tobacco, and co-infection with hepatitis C virus. HCC screening is usually done at 6±12 month intervals. Common modalities used include serum alpha fetal protein (AFP), ultrasound (US), and computed tomography (CT). The usefulness of AFP is limited by sensitivity (approximately 60%) and specificity (approximately 85%). Thus, US or CT must be used in conjunction with AFP. Ultrasound, the most widely-used modality, has a variable sensitivity determined by operator experience, reported to be 35% to 84%. CT, with less operator-dependent variability and sensitivity greater than 90%, is the most expensive of screening modalities. Given the limitations of existing screening methods and overall small survival-benefits of current widely-available treatments, the cost-effectiveness of HCC screening has been questioned. There are no randomized studies assessing cost-effectiveness of HCC screening. Non-randomized studies have shown conflicting results. Thus, although a recent survey of U.S. hepatologists showed that 84% of respondents screen for HCC in individuals with cirrhosis, no official guidelines have yet been provided by the National Cancer Institute (NCI).\nALONG CAME A SPIDER . . . . . . CAUSING UNUSUAL ULCERS. N. Correia 1 , R. Abou Jawde 1 , S. Baghdasarian 1 , S. Schmitt 1 ; 1 Cleveland Clinic Foundation, Cleveland, OH (Tracking ID #76813) LEARNING OBJECTIVES: To emphasize the extensive differential diagnosis of lower extremity (LE) ulcers, the incidence of pyoderma gangrenosum (PG) following trauma and the importance of accurate diagnosis, we present a case in which lesions that appeared following a spider bite were intially treated as infection. CASE INFORMATION: A 59 year old man presented with non-healing painful left LE ulcers. He was bitten by a spider after which he noted a small red tender spot over his shin that progressed to an ulcer with purulent drainage. He was treated with oral and intravenous antibiotics, local wound care and surgical debridement without improvement. During treatment he developed a new ulcer on the same leg and an inflamed nodule over the contralateral malleolus. IMPLICATIONS/DISCUSSION: PG, a deforming ulcerative skin disease, usually presents with erythematous nodules or vesiculopustules on the legs. The lesions undergo a rapidly destructive necrotizing process to form large ulcers with surrounding erythema. Although frequently associated with inflammatory bowel disease or immunosuppression, in adults PG can occur spontaneously, following trauma (including spider bites), or in association systemic diseases. Biopsy confirmed the presence of PG in our patient and he improved dramatically on prednisone.\nSARCOIDOSIS PRESENTING AS TESTICULAR MASS: CASE REPORT AND REVIEW OF THE LITERATURE. L. Corsino-Nunez 1 , M. Kam 1 , C. Kaye 1 ; 1 Wayne State University, Detroit, MI (Tracking ID #75657) LEARNING OBJECTIVES: To recognize sarcoid presenting as a testicular mass. CASE INFORMATION: Sarcoidosis a chronic multisystem disease of unknown etiology characterized by noncaseating granulomas. The most commonly involved organs are the lungs, lymph nodes, skin, uvea, liver and bone. Sarcoidosis of the male urogenital tract is uncommon. Physicians need to be aware of this occurrence especially in males with a known history of sarcoidosis. Case: A 31 y/o African American male presented to the Emergency Department complaining of dyspnea, fever and night sweats. Past medical history was significant for asthma. Admission physical exam was remarkable for bilateral, nontender, mobile, cervical lymph nodes and a right testicular mass. Chest-x-ray revealed mediastinal and bilateral hilar lymphadenopathy. Computarized Axial Tomography scan of the chest, abdomen, and pelvis was consistent for bilateral lymph nodes in the axillary and inguinal region. Ultrasonography of the right testicule was done and showed marked enlargement of the right epididymus and small hydrocele. Biopsy of one of the submandibular lymph node revealed non-caseating granulomas. A right orchiectomy was performed. The pathology report was consistent with non-caseating granuloma involving the epidiymis and focal testicular parenchyma. The spermatic cord was free of granulomatous inflammation. IMPLICATIONS/DISCUSSION: Discussion: Sarcoidosis with testicular involvement is rare. Only 0.2% of patients with sarcoidosis have urogenital involvement. Forty-three cases have been described in the English language medical literature. We want to enhance physicians' awareness of urogenital sarcoidosis and include in the differential diagnosis of a palpable testicular mass. The treatment for urogenital sarcoid is corticosteroids, identical to systemic sarcoid. Review of the literature shows that a great unnecessary percentage of patients with testicular sarcoid have undergone orchiectomy. Thus awareness of this condition may result in prevention of this radical approach in males who present with sarcoidosis and a testicular mass. LEARNING OBJECTIVES: To recognize Pott±Puffy Tumor as a complication of sinusitis. CASE INFORMATION: Pott±Puffy Tumor is defined as subperiostial abscess and osteomyelitis of the frontal bone seen as a complication of frontal sinusitis. With the advent of antibiotics, Pott±Puffy Tumor is a rarely recognized entity. A 31 y/o white female, previously healthy until one day prior to admission, presented with frontal headaches, dull in nature that progressively worsened, associated with remarkable forehead swelling, and dizziness. One week prior to admission she received a two-week course of cefuroxime for frontal sinusitis. Her past medical history was significant for fibrodysplasia of the ethmoidal sinus during childhood requiring five surgical interventions. Admission physical exam was remarkable for forehead swelling with tenderness over the frontal bone and absence of fever. Computerized axial tomography scan of the head revealed opacification of the frontal sinus. The frontal sinus was drained and a purulent discharge was obtained. Gram stain and culture showed no organisms or growth. Frontal headache and swelling improved after drainage. She was placed on ampicillin/sulbactam for a total of two months. She was scheduled for frontal sinus obliteration after completing the antibiotics. IMPLICATIONS/DISCUSSION: Pott±Puffy tumor represents a rare complication of frontal sinusitis. Only 21 cases have been reported after the antibiotic era, of wish 7 have presented in adults. The importance of making this diagnosis is to prevent expansion of the infection to the brain. We would like to highlight the importance of making an accurate diagnosis, especially in patients treated for frontal sinusitis. Five cases in which no organisms have been identified are reported in the literature. This entity can be successfully treated with antibiotics and drainage. LEARNING OBJECTIVES: Recognize the importance of secondary causes for hypertension. CASE INFORMATION: A 51 year old diabetic gentleman with obstructive sleep apnea treated with CPAP and a long-standing history of hypertension came to our clinic for a routine visit. Despite five anti-hypertensive medications, blood pressures remained near 180/100. This prompted a search for secondary hypertension. Studies were negative for renal vascular disease and pheochromocytoma as well as primary hyperaldosteronism. Despite preserved renal function, Nephrology consultation was obtained with the resulting suggestion that this may be a case of occult noncompliance. However, the patient regularly attended appointments and filled all of his antihypertensives in a timely manner. This convinced us to look further into additional possible rare causes for secondary hypertension. On one subsequent follow-up visit, the patient did complain of jaw changes and a thick tongue. He has coarse facial features, a large fleshy nose and large tongue along with multiple skin tags and large hands. The constellation of hypertension, sleep apnea, diabetes, and a documented history of carpal tunnel syndrome, along with his complaints and findings raised the possibility of acromegaly as a reason for refractory hypertension. For the initial workup, a screening insulinlike growth factor 1 level was then ordered, which returned at 2.5 times the normal range. A subsequent confirmatory glucose tolerance test with measurement of growth hormone levels then failed to show GH suppression with a glucose load, supporting the diagnosis of acromegaly. IMPLICATIONS/DISCUSSION: Acromegaly is a somatic growth and proportion disorder with elevated levels of GH and ILGF-1 found as hallmarks of this syndrome. It is most often caused by GH-secreting pituitary tumors. Some of its features include skeletal overgrowth, arthropathy, skin changes, cardiovascular disease and respiratory dysfunction. Hypertension is one important complication of acromegaly, with the prevalence of hypertension in acromegalic patients estimated to be about 35%, although there is no consensus in the medical literature regarding the exact mechanisms involved in its pathogenesis. While hypertension is a common condition treated by internists, fewer than 5% of cases are found to have secondary causes. This case highlights the need to consider rare causes of hypertension in patients resistant to treatment, and demonstrates why acromegaly should be considered in the differential.\nTHE TUMOR THAT MELTED AWAY. J. Cunningham 1 , M. Panda 1 ; 1 University of Tennessee±Chattanooga Unit, Chattanooga, TN (Tracking ID #76984) LEARNING OBJECTIVES: 1. To recognize the similarity in the radiographical and clinical presentation of pulmonary blastomycosis and neoplasms. 2. Stress the importance of a high index of suspicion to avoid misdiagnosis and unnecessary invasive diagnostic procedures. CASE INFORMATION: 57 year old white female with a 40 pack/year history of tobacco abuse presented with persistent cough, mucoid and occasionally bloody sputum for 2 months. She denied fever, chills, night sweats, dyspnea, or recent travel. Her vital signs and room air oxygenation were normal. Physical exam revealed diffusely diminished breath sounds with no accessory muscle use or clubbing. Labs were normal except for a normocytic anemia, and thrombocytosis. CXR showed a right upper lobe infiltrate. CT revealed a large right hilar structure surrounding the distal main stem bronchus with impingement, infiltrative changes surrounding the mass, and an enlarged precarinal lymph node. An endobronchial biopsy and aspiration of the mass revealed no neoplastic cells. Gram stain was negative, and cultures revealed no fungal growth after 28 days. Aspiration smears from the precarinal area were nondiagnostic, but smears from the postcarinal area revealed Blastomyces dermatitidis. She was treated with Itraconazole with complete resolution on repeat CT scan after 6 months. IMPLICATIONS/DISCUSSION: Blastomycosis is a pyogranulomatous infection that can involve many organ systems, but primarily affects the lungs. Infection results from inhalation of the Blastomyces conidia, which may be asymptomatic or present in ways indistinguishable from TB, bacterial pneumonia, ARDS, histoplasmosis, and most ominously bronchogenic carcinoma. Most cases occur in areas surrounding main waterways in North America. Demographics indicate no specific sex, age, race, occupation or seasonal predilection, but exposure to soil is a common factor linking infections. Blastomycosis radiographically mimics many lung diseases with carcinoma being the most common primary diagnosis. Definitive diagnosis is made by isolation in sputum smears or growth in sputum culture which may take up to 30 days. Bronchoscopy requires special considerations in order to obtain a higher yield of fungus. Treatment with lidocaine inhibits growth & limiting concentration to 1g/dL has been recommended. B. dermatitidis can be missed on H&E specimens, so silver or PAS stains should be used. If no diagnosis results invasive thoracoscopy or thorocotomy are often done hastily before the fungus has had time to grow in culture because of the common presentation of Blastomycosis and carcinoma. Blastomycosis infection has been known as``the great masquerader'', therefore, it is very important for physicians to be familiar with the epidemiology in order to develop an index of suspicion for the fungus in endemic areas. Thus, patients can be spared from the distress of undergoing unnecessary testing and invasive surgical procedures. year old Latino male presented to the emergency room with a five day history of severe sharp, pulsatile left sided neck pain, worsened with exertion and head movement with flexion and lateral rotation. This neck pain waxed and waned in intensity throughout the five days prior to admission. The patient reported weakness and numbness of the left arm, which he had used while driving to the emergency room. This weakness resolved with lowering of the left arm. He denied having associated headaches or visual disturbances. The patient was afebrile with a blood pressure of 127/76. Physical examination revealed marked tenderness upon palpation over the left carotid artery. Carotid artery bruits were not present on auscultation. No thyroid tenderness, enlargement, or nodularity was evident. Funduscopic examination, evaluation of cranial nerve function, and assessment of motor and sensory function in the upper and lower extremities were within normal limits. The patient was admitted for treatment of possible left carotid artery dissection and anticoagulation therapy with unfractionated heparin was initiated. An MRI/MRA of the neck was subsequently performed to evaluate for carotid artery dissection. No abnormal flow voids or evidence of dissection were noted. A sonogram of the neck failed to reveal evidence of dissection. ESR was 5, CBC was within normal limits. The diagnosis of carotidynia was made, and the patient's symptoms resolved with prednisone and NSAIDS. IMPLICATIONS/DISCUSSION: Carotidynia is an idiopathic neck pain syndrome arising from the cervical carotid artery resulting in unilateral neck pain that frequently radiates to the ipsilateral face and ear and sometimes to the head. Facial pain is a common primary symptom and carotid artery tenderness and overlying soft tissue swelling are the major physical findings. Two forms of this disorder have been described, an acute form that results in a monophasic illness usually lasting less than two weeks and tending not to recur, and a chronic recurring form which manifests as a vascular headache variant, responsive to antimigraine therapy. Both forms are responsive to the administration of corticosteroids. An evaluation for structural disease of the carotid arteries should be considered in patients with ipsilateral neck pain. MRI/ MRA of the neck has proven highly sensitive. Other imaging modalities to consider include ultrasound and angiography. The diagnosis of carotidynia should be considered only after careful evaluation for other etiologies of neck pain. Considerations for ipsilateral neck pain in the region of the carotid artery near its bifurcation include carotid dissection, migraine headache, thyroiditis, aneurysm of the carotid system, neck neoplasms, or rarely, temporal arteritis.\nAN UNUSUAL CAUSE OF CHEST PAIN: SPONTANEOUS PNEUMOTHORAX. S. Daud 1 , R. Granieri 1 ; 1 University of Pittsburgh, Pittsburgh, PA (Tracking ID #73682) LEARNING OBJECTIVES: 1) Recognize spontaneous pneumothorax as an etiology of chest pain. 2) Describe the typical presentation of spontaneous pneumothorax 3) Recognize the treatment options and prognosis of spontaneous pneumothorax. CASE INFORMATION: A previously healthy seventeen year-old male was seen in the emergency department for complaints of chest pain. His pain was described as a sharp pain above his nipples, bilaterally, which initially had occurred at rest. The pain was neither exacerbated nor relieved by movement or deep inspiration. There were no associated palpitations nor diaphoresis or vomiting. He was taking no medications, but smoked marijuana weekly for the past few months. He occasionally smoked cigarettes, but never consumed alcoholic beverages. There was no family history of coronary artery disease, hypercoagulable states, or vasculitis. Examination revealed a 5 H 11 HH young man in mild distress. His weight was 60 kg, his respiratory rate was 16, and his heart rate was 84. He had no oral lesions and had normal dentition. The cardiac exam revealed normal heart sounds and no murmurs. The lungs were clear to auscultation and had a normal percussion note. The abdomen was scaphoid and non-tender. There was no peripheral edema. Electrocardiogram and pulse oximetry were normal. A chest roentgenogram revealed bilateral apical pneumothoraces. They were estimated to be 10% each. Bilateral apical drainage catheters were placed and the patient's symptoms resolved. The patient was admitted to the hospital for observation. IMPLICATIONS/DISCUSSION: Primary spontaneous pneumothorax is defined as a pneumothorax without a precipitating factor such as trauma, in a patient without clinically evident lung disease. It typically occurs in tall, thin males between 10 and 30 years of age. The use of smoking tobacco has been reported to increase the risk in men by as much as a factor of 20 in a dose dependent manner. Subpleural bullae are found in virtually all patients; their mechanism of formation is not completely understood, but is thought to be related to degradation of elastic fibers in the lung. This process is thought to be accelerated by smoking. The primary spontaneous pneumonthorax results in a decrease in vital capacity, and an increase in the alveolar-arterial oxygen gradient, leading to hypoxemia. Because underlying lung function is normal, hypercapnia is not usually seen. Most patients present with ipsilateral pleuritic chest pain occurring at rest. Tachycardia is frequently the only abnormal physical finding. The diagnosis is confirmed by the identification of a thin, visceral pleural line displaced from the chest wall on chest roentgenography. Treatment with observation, needle aspiration, or drainage through a chest tube all have been advocated. The average rate of recurrence is about 30%, with pulmonary fibrosis, asthenic habitus, and history of smoking all thought to be risk factors for recurrence. (Tracking ID #73698) LEARNING OBJECTIVES: 1) Recognize the common causes and workup of diffuse pulmonary infiltrates. 2) Recognize the usual presentation of desquamative interstitial pneumonitis. CASE INFORMATION: A 45 year-old male with a history of COPD and multiple pneumonias within the past year was admitted to the hospital for complaints of cough productive of yellow sputum, fevers, chills, and worsening shortness of breath. He was found to be febrile and hypoxemic, and have a leukocytosis with a predominance of neutrophils. A chest x-ray showed the presence of diffuse interstitial infiltrates. He was admitted to the hospital and treated for pneumonia with antibiotics and bronchodilators for 11 days without significant improvement. Sputum and blood cultures were negative for bacterial pathogens. Computed tomography (CT) of the thorax showed ground-glass opacities in all lung fields, worse on the right. Pulmonary function testing revealed mild restrictive disease with decreased lung volumes and decreased DLCO. He did not improve clinically and was scheduled for open lung biopsy. Pathology revealed the diagnosis of desquamative interstitial pneumonitis. He was started on Prednisone 60mg daily, had significant clinical improvement and was discharged from the hospital. CT of the thorax performed one month later showed near-complete resolution of the ground-glass infiltrates. IMPLICATIONS/DISCUSSION: The differential diagnosis of a patient with diffuse pulmonary infiltrates is broad. Acute diffuse lung diseases (less than 4±6 weeks in duration) include infection, pulmonary edema, pulmonary hemorrhage, hypersensitivity pneumonitis, and acute eosinophilic pneumonia. Chronic diffuse lung diseases include idiopathic pulmonary fibrosis, sarcoidosis, pneumoconioses, and interstitial lung disease associated with connective tissue diseases. Desquamative interstitial pneumonitis (DIP) is one of the distinct clinical and histopathological entities belonging to the family of idiopathic interstitial pneumonias. The average age of onset is about 45 years of age, and a male preponderance has been noted. Cigarette smoking has been linked consistently to this diagnosis. The typical presentation is an insidious onset of dyspnea and cough. Chest roentgeography often shows a diffuse pattern of reticulonodular opacities, with CT showing a ground-glass appearance. Pulmonary function testing shows a restrictive pattern. Diagnosis is best made with lung biopsy demonstrating the characteristic appearance of alveolar filling by mononuclear cells and thickening of the alveolar septa. Pateints are generally treated with corticosteroids until symptoms are improved at which point they are tapered slowly. Data are lacking regarding the optimal duration of treatment and prognosis of DIP. A 44 year old African American woman presented with the acute onset of shortness of breath and substernal chest pain with radiation of the pain to the neck and left arm. She was diagnosed with acute myocardial infarction with positive CK and isoennymes and troponin-T. Initial ECG did not indicate an acute injury pattern, however ST-T wave change in V5 and V6 suggested lateral ischemia. Platelet count on initial presentation was 36,000. Her initial Hgb was 9.2. No previous laboratory data were available to review. She was initially treated with aspirin, low molecular weight heparin, and GIIB/IIIA inhibitors. Subsequent to this her blood count was repeated and platelet count had dropped to 11,000. At this time her peripheral smear was reviewed and 20±25 schistocytes were identified per high power field. The case was discussed with the Hematology division and plasmapheresis was initiated within 12 hours. After two double volume exchange transfusions, her platelet count had rebounded to 50,000 and her LDH was falling as well. Her platelet count slowly continued to improve over the course of her hospitalization. At discharge on hospital day #8, her platelet count was 236,000. At no point did the patient have renal dysfunction, neurologic symptoms, or fever. IMPLICATIONS/DISCUSSION: TTP is characterized by arteriolar lesions in various organs that contain platelets and fibrin. This results in MHA, and thrombocytopenia. The tissue hypoxia that ensues is responsible for the symptoms. Myocardial vessel involvement may be a cause of sudden death in some patients. However the isolated involvement of myocardial vessels is rare, especially when there is no of involvement of renal and cerebral vessels. TTP is treated with plasmapheresis initially. The disease is now 90% survivable if therapy is initiated promptly. Before this therapy was available, TTP had a 90% mortality. Plasmapheresis is conducted until platelet count, LDH, and MHA resolves. At that time, pheresis is gradually weaned. Those patients who relapse can be treated with corticosteroids, cyclophosphamide, or vincristine. Splenectomy is also an option. LEARNING OBJECTIVES: 1. Diagnose B12 deficiency on clinical grounds 2. Recognize the relationship between H. pylori infection and B12 deficiency 3. Assess potential complications of B12 deficiency. CASE INFORMATION: A 65 year old male was admitted to our hospital with a two month history of progressive gait instability, weakness and bilateral lower extremity numbness. He was unable to walk without assistance, was totally dependant for activities of daily living, had recently become incontinent of urine and had developed severe constipation. He also complained of mild numbness, tingling and weakness in his upper limbs, and had difficulties with hand motor control. He denied fevers, myalgias, pain, involuntary limb movements, headaches, or trauma, however he did have a forty pound weight loss over the previous three months. He had no significant past medical history and was on no medications. Physical exam showed no cranial nerve defects, mild proximal weakness and proprioceptive loss, with severe, diminished lower extremity strength, proprioception, vibration and sensation. Lower extremity tendon reflexes were brisk with positive Babinskis. Bilateral crackles at mid lung fields were evident on chest exam. Laboratory values on admission were significant for hemoglobin of 10.8 g/dl, mean corpuscular volume of 106.8 fL, and B12 of 75 ng/L (190±900). Arterial blood gas on 2L of oxygen showed pH of 7.456, PCO2 of 40.4, and PO2 of 69.0. With the laboratory diagnosis of B12 deficiency, a homocysteine level was obtained and was significantly elevated at 188 mmol/L (0±13). Chest X-ray showed extensive bilateral reticular infiltrates and small bilateral effusions. Due to these findings, a computed tomography of the chest was done which revealed left apical bullae, extensive ground glass opacities, and multiple filling defects in the interlobar and branch arteries consistent with pulmonary emboli. Lower extremity Doppler ultrasound confirmed deep vein thrombosis (DVT). An esophagogastroduodenoscopy was ordered to help determine the cause of the B12 deficiency. This showed mild chronic gastritis and H. pylori organisms. Serum studies for intrinsic factor and parietal cell antibodies were negative. He was treated with intravenous B12, triple therapy for H. pylori, anti-coagulation and oxygen support, however his respiratory status declined over the next three to four weeks and he died approximately one month after admission. Autopsy confirmed sub acute combined degeneration in the spinal cord and organizing thromboemboli with diffuse alveolar damage of the lungs. IMPLICATIONS/DISCUSSION: This case demonstrates a cascade of events that most likely began with the relatively common finding of H. pylori gastritis. Our patient then developed B12 deficiency, which has a known association with this infection. This in turn contributed to elevated homocysteine by interfering with its conversion to methionine. This contributed to a hypercoagulable state and in combination with immobility from the B12 deficiency induced sub acute combined degeneration, led to the DVT and pulmonary emboli. This directly contributed to the patient's respiratory decline. This case demonstrates that when H. pylori gastritis is confirmed, the association between this infection, B12 deficiency and hyperhomocysteinemia should be considered, as this can have profound consequences. 3) Discriminate between opiate dependence and addiction. CASE INFORMATION: A 29-year-old male with a history of hemophilia and hepatitis C presented with recurrent episodes of crampy abdominal pain over the past seven months. He denied fevers, chills, nausea, vomiting. There was no change in bowel habits or alteration of the pain with food or defecation. He denied previous surgeries or intravenous drug use. Physical exam showed no fever, heart rate 106, dilated pupils, diaphoresis, and a soft abdomen with voluntary guarding. The patient had undergone an extensive prior workup for these symptoms including CT scanning, colonoscopy, and upper endoscopy-all of which were normal. On our further review we learned that the patient had been taking two narcotic medications prescribed from different doctors. He described occasionally taking these medications for pain control, but was unaware both were narcotics. His symptoms completely resolved after starting a low dose methadone taper, and the patient was grateful for ending a seven-month cycle of pain. IMPLICATIONS/DISCUSSION: The symptoms of opiate withdrawal can include sweating, headache, nausea/vomiting, abdominal cramping, diarrhea, dilated pupils, piloerection, insomnia, agitation, and anxiety. Opiate withdrawal can occur from chronic prescription narcotic use, but may go unrecognized by patients and physicians. In our patient with hemophilia, prescription narcotics rather than NSAIDS were given for minor pains. His abdominal pain along with dilated pupils and diaphoresis suggested withdrawal leading to a more extensive review of his medication use. Despite using his medications as prescribed, his chronic use led to physical dependence. Addiction is defined as the compulsive use of a substance despite negative consequences. Dependence, however, does not usually have the psychological associations that are seen in addiction, making it harder to recognize. Nonetheless, patients with physical dependence can experience severe distress, as did our patient. Opiate withdrawal causing abdominal pain remains a diagnosis of exclusion and other serious causes should always be ruled out. LEARNING OBJECTIVES: 1. Discuss approach to fever in travelers 2. Recognize Dengue fever presentation 3. Realize importance of controlling mosquito vectors. CASE INFORMATION: 23 year old male with no prior history presents with 3 days of fever, malaise, headache and body aches. He returned to the US 6 days prior after a month-long backpacking trip in Brazil. His fever has no pattern and is relieved with Ibuprofen. His headache is bitemporal with some associated photophobia and neck pain. His body aches are generalized but most severe in the lower back. He denied HIV risk factors and review of systems is negative. He did not receive any vaccines or prophylactic medications. His bp is normal, pulse 70, and temperature 38.6 degrees Centigrade with malaise and a maculopapular rash over the trunk. Otherwise exam was negative. At this point the differential included flu, malaria, typhoid fever, leptospirosis, acute HIV, and Dengue fever. Laboratory investigation revealed a mild leukopenia with normal platelet count. CSF, HIV p24 Ag, thick and thin smear, RPR were all negative. Dengue titer came back positive for serum ELISA IgM. He was treated symptomatically with resolution of symptoms within a week's time. IMPLICATIONS/DISCUSSION: Dengue fever is caused by a single stranded RNA virus in the family Flaviviridae (found throughout the tropics) and transmitted to humans by the Aedes aegyptia mosquito. With urbanization and subsequent inadequate waste and water management, Dengue incidence is increasing in South America and the Caribbean. There is a disease spectrum with Dengue ranging from a relatively non-specific illness, to Dengue fever (acute illness with leukopenia, headache, malaise, myalgias/arthralgias) to Dengue hemorrhagic fever (the above plus positive tourniquet sign, thrombocytopenia, and increase vascular permeability) to Dengue shock syndrome (the above plus hypotension and narrow pulse pressure). Diagnosis of Dengue is usually by isolation of the virus or serologic testing such as ELISA for IgM. Dengue fever is typically self-limited with symptoms lasting appoximately 7 days. Warning signs for Dengue shock syndrome include sustained abdominal pain, persistent vomiting, change of level of consciousness, and a sudden decrease in platelet count. Dengue has 4 serotypes and repeat infection with another serotype is more likely to casue severe disease. In addition to improved mosquito control, a vaccine is currently being developed.\nBLOOD IS THICKER THAN WATER: THE MANAGEMENT OF HYPERVISCOSITY IN ADULTS WITH CYANOTIC HEART DISEASE. A. DeFilippis 1 , S. Aaberg 1 ; 1 Emory University, Decatur, GA (Tracking ID #76037) LEARNING OBJECTIVES: 1. Recognize the clinical presentation of hyperviscosity syndrome in patient with chronic hypoxia. 2. To recognise the complications of phlebotomy in the treatment of erythrocytosis of cyanotic heart disease. 3. To recognise the appropriate use of hydroxyurea in the treatment of erythrocytosis of cyanotic heart disease. CASE INFORMATION: A 19-year-old male with uncorrected tetralogy of Fallot presented to the emergency room after a ground level fall after climbing two steps. The fall was preceded by two episodes of lightheadedness. He denied vertigo, bowel or bladder incontinence, chest pain, dyspnea, or palpitations. At presentation, he was afebrile with a pulse of 90 beats per minute, blood pressure of 117/63, 30 respirations per minute and an oxygen saturation of 79%. Physical examination was remarkable for a well developed, alert and oriented male in no distress, clubbing in all digits, normal S1 and S2 heart tones with a II/VI early systolic ejection murmur heard throughout the chest; a cardiac heave over the left side of the chest, no jugular venous distention, no peripheral edema or crackles. Chest radiograph revealed a large bootshaped heart and right-sided aortic arch. EKG was notable for a normal sinus rhythm with left axis deviation and right atrial enlargement. Remarkable laboratory values were hemoglobin of 24g/dL and a hematocrit of 76 %. The patient was phlebotomized 500ml of blood and discharged from the hospital after remaining in normal sinus rhythm and asymptomatic for 24 hours. IMPLICATIONS/DISCUSSION: Complications of chronic hypoxia including erythrocytosis, hyperviscosity, abnormalities of hemostasis, cerebral abscesses or stroke and endocarditis are among the most common consequences of cyanotic heart disease in adults. The compensatory erythrocytosis of cyanotic heart disease can become pathologic by causing an increase in blood viscosity, which decreases perfusion, resulting in a decrease in total oxygen delivery and an increased risk of veno-occlusive/hyperviscosity syndrome. Treatment of hyperviscosity secondary to erythrocytosis in cyanotic heart disease is controversial. Data suggest that phlebotomy has the potential to increase exercise capacity, reduce the symptoms of hyperviscosity syndrome and reduce the potential increased risk of vasoocclusive disease in selected patients with polycythemia secondary to cyanotic heart disease. Unfortunately, repeated phlebotomy can quickly lead to iron deficiency. Microcytic erythrocytes produce higher viscosity than normocytic erythrocytes at comparable hematocrits putting patients at high risk for vasoocclusive events. There is limited data on the use of hydroxyurea to suppress erythrocytosis in this patient population. Thus we concluded that until newer approaches to decreasing the hematocrit, without causing iron deficiency anemia, are shown to be safe and efficacious, phlebotomy should only be used for the acute resolution of hyperviscosity symptoms; and the use of hydroxyurea should be limited to recurrently symptomatic patients.\nCARDIAC SARCOID AND RISK OF SUDDEN DEATH. A. DeFilippis 1 , A. Palacio 1 , D.W. Brady 1 ; 1 Emory University, Decatur, GA (Tracking ID #76794) LEARNING OBJECTIVES: 1. Recognize the clinical presentation of cardiac sarcoid. 2. Treat cardiac sarcoid and its inherent risk for sudden cardiac death. CASE INFORMATION: A 58-year-old female with cutaneous sarcoid presents to the emergency room with a 3 month history of progressive dyspnea on exertion. She also reports a 2-week history of proximal nocturnal dyspnea, cough, 2-pillow orthopnea and lower extremity edema. She denies chest pain, fever, chills, or palpitations. Her past medical history includes hypertension, diabetes, hypercholesterolemia and obstructive sleep apnea. On presentation, she was afebrile, pulse 106, blood pressure 120/60, respiratory rate of 28 and oxygen saturation of 88% on room air. Physical exam was significant for S3 gallop, normal pulmonary exam, lower extremity edema and several silver dollar size hyperpigmented lesions on her legs. Routine laboratory testing was unremarkable, chest radiograph revealed mild cardiomegaly as compared to a normal radiography 1 month earlier. EKG revealed a new right conduction system block as compared to a normal EKG 2 years earlier. High resolution CT scan was negative for pulmonary embolus and deep venous thrombosis but revealed multiple enlarged mediastinal lymph nodes. Echocardiogram was notable for hypokinetic lateral and inferior wall motion, new from a normal dobutamine stress echocardiogram 2 years earlier. Cardiac catheterization revealed normal coronary angiography, an ejection fraction of 20% and regional wall motion abnormalities, which lead to a cardiac biopsy that revealed non-caseating granulomas consistent with active cardiac sarcoid. The patient improved with diuresis and systemic afterload reduction. She was started on a 6-month course of prednisone and equipped with an automated internal cardiac defibrillator with rescue pacing, prior to discharge from the hospital. IMPLICATIONS/DISCUSSION: Dyspnea and cough are amongst the most common clinical presentations of sarcoid but are attributed to pulmonary rather than cardiac pathology. Although less than 10% of sarcoid patients demonstrate symptoms secondary to cardiac sarcoid, cardiac involvement is found in up 76% of sarcoid patients at autopsy and accounts for 50% of the mortality in this patient population. The most common presentation of cardiac sarcoid, found in approximately 50% of patients, is arrhythmias or cardiac conduction abnormalities as a result of granuloma formation in the conduction system. Sarcoid granulomas in the ventricular myocardium may lead to ventricular arrhythmias in as many as 22% of patients with sarcoid. Granulomas in myocardium can result in diastolic or systolic dysfunction and has accounted for 25±75% of the deaths in some studies of cardiac sarcoid patients. Endomyocardial biopsy is highly specific but due to the non-homogeneous pattern of infiltration will only lead to a diagnosis of cardiac sarcoid in approximately 20% of cases. With arrhythmias or cardiac conduction abnormalities accounting for 30±65% of the deaths inpatients with cardiac sarcoid, automated internal cardiac defibrillators (AICD) with rescue pacing are standard of care in survivors of deadly arrhythmias. Although no trials have assessed the efficacy of AICDs in the absence of ventricular tachyarrhythmias, many advocate their use in all cases of known cardiac sarcoid; others reserve the use to patients with EKG abnormalities by standard of Holter monitor testing. Treatment of the underlying pathological process with immunosuppression is controversial and void of any large randomised trials to access efficacy. A small uncontrolled study demonstrated improvement and/or reversal of EKG abnormalities in greater than 50% of patients treated with corticosteroids. In one retrospective study of patients with cardiac sarcoid, 5-year survival was increased from 10% to 75% with the use of corticosteroids. Opponents to the use of corticosteroids raise concern that corticosteroids convert granulomas to scar tissue increasing the risk of aneurysms. Cardiac transplant has been successful with recurrent disease possible but rare.\nA WOMAN FROM CAMEROON WITH FEVER, ABDOMINAL PAIN AND EOSINOPHILIA. K. Dendrinos 1 ; 1 Boston University, Boston, MA (Tracking ID #75960) LEARNING OBJECTIVES: Review the Differential Diagnosis of Eosinophilia, Evaluate Fever of Unknown Origin. CASE INFORMATION: A 70 year old woman, recently emigrated to the U.S., presents to this hospital complaining of fever and chills for the past 3 months. She also complained of right and left upper quadrant pain. These complaints originally began in her home country of Cameroon, she came to the U.S. one week prior to her initial admission. During her first admission, she was febrile daily to 101 degrees Fahrenheit. Her exam was notable for right upper quadrant abdominal tenderness. Her laboratory examinations were significant for a CBC with an eosinophilia of 43%, that would peak later that month at 60%. Suspicion was high for a parasitic infection. Serologies would later be positive for multiple parasites endemic to Cameroon. She was treated with a wide array of antibiotics, including anti-tuberculosis medications, anti-fungals, and anti-helminthics. Her clinical course deteriorated and she died in the intensive care unit. On autopsy, she was found to have widely disseminated lymphoma involving nearly every organ system. IMPLICATIONS/DISCUSSION: This case initially appeared to be a classic presentation of a parasitic infection: a patient with fever and eosinophilia from an endemic area with positive serologies. It presents a chance to review the differential diagnosis of eosinophilia, and to again rethink the work-up of a fever of unknown origin. This case again demonstrates that``common things occur commonly'' (even when the patient comes from an unfamiliar part of the world). It also demonstrates the importance of keeping the differential diagnosis open, especially when the therapy instituted according to the initial dianosis fails.`L OCKING'' IN THE CASE FOR PREVENTIVE IMMUNIZATIONS. J. Derby 1 , C. Manhart 1 , C. Murphy 1 ; 1 Creighton University, Omaha, NE (Tracking ID #74723) LEARNING OBJECTIVES: Recognize that there are many patients at risk who have not received a primary series in tetanus. Distinguish a differential diagnosis of tetany. Manage a tetanus infection. CASE INFORMATION: A 71 year old female with no significant past medicial history and on no medications presented to the ER complaining of waking up in the middle of the night and being unable to move for the last 24 hours due to muscle stiffness. On initial exam she had marked neck stiffness with muscle spasms triggered by light touch and deliberate speech. A CT scan was unremarkable. A trial of IV diphenhydramine was administered to rule out a dystonic reaction. Stychnine levels and tetanus antibodies were drawn ( both later reported as negative). Over the next few hours obvious trismus developed. Her muscle spasms spread inferiorly to involve her abdominal wall, and her upper and lower extremities demonstrated lead pipe rigidity. The patient was intubated prophylactically and IV diazepam was begun. The patient was given tetanus immunoglobulin as well as a Td vaccination (since the illness is not protective). Metronidazole was administered IV. IMPLICATIONS/DISCUSSION: The majority of reported cases of tetanus in the US are in patients over the age of 60 years old. Currently, nearly 50% of elderly patients in the United States have non-protective titers. This is attributed to waning immunity in patients who have not received booster shots. Additionally, many elderly never received the primary series as children (which was not available until the 1950's). This patient had received one booster without a primary series which did not protect her,but may have prevented her mortality. Many immigrants as well, may have never received the primary tetanus series. Physicians must be diligent in immunizing at-risk groups and ensure that all patients whos tetanus immunization status is unknown receive a primary series. LEARNING OBJECTIVES: To analyze an interesting presentation of rhinocerebral mucormycosis and review current diagnostic and treatment modalities. CASE INFORMATION: The patient is a 26 yo African American male with a past medical history significant for Type I Diabetes Mellitus who presented with a chief complaint of leftsided facial swelling for nine days. He stated that the onset of facial swelling occurred over few hours and that the progression was rapid. Pt reported subjective fevers and chills. He denied upper respiratory or sinus-related symptoms. He initially presented to an outside hospital after two days of facial swelling. There he underwent a sinus CT that revealed mucosal thickening involving the left ethmoid, maxillary and ethmoid sinuses. He was treated with a seven day course of Rocephin and Cleocin. The patient was in DKA at the time of presentation and was treated for two days per hospital DKA protocol. On the day of discharge from this outside hospital the patient presented to our institution reporting that the facial swelling had not improved with prior therapies. Additionally, he complained of bilateral blurry vision with increased sensitivity to light. On physical examination the patient was afebrile. HEENT examination was significant for left-sided facial swelling with infraorbital involvement, a greenish-yellow discharge from the left medial canthus and left nares, photophobia and a necrotic plaque of the hard palate. The patient was unable to medially deviate his left eye. Laboratory work revealed a normal white cell count and differential, and a normal chemistry panel with the exception of an elevated serum glucose of 256. The differential diagnosis at this point included among other conditions fungal sinusitis, bacterial sinusitis, lymphoma and Wegener's granulomatosis. MRI of brain revealed extensive paranasal sinus opacification with abnormal dural enhancement extending from the left sphenoid sinus to involve the left cavernous sinus, left middle cranial fossa and left fifth cranial nerve with no intracranial, parenchymal or orbital involvement. The patient underwent a nasal biopsy that revealed multiple hyphal elements consistent with Mucormycosis. The patient was started on Amphotericin B at 1.5/mg/kg/day and underwent extensive surgical debridement. Although no orbital involvement was confirmed on MRI, the patient's cranial nerve palsy and opthalmoplegia were understood to be a manifestation of rhino-orbital mucormycosis. The hospital course was complicated by acute renal failure, septic shock, diabetic ketoacidosis and cardiac arrest. While serial MRIs showed no regression of disease, the patient was medically stabilized, and showed clinical improvement over an eight week hospital course. However, the patient's vision loss and opthalmoplegia did not improve with treatment. IMPLICATIONS/DISCUSSION: Rhizopus oryzae accounts for 90% of rhinocerebral mucormycosis. Patients in DKA are highly susceptible because the acidic, glucose rich environment provides a perfect medium for growth. The clinical presentation includes classic sinusitis symptoms and signs along with nasal necrosis (38%) and palatal or gingival necrosis (14%) as in the case of our patient. The diagnosis of mucormycosis is histologic, and it is the only fungus that can be seen with H & E stain. The mainstays of treatment are surgical debridement and reversal of predisposing factors. While lipid containing formulations allow for higher doses to be given due to reduced toxicity there may be problems with delivering the complex to infarcted tissue and obtaining effective concentrations. Administration of intravenous Amphotericin B along with alternative routes such as aerosolized and CSF perfusion pathways is an aggressive treatment modality. Hyperbaric oxygen is an accepted, but not commonly utilized adjuvant to treatment. Even with early detection and treatment the overall mortality rate in patients with rhinocerebral mucormycois remains high. A 59 year old homosexual Caucasian male presented with complaints of b/l hearing loss, tinnitus and vertigo for several months prior to presentation. Patient had an audiogram performed which showed moderate to severe symetrical sensorineural hearing loss. Patient did not have a history of recent aminoglycoside use and was not a diabetic. He was worked up with thyroid stimulating hormone, sedimentation rate, coagulation tests, rheumatoid factor and anti-nuclear antibody titers which were all within normal limits. PPR was positive at 1:16 and MHA-TP was positive at 4+. He refused an HIV test. Patient subsequently received a lumbar puncture which was positive for VDRL . He was thus diagnosed with neurosyphilis and admitted to the inpatient medicine wards where he received a 14 day course of IV Penicillin. Hearing loss improved with treatment of the neurosyphilis. IMPLICATIONS/DISCUSSION: Neurological manifestations of syphilis can occur during any phase of this disease as the central nervous system is seeded during spirochetemia. Up to forty percent of patients infected with Treponema pallidum will have cns involvement. Although neurosyphilis can be aymptomatic, symptomatic neurosyphilis can present earlier as syphilitic meningitis or later in the infection, can have a meningovascular or parenchymatous pesentation. Cranial nerve involvement, especially of cranial nerves II±VIII, can be seen. Syphilitic otitis is a form of neurosyphilis that can present as deafness, vertigo and/or tinnitus and should considered in patients who present sensorineural hearing loss. If treated early, syphilitic otitis may be curable, but can lead to irreversible damage if untreated. CASE INFORMATION: 78 years old man with coronary artery disease, emphysema and alcoholism presents with short of breath and palpitations. He had clear chest film, but was found to be in rapid atrial fibrillation and controlled with diltiazem. On day 3 of hospital stay, his mental status deteriorated and rapidly went into delirium tremens. He subsequently had temperature spike to 103.0(F) with associated leukocytosis. His oxygen saturation became very unstable and eventually requiring mechanical ventilation. He repeat chest X-ray showed right lower lobe consolidation. His sputum gram stain showed 4+ small gram negative rods which was identified to be Pasteurella Multocida. He had been a long time avid deer hunter and has three dogs which he is in close contact with. He was treated with a third generation cephalosporin and his condition slowly improved. His subsequent CT of chest revealed left lung nodule. The biopsy of the nodule was consistent with squamous cell carcinoma of the lung. IMPLICATIONS/DISCUSSION: Pasteurella Multocida is common organism in many domestic and wild animals. Human Pasteurella is a major pathogen in wound infections due to animal bites. Greater than 70% of the reported cases of Pasteurella Multocida pneumonia occurred in patients with non-traumatic exposure to colonized animals.\nRespiratory infection caused by Pasteurella Multocida can range from asymptomatic colonization to otitis media, sinusitis, epiglottitis, pneumonia, empyema and lung abscess. The degree of pulmonary infection depends on the immunity state of the host and can carry a very high mortality. Treatment of choice for Pasteurella Multocida infection is either penicillin, second or third generation cephalosporins, or doxycline. Prolonged antibiotic treatment is warranted in immnocompromised host. 2) To consider macrophagic myofasciitis in patients presenting with proximal muscle weakness and myalgias. CASE INFORMATION: Macrophagic myofasciitis is a syndrome of proximal muscle weakness, pain and fatigue. Clinical presentation is very similar to polymyositis, with proximal muscle pain, weakness, fatigue, fever and ternderness. Macrophagic myofasciitis is distinguished from polymyositis microscopically, with infiltration of the fascia with macrophages in MM. Cardiac involvement is found in over 70% of patients with polymyositis. We report a case of macrophagic myofasciitis presenting with proximal muscle weakness and ventricular tachycardia. A 49 year-old African American female with several months of progressive muscle weakness presents with palpitations and dizziness. Physical examination demonstrated significant proximal muscle weakness with no evidence of a skin rash. Laboratory data revealed a creatinine kinase of 1301 u/liter (normal <2oo), a CK-MB of 41.7 (normal 0± 60) and an aldolase of 40.2 (normal 1.5±8.1). An EKG showed a wide complex tachycardia with a rate of 220. A coronary angiogram was normal and electrophysiologic studies failed to induce ventricular tachycardia. Electromyopathy was consistent with a myopathy and her muscle biopsy revealed macrophagic infiltration of fascia, consistent with a diagnosis of macrophagic myofasciitis. Her ventricular tachycardia was postulated to have arisen from a myofasciitis. After initiation of high dose steroids, her muscle enzymes normalized and no further episodes of tachyarrhythmia occurred. IMPLICATIONS/DISCUSSION: MM is a relatively new muscle disease, with a first case report in 1993 in France. In several ways, it is similar to polymyositis, with bilateral muscle weakness and pain. Both are associated with an altered immune system. Cardiac arrhythmia in polymyositis is fairly common, occurring in over 70% of patients. These arrhythmias are due to an increase in spontaneous and disorganized myopathic contraction. There are no previous reports of cardiac disrhythmias in macrophagic myofasciitis. Most patients respond well to treatment with steroids and/or antibiotics in a few weeks. LEARNING OBJECTIVES: To recognize rare presentations of severe hypothyroidism. CASE INFORMATION: 18 year old African American female presented to the teen pregnancy center secondary to increased abdominal girth and amenorrhea. She also reported fatigue, hair loss, dyspnea on exertion, and sadness. Physical exam was significant for alopecia, a large goiter, markedly distended abdomen with fluid wave, tense edema of the hands, and delayed deep tendon reflexes. Labs were remarkable for a TSH of 240 with undetectable T4 and T3, marked microcytic anemia and an erythrocyte sedimentation rate of 140. CXR revealed a globular pericardial silhouette and ultrasound of the abdomen showed a large amount of ascites. Echocardiogram was unremarkable except for a moderate pericardial effusion. Pelvic ultrasound showed enlarged cystic ovaries, consistent with Van Wky Grumbach syndrome. Peritoneal fluid studies were consistent with myxedematous ascites. Upon further questioning, the patient stated that she had been treated for hypothyroidism as a young teen and had been lost to follow-up for two years. She also had a history of prior suicide attempts and depression. IMPLICATIONS/DISCUSSION: The manifestations of thyroid disorders are many, myxedematous ascites are a well-known but not often seen presentation. Serositis from hypothyroidism may be caused by several things: a capillary leak syndrome resulting from the dearth of thyroid hormones, an SIADH-like picture causing body cavity fluid accumulation, or high hyaluronate concentrations in the body fluid acting as an osmotic attractant. The anemia of hypothyroidism is thought to be caused by either direct bone marrow toxicity, lack of stimulation of erythropoiesis, decreased RBC survival time, and increased 2,3-DPG concentration. Van Wky Grumbach syndrome is a rare condition in which the TSH at high levels acts as an FSH analogue to cause ovarian hyperstimulation/cyst formation. This should be considered when a patient with no known thyroid disease presents with cystic ovaries. year old Hispanic male with no significant past medical history presents to our clinic with a one year history of intermittent bright red blood per rectum and two week history of mild dyspnea on exertion. He has previously been evaluated for the rectal bleed at various clinics and treated with topical hemorrhoidal ointments without relief. Patient denies chest pain, orthopnea, paroxysmal nocturnal dyspnea, abdominal pain, bowel changes, fever, chills, and weight loss. He is a sexually active homosexual. Family history is negative for colon cancer and polyps. Vital signs show temperature 100.3, BP 138/76, HR 75, RR 14. Physical exam is significant for mildly decreased breath sounds in left upper lobe and guaiac positive stool but no palpable rectal mass. Lab results show hemoglobin 10.9, MCV 78.0, and WBC 6.4. HIV test is negative. Colonoscopy with biopsy shows squamous cell cancer. Staging studies including chest x ray and a CT scan of the thorax display a 3.5 cm mass in the left upper lobe and CT pelvis confirms anorectal mass. Biopsy of the lung mass is consistent with a metastatic lesion. He is treated with chemotherapy and undergoes resection of the solitary lung mass. IMPLICATIONS/DISCUSSION: Anal canal carcinoma can be overlooked in young otherwise healthy patients as rectal bleeding is often attributed to hemorrhoids. Squamous cell cancer of the anal canal has a high association with human papilloma virus (HPV) particularly HPV-16. HPV DNA is isolated in 88% of anal cell cancers. Other risk factors include chronic immunosuppressive therapy, HIV, and smoking. Past treatment for anal canal carcinoma involved abdominoperineal resection with permanent colostomy. Cure rates with surgical resection ranged between 40±70%. However, in recent studies, chemotherapy with 5-FU and mitomycin combined with radiotherapy offer a 66±70% cure rate and lower rate of complications. Prognosis is dependent on the size of tumor, distant metastases, and nodal involvement. In conclusion, recurrent rectal bleeding in young patients with associated risk factors warrants a more thorough investigation. year old male presented with ten days of progressive difficulty walking, stating he had weakness in the lower extremities and in his right hand. He also reported slurred speech. Pt had no known medical problems and denied the use of medications, illicit drugs, tobacco or alcohol. Physical examination was significant for 3/5 right lower extremity strength with absent reflexes of the right leg. Sensory exam was grossly intact. The patient had poor performance of finger to nose testing and difficulty with rapid alternating movements. There was severe gait instability. Babinski's sign was bilaterally equivocal. CBC, chemistry panel, ESR, TSH, RPR, EKG were normal. After an unremarkable head CT, an MRI of the head showed a demyelinating pattern involving the middle cerebellar peduncles. The CSF protein, gram stain, culture, cell count, and glucose were within normal limits. On hospital day 4, HIV serology was positive and thus confirmed the clinical diagnosis of PML. CSF testing for JC virus later returned positive. IMPLICATIONS/DISCUSSION: PML is a demyelinating disease associated with infection of the JC virus. An AIDS defining condition, PML may present with rapid or progressive focal neurological deficits, including aphasia, ataxia, hemiparesis, hyporeflexia, and impaired cognition. In AIDS, PML implies a very poor prognosis with a survival expectancy of about six months. The diagnosis of PML is made through a clinical constellation which includes HIV positivity, characteristic MRI findings, and positivity for JC virus in the CSF. MRI findings of PML are similar to those seen in Acute Disseminated Encephalomyelitis (ADEM), a demyelinating process associated with infections or vaccinations in children, but they differ from those seen in Multiple Sclerosis (MS). The MRI with MS typically shows plaques in the periventricular region, corpus callosum, centrum semiovale, and less commonly in the deep white matter structures. There is no specific therapy for PML, and improvement of symptoms relies on increasing CD4 count via antiretroviral therapy. There has also been suggestion of improved clinical response with the addition of Cidofovir, a drug typically used to treat CMV retinitis.\nABDOMINAL PAIN: A CASE OF NARROWLY±FOCUSED DIFFERENTIAL. A.K. Duncan 1 , W.E. Wysokinski 1 , A.K. Ghosh 1 ; 1 Mayo Clinic, Rochester, MN (Tracking ID #74267) LEARNING OBJECTIVES: 1) Recognize the need to expedite the work-up of poorly responding abdominal pain; 2) Recognize that vasculitis could present with diarrhea. CASE INFORMATION: A 72-year-old women was transferred to our hospital for management of her diarrhea and weight loss. Three months prior to admission, she had an episode of upper respiratory tract infection and was treated with a 10 days course of antibiotics. One month prior to entry, she developed profuse diarrhea and malaise. EGD reveled mild gastritis, small bowel follow through and colonoscopy was normal and she was empirically treated with metronidazole for pseudomembranous colitis. She complained of increasing abdominal pain and was found to have air on an upright abdominal film. Exploratory laparotomy revealed necrosis, inflammation and perforation in the small bowel and nodular lesions in the liver. She underwent extensive small bowel resection, cholecystectomy, and liver biopsy. Liver histology was reported to reveal stellate granulomata consistent with Yersinia. However, cultures and serology for Yersinia were negative. She was empirically treated with ciprofloxacin and transferred to our hospital. Examination revealed an elderly woman, with mild dyspnea, and atrial fibrillation with HR of 110/mt. She was mildly icteric, with skin breakdown in the sacral and inter-scapular region. Chest examination revealed bilateral basilar crackles. Abdomen was mildly tender and distended. Neurological examination revealed generalized but asymmetric neuropathy. Re-evaluation of the small bowel biopsy revealed transmural fibrinoid necrosis with granulomatous inflammation typical of polyarteritis nodosa (PAN). EMG was consistent with mononeuritis multiplex. ANCA was negative. Visceral angiogram revealed micro-aneurysms in the spleen, kidney, jejunal and ileal branches. She was initiated on prednisone and pulse cyclophosphamide therapy with rapid improvement in symptoms. On follow-up at 3 months she continues to gain weight and has no new symptoms. IMPLICATIONS/DISCUSSION: PAN affects the gastrointestinal tract in over 40% of cases. PAN cases presenting with acute abdomen has been associated with a high mortality (60%). GI bleeding, perforation, pancreatitis, renal insufficiency, proteinuria, cardiomyopathy and CNS vasculitis are poor prognostic factors. A high index of suspicion and re-examination of a broad differential diagnosis in cases of acute abdomen, could lead to earlier detection of PAN and successful therapy of this potentially fatal condition.\nHYPOGLYCEMIA AND HYPONATREMIA: A HIGHER CAUSE. E.J. Dzielak 1 , N.C. Chiappetta 2 ; 1 Dunmore, PA; 2 Moses Taylor Hospital, Scranton, PA (Tracking ID #77122) LEARNING OBJECTIVES: DDX of hypoglycemia W/U of and DDX of primary, secondary and tertiary causes of adrenal insufficiency. CASE INFORMATION: A 74 year-old man with past medical history of hypothyroidism, prostate carcinoma, iron deficiency anemia, paroxysmal atrial fibrillation, and hyponatremia, presented nausea and vomitting. Physical exam was normal. Abnormal laboratory data included a sodium of 125, glucose of 40, and mild normocytic anemia. His hypoglycemia did not correct with exogenous glucose administration. An A.M. cortisol level returned as 1.3. ACTH level was then drawn and returned as 20 pg/ml. Further evaluation to distinguish between secondary and tertiary adrenal insufficiency was preformed with a corticotropin stimulation test. The results of this test confirmed the diagnosis of secondary adrenal insufficiency. IMPLICATIONS/DISCUSSION: Secondary adrenal insufficiency is a deficiency of ACTH from the anterior pituitary gland resulting in a decreased production of cortisol . In the adrenal gland, ACTH stimulates the zona fasiculata, while having minimal affect on the zona glomerulosa. Therefore, manifestations of aldosterone deficiency (hyperkalemia and hypotension) are not present, as this case demonstrated. To confirm adrenal insufficiency, an AM cortisol level of <1.3 is needed. If the cortisol level is greater than 1.3, the diagnosis of adrenal insufficiency could be confirmed by a cosyntropin stimulation test. To distinguish between primary and secondary adrenal insufficiency, an ACTH level should be drawn which should be >100 pg/ml in primary adrenal insufficiency. The Metyrapone and Insulin Induced Hypoglycemia tests confirm secondary adrenal insufficiency. To distinguish between tertiary and secondary adrenal insufficiency, the corticotropin stimulation test is indicated. An absent response from baseline with ACTH and cortisol confirms secondary adrenal insufficiency, while an prolonged and exaggerated ACTH repsonse is consistent with tertiary adrenal insufficiency.\nA GASTROINTESTINAL LINK TO OSTEOPOROTIC FRACTURES. L. Eck 1 , C. Jachna 1 ; 1 Kansas University Medical Center, Kansas City, KS (Tracking ID #74458) LEARNING OBJECTIVES: To recognize that celiac disease is commonly associated with low peak bone mass and adult osteoporosis leading to disabling fractures. CASE INFORMATION: A 49 year old white male with a history of multiple atraumatic fractures including a hip fracture at age 22, wrist fracture at age 16, multiple rib fractures and a recent wrist fracture presented for evaluation of osteoporosis. Bone mineral density studies revealed a T-score of À3.1 at the spine, consistent with osteoporosis. On clinical history, the patient reported a long-standing history of approximately eight loose bowel movements per day associated with abdominal cramping. He also reported low body weight through young adulthood and lactose intolerance. Lab studies were notable for an elevated IgG antigliadin antibody of 132 Units, with a reference range of >30 Units being strongly postive for a diagnosis of celiac sprue. Follow up small bowel biopsy revealed villous atrophy consistent with the diagnosis of celiac sprue. IMPLICATIONS/DISCUSSION: Celiac disease is caused by a genetically based inability to digest gluten, a major protein commonly found in grains. Due to the availability of new highly sensitive and specific serologic diagnostic tests, it is increasingly recognized in the United States as a cause of malabsorption. Celiac disease is often asymptomatic but symptoms suggesting the diagnosis include bloating, flatulence, chronic diarrhea and lactose malabsorption. Other than gastrointestional symptoms, celiac disease also has a wide spectrum of extraintestinal manifestations including iron deficiency anemia, low bone mineral density, and dermatitis herpetiformis. In a study of North American adults with celiac disease, 70% of subjects had low bone mineral density, predisposing them to fractures. Other studies have suggested that in subjects with celiac disease, bone manifestations may be more severe in men than in women possibly due to estrogen's protective effects on the bone. Historically, initial screening for celiac disease involved detection of antigliadin antibody. However, more specific serologic tests, including antiendomysial and antitissue transglutaminase antibodies, are now available. Histologic identification of gluten sensitive enteropathy, with characteristic small bowel mucosal abnormalities including villous atrophy, crypt heperplasia, and an increased density of inflammatory cells, still remains diagnostic. The pathogenesis of low bone mass and osteoporosis in celiac disease is multifactorial. The reduction in surface area of the intestinal mucosa may contribute to calcium malabsorption and subsequent secondary hyperparathyroidism leading to increased bone resorption. Vitamin D deficiency is also believed to be common. Those who are undiagnosed during childhood and young adulthood may never achieve peak bone mass. In addition, some researchers suspect that systemic effects of inflammatory cytokines involved in the intestinal mucosa inflammation may also contribute to the development of bone disease. Not only should patients with celiac disease be screened for osteoporosis, but patients with low bone mineral density and no evident osteoporosis risk factors should be evaluated for celiac sprue.\nA. El-Sharkawi 1 , S. Kripalani 1 ; 1 Emory University, Atlanta, GA (Tracking ID #75179) LEARNING OBJECTIVES: Hepatitis due to herpes simplex virus (HSV) is an uncommon, but potentially fatal cause of hepatitis that is often not considered in the differential diagnosis of acute hepatitis during pregnancy. Serological tests have limited value in establishing an early diagnosis and a high index of suspicion and early diagnostic tools, such as HSV DNA detection are essential. Early treatment with acyclovir decreases mortaility from herpes simplex hepatitis. CASE INFORMATION: A 19 y/o woman, G2 P1, with no significant PMHx, presented with premature labor at 32 weeks gestation. The initial symptoms were controlled, but over the next several days, the pt developed a cough, dyspnea, RUQ abd pain, and fever of 39C. Evaluation of blood, urine, and amniotic fluid cultures was unremarkable. CXR revealed bibasilar atelectasis versus infiltrate, and she received azithromycin and ceftriaxone for presumptive pneumonia. The pt remained febrile with increasing RUQ pain. Labs revealed declining Hb (7.1), WBC (5.6 with 56% bands), and Plt counts (62,000), with rising liver transaminases (AST 2490, ALT 796), INR 2.5, T. Bili 2.2. RUQ U/S showed hepatomegaly and fatty liver. Considering her worsening condition, obstetrics performed a C-section on hospital day 5.\nPost-operatively, the pt was emergently intubated for acute respiratory failure. Physical exam revealed Temp 39.2, HR 146 (reg), BP 107/38, slight RUQ tenderness, liver span 13 cm, no fluid wave or shifting dullness, and 2 vesicular lesions on the Rt upper inner thigh. DDx at this point included HELLP syndrome, acute fatty liver of pregnancy, acute viral hepatitis, pyelophlebitis, portal vein thrombosis, and acute hepatic failure secondary to sepsis. Antimicrobials were changed to ceftazidime, vancomycin, and acyclovir. On hospital day 11, PCR for HSV type 1 and 2 returned positive. In addition, cultures of the vesicular lesions on the patient's right thigh grew herpes simplex. Further questioning of the patient's partner revealed a hx of genital herpes. With the initiation of acyclovir, the pt defervesced and her transaminases decreased. However, she suffered multiple ICU complications, as well as herpes encephalitis. The newborn infant was in the NICU with herpes meningitis. IMPLICATIONS/DISCUSSION: Hepatitis due to herpes simplex virus (HSV) is an uncommon, but potentially fatal cause of hepatitis that is often not considered in the DDx of acute hepatitis during pregnancy. HSV can affect the liver during primary or recurrent infection, although hepatitis during recurrent infection has not been documented in immunocompetent hosts. Review of the literature suggests that HSV hepatitis can present with wide clinical spectrum from mild symptoms to fulminant hepatic failure. However, the initial symptoms are often nonspecific. Therefore, a high index of suspicion and early diagnostic tools, such as HSV DNA detection are essential. Unfortunately, serological tests have limited value for establishing an early diagnosis, though they may be used retrospectively to support the diagnosis. Definitive diagnosis is made with a liver biopsy, but this is frequently not possible because of severe coagulopathy.`J UST A MED REFILL''. S. Elad 1 , G. Applebaum 1 ; 1 University of California, Los Angeles, Sylmar, CA (Tracking ID #74705) LEARNING OBJECTIVES: 1. To recognize the impact the closing of community clinics has had on uninsured and indigent patients. 2. To raise the possibility that short term economic gains resulting from the closure of primary care clinics may result in preventable hospitalizations. CASE INFORMATION:`Maria' is a 56 -year old woman with a past medical history of diabetes, hypertension and hyperlipidemia that came to the hospital urgent care for a`med refill'. She ran out medications 2 months ago after her community clinic was closed secondary to`budget cuts'. Today Maria reports she has noticed increased urination and thirst for the past two weeks and a frontal headache with blurry vision for the past three days. She denies chest pain or shortness of breath. She has not been febrile. Maria had been on two medications for her blood pressure and diabetes, and a pill for high cholesterol. Presently she is completely out of her medications. When questioned why she waited two months to get her medications refilled Maria stated the two hour bus ride and 6 hour wait in the emergency room required her to take time off work she could not afford. On exam, Maria's blood pressure was 190/108 with a pulse of 83. The rest of her cardiac, pulmondary and nuerologic exam was unremarkable except for bilateral decreased visual acuity. Her blood sugar was 275 with sugar and protien in her urine but no ketones. Her anion gap was normal as was her blood count. Maria was given a dose of lisinopril and her headache resolved once her blood pressure decreased to 160/90. She was restarted on her oral diabetes medication. Her medications were refilled and she was instructed to return to urgent care in one week to have her blood pressure and blood sugar rechecked. IMPLICATIONS/DISCUSSION: The national health care crisis has been the topic of many news programs and newspaper articles for the past year. According to the Kaiser Family Foundation approximately 19% of Californians are uninsured. In California, which faces a multi-billion dollar deficit, politicians have been discussing ways to trim costs which have included proposals to close hosptials and clincs. The recent closure of several primary care clinics in Los Angeles County has left many patients like Maria without access to health care. By the time Maria was able to visit the hospitals' urgent care clinic she had run out of medications and both her diabetes and hypertension were poorly controlled. Had Maria required hospitalization at the time of her visit the cost would have been much higher. Maria's primary care visit to the emergency room was entirely avoidable had her clinic not been closed. Much of the short term economic gains obtained by closing county subsidized clinics may later be lost by patients utlizing our emergency rooms for primary care visits. Even in the context of our current financial situation steps need to be taken to ensure that uninsured patients have reasnable access to primary care services outside the hospital setting. LEARNING OBJECTIVES: 1-To recognize fungal vertebral osteomyelitis as an etiology for worsening back pain in patients with history of multiple back surgeries. 2-To consider secondary bacterial infection in patients who fail to respond to appropriate antifugal therapy. CASE INFORMATION: A 48-year-old male presented in January 2001 with worsening back pain, bilateral leg weakness and urinary incontinence. Condition started in 1993 after back injury during demolition job that required multiple back surgeries. Examination was remarkable for bilateral leg weakness and local tenderness over lumbar spine. MRI examination revealed disc space infection at the L3-4 level with adjacent vertebral osteomyelitis. CT guided aspiration of L3-4 space was performed and cultures grew Candida albicans. Treatment was instituted with IV Amphotericin B via Mediport. Therapy was switched to oral fluconazole 400 mg daily. In March 2001, he developed Mediport related Staphylococcus aureus sepsis and was treated with cefazolin for two weeks. Patient was re-admitted in April with worsening back pain. MRI examination revealed worsening inflammatory changes within L3 and L4 vertebral bodies and small epidural abscess. CT guided aspiration was performed and cultures grew methicillin sensitive Staphylococcus aureus. We started treatment with Nafcillin 2 gm IV every 4 hours in addition to fluconazole. Surgical debridement followed by physical therapy and long term IV antibiotics controlled our patient's symptoms. IMPLICATIONS/DISCUSSION: Candidal vertebral osteomyelitis is a rare disease with 65 cases identified in literature. Risk factors have been associated with hyperalimentation, surgical procedures, abdominal surgeries, immunosuppressive therapy and IV drug abuse. The usual mode of spread is hematogenous during episodes of fungemia. The most frequent mode of presentation is severe back pain and diagnosis can reliably be made only when the organism is isolated from the lesion. Both medical and surgical treatment resulted in more favorable outcome for most patients. This case demonstrates the importance of recognizing fungal vertebral osteomyelitis as a cause of worsening back pain in patients with history of back trauma and multiple back surgeries even in the absence of risk factors.\nA CASE OF STERNOCLAVICULAR JOINT SEPTIC ARTHRITIS. E.H. Elbadawy 1 , K. Gopal 1 ; 1 Fairview Hospital, Cleveland, OH (Tracking ID #76754) LEARNING OBJECTIVES: 1-To recognize the clinical picture of sternoclavicular septic arthritis. 2-To emphasize the importance of early diagnosis and treatment in preventing devastating complications. CASE INFORMATION: A 43-year old white male admitted with 2 days history of worsening dull aching pain, swelling and erythema at the right sternoclavicular joint (SCJ) area. Condition started 2 days after he sustained a fall as he was wrestling with his 8-year old son. Patient denied fever, chills or dyspnea. Past medical history was significant for hypothyroidism and depression. Patient denied IV drug abuse, alcohol intake or smoking. On examination he was febrile at 103 F with pulse of 125 and blood pressure of 146/81. He appeared to be in pain with significant soft tissue swelling, erythema and marked tenderness over the right SCJ. Range of motion of the right shoulder was severely limited due to pain. Initial laboratory evaluation revealed WBCs of 12.5 k/AL and ESR of 56. Therapy was initiated with imipenem 500 mg IV every 6 hours for possible SCJ septic arthritis because of history of penicillin allergy. SCJ aspiration was unsuccessful in obtaining any fluid. Computed tomography scan revealed soft tissue swelling around the right SCJ with no evidence of fluid collection. Three-phase bone scan showed increased soft tissue activity and delayed bony uptake involving the right SCJ area. Blood cultures drawn on admission grew methicillin-sensitive Staphylococcus aureus and therapy was switched to cefazolin 2 grams IV every 8 hours. Patient reported significant improvement is his symptoms after 3 days of intravenous antibiotics and was discharged home on cefazolin 2 gm IV for total of 4 weeks. Follow-up one week later revealed complete resolution of the soft tissue swelling, tenderness and erythema with painless full range of motion of his right shoulder. IMPLICATIONS/DISCUSSION: Sternoclavicular septic arthritis accounts for 1±9% of septic arthritis cases. Predisposing factors include IV drug abuse, DM, central venous catheters and trauma. Aspiration of the joint can be helpful in confirming the diagnosis and guiding treatment. Unfortunately, the failure rate with this method is high due to the technical difficulty in aspirating the small joint space, as in our patient. Clinical diagnosis requires a high index of suspicion in any patient presenting with swelling and pain around the SCJ. Spread of infection can lead to superior vena cava obstruction, mediastinitis and septic shock. Early diagnosis is critical in order to prevent life threatening complications.\nM. Eskildsen 1 , K. Lasser 1 ; 1 Cambridge Hospital, Cambridge, MA (Tracking ID #74000) LEARNING OBJECTIVES: 1. To recognize the common side effects of atypical antipsychotics. 2. To learn about a previously unreported complication of these drugs. CASE INFORMATION: A 39-year-old man with a history of hepatitis C, schizophrenia, and past alcohol abuse was admitted with worsening abdominal pain and distension over one month, accompanied by vomiting and watery diarrhea. He stated that he had been sober for two years. His only medications were risperidone at 8 mg daily, and olanzapine at 10 mg daily. His psychiatrist had decreased the olanzapine dosage and attempted to discontinue his prescription one year prior to admission due to poor response, but the patient had refused to do so for unclear reasons. Physical exam revealed a temperature of 37.8C; and a distended, diffusely tender abdomen, without signs of ascites or peritoneal irritation. Laboratory evaluation was remarkable for triglycerides that were over 3800 mg/dl, our lab's upper limit of measurement, and a lipase of 250 U/l. Serum glucose was 553 mg/dl, with no prior readings in the diabetic range. A computed tomography of the abdomen indicated mild pancreatitis with no biliary obstruction. On admission, after discontinuing both medications, we gave him intravenous fluids and nothing by mouth, as well as patient-controlled analgesia with intravenous morphine. Upon discharge, one week later, he was pain-free, and his triglycerides and glucose decreased to 308 and 250 mg/dl, respectively. Of his antipsychotics, risperidone alone was resumed before discharge, up to a dose of 8 mg daily two months later. He has remained without abdominal pain, with serum triglycerides in the 300 range, and persistent diabetes mellitus treated with metformin. IMPLICATIONS/DISCUSSION: A MEDLINE search yielded two reports of pancreatitis associated with olanzapine, and two with risperidone. Hyperlipidemia, including elevated triglycerides, is a side effect of both drugs, although it is more commonly observed with olanzapine. However, this is the first described case of pancreatitis in relation to either of these two drugs that has been associated with massive hypertriglyceridemia, and we have reported it as an adverse event to the FDA. Because he has tolerated risperidone well after discharge, olanzapine seems more likely to have precipitated his pancreatitis, although a synergistic mechanism between the two drugs is another possibility. This case illustrates the need for close monitoring of lipids and glucose in patients taking atypical antipsychotics. \nA 28 year old white male with no significant past history was referred for preoperative clearance for left foot amputation. Patient had non-healing ulcers on his left heel and ankle for 1 month with no response to antibiotics. One year prior, he had a flulike illness, hematuria and painful left testicle which resolved spontaneously except for a persistent dry cough. CXR revealed a spot on the left lung. His TB skin test was negative. The patient denied hematospermia, weight loss, night sweats, trauma, neurologic or GI symptoms. On exam he was a healthy appearing male with previously unnoticed 2 crusted, raised lesions measuring 2.5  2.5 cm on the left arm. These lesions had not been documented during his earlier visit notes though patient admitted to having these lesions for many months. He also had 3 left foot lesions, 1 on the heel and 2 draining lesions on the left malleolus. A plain film of left ankle revealed a lytic area in the distal tibial metaphysis. Bone scan was consistent with acute osteomyelitis. Biopsy of left bony lesion revealed chronic inflammation & giant cells but negative AFB stains and routine cultures. On H & E stain, a multinucleated, thick double contoured wall of yeast with single, broad-based buds was visualized consistent with BD. The patient had complete resolution of all skin lesions and CXR after 6 months of itraconazole treatment. IMPLICATIONS/DISCUSSION: BD is a fungus found in the soil and causes multisystemic pyogranulomatous disease. Blastomyces in endemic in areas bordering Mississippi and Ohio river basins and the Great Lakes. Initial infection is usually via the lungs, can be asymptomatic in 50% of patients or be mistaken for the flu or pneumonia. Pulmonary manifestations include involvement of upper lobes with cavitation, mass lesion with productive cough, hemoptysis, dyspnea, pleuritic chest pain, low grade fever, night sweats, and weight loss. Skin lesions are usually painless, nonpruritic, hyperkeratotic, ulcerative plaques on the face or extremities and may involve mucosa of nose, mouth and larynx. Other common sites of involvement include bone, genitourinary tract or CNS. If untreated, the clinical course can be months to years with remissions, exacerbations and progression in size. The rapid method of diagnosis is visualization of budding yeast in KOH mounts of pus from lesions. Definitive diagnosis is by growth of organism in culture. Treatment consists of 6 months of itraconazole in immunocompetent patients. Amphotericin B is used in patients who are pregnant, seriously ill & immunocompromised, with CNS infection or progressive disease on itraconazole. Our case stresses the importance of a thorough history and physical for appropriate management especially in a disease that is known as the great masquerader.\nNECROTISING FASCIITIS. C. Eze 1 , J. Wiese 1 ; 1 Tulane University, New Orleans, LA (Tracking ID #77055) LEARNING OBJECTIVES: 1. Recognize the signs of deep fascial infections. 2. Use the physical examination to prompt timely surgical intervention for necrotizing fasciitis. CASE INFORMATION: A 21 year-old Hispanic-speaking man presented with four days of left leg pain, swelling and fever after a spider bite. He had a temperature of 103 F, a heart rate of 128, and a blood pressure of 128/70. His left lateral leg was warm, edematous and tender. There was a 1 cm ulcer without drainage or necrosis. The pulses, sensation and range of motion of were normal. A plain film of his leg showed no soft tissue air or bone involvement. His WBC count was 22,000; the remaining laboratory values were normal. Blood cultures were obtained and he was empirically started on gatifloxacin due to a penicillin allergy. After 48 hours, he showed no improvement. Bullae and ecchymosis were noted on the second day; he lost sensation of the overlying skin on the third day. His antibiotic coverage was broadened to include coverage for MRSA. An MRI revealed fluid collections between the muscle and fascia consistent with necrotizing fascitis. An emergent fasciotomy and posterior compartment release were peformed. Wound cultures grew group A Strep. and Strep. milleri. IMPLICATIONS/DISCUSSION: The timely diagnosis of necrotizing fasciitis is important to preventing tissue damage, systemic toxicity, limb loss or death. Cellulitis not responsive to antibiotics, or the appearance of ecchymosis, bullae or anesthesia should prompt consideration of this diagnosis. The physical examination reflects the deep fascial structured damaged by the advancing infection. Ecchymosis, bullae and anesthesia reflect the damaged vessels, skin planes and nerves. While MRI is useful, establishing a timely pre-test probability prompting surgical exploration is the gold standard for making the definitive diagnosis. The closed space of deep fascial infections renders them immune from systemic antibiotics; treatment requires immediate surgical release. Antibiotics only buy time for surgical intervention.\nAUTONOMIC DYSFUNCTION AFTER HEAD TRAUMA. C. Eze 1 , J. Wiese 1 ; 1 Tulane University, New Orleans, LA (Tracking ID #77084) LEARNING OBJECTIVES: 1. Identify the subtle presentation of autonomic dysfunction in the elderly. 2. Recognize the association between autonomic dysfunction and spinal cord lesions. CASE INFORMATION: An 81-year-old man presented with acute confusion following six months of progressive lower extremity weakness. He had fallen two weeks prior resulting in a periorbital hematoma. A CT had been obtained on two prior ER visits to exclude a sub-dural hematoma, and was negative on both occasions. His initial blood pressure was 122/60 mmHg with a pulse of 84. Fifteen minutes later his blood pressure dropped to 64/42 with a pulse of 134. Intravenous fluids resulted in a blood pressure of 175/104 mmHg with fifteen minutes. Ten minutes later his blood pressure again declined to 55/39 mmHg He was placed on an epinephrine drip with an immediate and sustained response of his blood pressure to 169/101 mmHg. His examination was notable for flat neck veins, no S3, and normal lung sounds. His reflexes were absent in both lower extremities without a Babinski's response; his upper extremity reflexes were 1+. His laboratory values, including cortisol, were normal. The EKG revealed atrial fibrillation. There was no subdural hematoma on repeat head CT. Owing to the suspicion of autonomic insufficiency, cervical-spine X-ray was obtained, revealing compression fractures involving the C4 to C7 vertebrae. He was admitted to the ICU and neurosurgery was consulted. IMPLICATIONS/DISCUSSION: Wide hemodynamics variations should prompt consideration of autonomic dysfunction, that should in turn prompt suspicion for an occult spinal cord lesion. This is especially true in elderly patients who may not be able to verbalize cervical pain. Hypertention and bradycardia may result from spinal cord reflexes unopposed by central regulatory mechanisms. Support of vascular tone with vasopressors is critical to resuscitation. This case emphasizes the importance of a careful cervical spine evaluation in all falls involving the elderly. LEARNING OBJECTIVES: To recognize that high dose simvastatin monotherapy, although having an excellent safety profile, can cause significant rhabdomyolysis, acute renal failure and hepatitis in the right patient population. To recognize that the addition of a potent or weak cytochrome P450 inhibitor is not needed for the significant adverse effects of simvastatin to occur. CASE INFORMATION: A 62 y.o. woman developed increasing myalgias, malaise, shortness of breath and dyspnea on exertion over 1 week and left lower extremity swelling. Medications included simvastatin 80mg daily with normal baseline transaminase levels and diltiazem for the past year. CXR revealed mild bilateral pulmonary edema and an ultrasound of the left lower extremity revealed no deep vein thrombosis. Exam revealed in ill appearing female in moderate distress secondary to myalgias, mild rhonchi bilaterally, normal heart tones and bilateral lower extremity dependent edema (Lt>Rt). Laboratory studies: alkaline phosphatase 177, AST 1511, ALT 224, LDH 1396; creatinine 5.6 (baseline 1.4±1.7); BUN 92; potassium 5.6; phosphate 6.5; calcium 8.8; creatinine phosphokinase >20,000; troponin <0.05; CKMB 171; TSH wnl; uric acid 8.0. Urinalysis revealed a large amount of blood with only 4±10 red blood cells. Simvastatin was discontinued. Aggressive treatment with sodium bicarbonate, intravenous hydration and hemodialysis was begun. Over a sixty-plus day hospital course, the patient's renal failure resolved (baseline creatinine now 2.2±2.4) and transaminases normalized. The patient was eventually discharged to home to continue physical rehabilitation with no permanent requirement for hemodialysis. IMPLICATIONS/DISCUSSION: Simvastatin's long term safety profile is excellent. Simvastatin induced rhabdomyolysis is reported with low incidence (0.6% in one study involving 80mg of simvastatin) and it is seldom associated with acute renal failure or hepatitis. Myopathy and rhabdomyolysis increases with dose and with concomitant use or addition of potent cytochrome P450 3A4 (CYP450) inhibitors, i.e. fibrates, cyclosporine, azathiaprine. A baseline severe renal insufficiency (creatinine clearance by the Cockroft-Gault method, 17cc/ min) and a urinary tract infection treated with a fluoroquinolone 2 days prior, combined to increase the risk of this patient developing rhabdomyolysis. Published reports of simvastatin induced myopathy involve the addition of potent or weak CYP450 inhibitors. Our patient had been stable on high dose simvastatin and diltiazem (a weak CYP450 inhibitor) for over 1 year prior to presentation and did correlate with the addition of known CYP450 inhibitors. Additionally unique, this is the first reported case involving the combined toxicities of rhabdomyolysis, acute renal failure and hepatitis with high dose simvastatin monotherapy. 2) Recognize the dangers of rapid correction of hyponatremia. CASE INFORMATION: A 56 year-old man with a history of alcohol abuse was admitted to an outside hospital with altered mental status. On exam, the patient was disoriented, but neurological exam was otherwise nonfocal. Computerized tomography (CT) of the head was unremarkable. Laboratory results revealed hyponatremia with sodium of 104 mmol/L. The patient was given hypertonic saline, and the sodium was corrected to 123 within the first 24 hours. The patient was then transferred to our facility for continuation of his care. Physical examination on arrival revealed multiple focal neurological findings including bilateral eye deviation to the left, neck deviation to the left, absent gag reflex, and poor motor coordination with spastic movement of all extremities. The patient's speech was severely dysarthric and unintelligible. Magnetic resonance imaging (MRI) of the brain showed enhancement of the motor cortex of both cerebral hemispheres and enhancement of the frontal eye fields on the left with sparing of the adjacent sensory cortex. In consideration of the MRI findings, neurology consultation was sought. The patient's severe dysarthria, dysphagia, ataxia, and optic apraxia were attributed to extrapontine (as opposed to central pontine) myelinolysis from rapid correction of the patient's hyponatremia. IMPLICATIONS/DISCUSSION: Severe symptomatic hyponatremia should be treated with hypertonic saline, but with a correction in plasma sodium of not more than 1±2 mmol/liter/ hour and a maximum increase of 8 mmol/liter during the first 24 hours. Our patient's sodium was corrected from 104 to 123, with an increment of 19 within 24 hours. Rapid correction may result in osmotic demyelination or central pontine myelinolysis, which is characterized by flaccid paralysis, dysarthria, and dysphagia. Isolated extrapontine myelinolysis is extremely rare, with only two cases reported in the literature. The neurologic deficits depend on the anatomical location of osmotic demyelination. Treatment is supportive, and recovery is slow within weeks to months. Gradual correction of hyponatremia is important because aggressive correction can cause not only central pontine myelinolysis but also the unusual manifestation of extrapontine myelinolysis. LEARNING OBJECTIVES: 1) Construct a differential diagnosis of chronic ataxia. 2) Review the etiology, presentation, and prognosis of paraneoplastic cerebellar degeneration. CASE INFORMATION: A 55-year-old male ex-smoker with no significant past medical history presented to clinic with paresthesias of the right upper and lower extremities, followed by gradual onset of ataxia, diplopia, and dysarthria over a period of five months. The patient also reported weight loss and a chronic dry cough. The patient had no history of toxic exposures or family history of inherited ataxias. Physical exam revealed optical dysmetria, truncal ataxia, finger-to-nose dysmetria, and dysdiadochokinesis. The patient's speech was severely dysarthric, and sensation was decreased on the right upper and lower extremities. Magnetic resonance imaging (MRI) of the brain was normal. HIV and RPR serologies were negative, and TSH was within normal limits. Lumbar puncture revealed paraneoplastic antibodies, including anti-Hu, anti-P/Q type calcium channel, and anti-acetycholine receptor. Computerized tomography (CT) of the thorax demonstrated a 2 cm left hilar mass. Bronchoscopy and endobronchial biopsy were negative, but subsequent thoracotomy and biopsy revealed small cell lung carcinoma (SCLC). IMPLICATIONS/DISCUSSION: The differential diagnosis of chronic ataxia includes primary and metastatic neoplasms, paraneoplastic syndromes, progressive multifocal leukoencephalopathy, multiple sclerosis, hypothyroidism, tabes dorsalis, inherited ataxia syndromes, vitamin E deficiency, toxic exposures, and stable gliosis due to stroke or demyelination plaque. In approximately 80% of cases with paraneoplastic cerebellar degeneration, the underlying tumor is SCLC. Fifty-one percent of patients with cerebellar degeneration and SCLC have detectable anti-Hu antibodies. Other antibodies found in paraneoplastic cerebellar degeneration include anti-P/Q calcium channel, anti-Yo, anti-Ma1, anti-Tr, and anti-CV2, but their exact sensitivities and specificities are not yet known. Therapy is supportive, and symptoms usually do not improve with immunosuppression or treatment of the tumor. In conclusion, paraneoplastic cerebellar degeneration with paraneoplastic antibodies should be considered in all patients with chronic ataxia without other predisposing etiologies. LEARNING OBJECTIVES: 1. Discuss the presentation of primary bacterial peritonitis. 2. Recognize group A beta-hemolytic Streptococcus as a rare but serious cause of primary peritonitis. CASE INFORMATION: A 52 year old woman presented to the Emergency Room with a four day history of fevers, chills, and profuse watery diarrhea with associated crampy lower abdominal pain. She denied any nausea, vomiting, cough, dysuria, or headaches. She denied any recent travel or sick contacts, but noted that the symptoms began hours after having eaten at a Chinese restaurant. Her past medical history was remarkable for a prior appendectomy, she was on no medications, and her family history was significant only for colon cancer in her mother. Her exam revealed a temperature of 38.7 C, a blood pressure of 72/40, and a pulse of 112, with abdominal tenderness and guarding in both lower quadrants. Labs showed a white cell count of 5.7 (with a bandemia of 52), a potassium of 2.4, a bicarbonate of 15, a Blood Urea Nitrogen of 52, and a creatinine of 3.7. Liver function tests, amylase, lipase, and urinalysis were all normal. Fecal leukocytes were absent and her abdominal film showed only a few air-fluid levels. It was the impression of all clinicians involved that the patient's presentation was most consistent with acute infectious diarrhea and profound dehydration complicated by renal failure and electrolyte derangement. Following aggressive hydration with IV crystalloids, the patient's blood pressure improved to 120/80 and her heart rate slowed to 88. After blood, urine, and stool cultures were taken, she was started on IV ciprofloxacin for empiric treatment of her presumed acute infectious diarrhea. She continued to have diarrhea over her hospital course, but her abdominal pain and temperature curve lessened, and she appeared to be improving clinically. All cultures (including c. difficle and giardia screens) had returned negative. On hospital day four, her fever and abdominal pain worsened and her abdomen became distended, rigid, and equisitely tender. An abdominal CT showed diffuse thickening of her colon with ascites and dilated loops of small bowel. An emergent exploratory laparotomy was performed and several liters of cloudy yellow ascites were drained. A nectrotic mesenteric lymph node filled with pus was resected and her abdomen was irrigated with several liters of saline. Gram stains of both the pus from the lymph node and the ascitic fluid revealed gram positive cocci in chains, and she was promptly started on IV clindamycin. Cultures of the ascites and pus grew group A betahemolytic Streptococci, and she was continued on the clindamycin. She continued to improve over her hospitalization and made a full and healthy recovery. IMPLICATIONS/DISCUSSION: Primary bacterial peritonitis occurs as a result of bacterial infection of the peritoneal cavity and is distinguished from secondary peritonitis by the absence of any underlying cause or demonstrable abdominal source. The onset is often rapid and insidious, and is marked by the appearance of fever, nausea, abdominal pain, and diarrhea. The patient typically has abdominal rigidity and rebound with features of systemic inflammation (tachycardia, hypotension, tachypnea, etc.) which may progress to frank shock. Streptococcus pyogenes, or group A Streptococcus, is a very common human pathogen, with up to 15% of asymptomatic individuals carrying the bacterium (typically in the upper respiratory tract). However, primary bacterial peritonitis due to Streptococcus pyogenes is exceptionally uncommon with very few adult case reports documented in the literature. It may be slightly more common in women, with asymptomatic colonization of the genital tract acting as a portal of entry. The diagnosis is often made only at laparotomy and despite the profound morbiditiy associated with the disease, rapid initiation of the appropriate antibiotics usually results in a successful cure.\nEUREKA! A TREATABLE CAUSE OF DIZZINESS. E.J. Favus 1 , R. Ambrosino 1 , R. Granieri 1 ; 1 University of Pittsburgh, Pittsburgh, PA (Tracking ID #73835) LEARNING OBJECTIVES: 1. To recognize and treat non-sustained ventricular tachycardia (NSVT). 2. To recognize the differential diagnosis of dizziness.\nA 70 year-old male with a history of gout, hypertension, chronic renal insufficiency, and hypercholesterolemia presented to his primary care physician complaining of intermittent dizziness. He had approximately three episodes per week, each lasting ten minutes, and resolving spontaneously. He described feeling``lightheaded'' during each episode, and denied a positional component, vertigo, visual disturbances, tinnitus, history of seizures, aura, post-ictal state, loss of consciousness, palpitations, chest pain, and shortness of breath. An EKG shortly after the first episode showed no rhythm abnormalities, ischemia, or infarction. His chronic medications were simvastatin, allopurinol, and lisinopril. Physical examination revealed a blood pressure of 156/84mmHg, pulse 66, and respiratory rate 16. He did not have orthostatic hypotension. Cardiac exam revealed a non-displaced PMI, regular rate and rhythm, normal S1 and S2 with no murmurs, gallops, or rubs. Holter monitor testing revealed multiple episodes of multifocal ventricular ectopy including short runs of ventricular bigeminy, trigeminy, and quadrigeminy with a heart rate of 80 beats per minute. He was diagnosed with non-sustained ventricular tachycardia (NSVT). A review of the patient's log revealed that each tachyarrhythmic event correlated with an episode of dizziness. He underwent echocardiography, which showed a structurally normal heart and an ejection fraction of 55%. He was prescribed metoprolol 12.5mg po qam, and at a one-month follow-up visit reported improvement in his symptoms. IMPLICATIONS/DISCUSSION: Dizziness is a common complaint heard by primary care physicians, yet only a small percentage of patients receive a firm diagnosis and an effective treatment. In this patient, the discrete nature of these episodes hinted at the possibility of a cardiac arrhythmia. Clinical suspicion for a cardiac problem prompted an evaluation by Holter testing. The results showed a temporal relationship between NSVT and dizziness, and provided evidence for a firm diagnosis. Patients with NSVT and a structurally normal heart are not at risk for sudden death, and may be treated with daytime oral beta-blocker therapy to decrease symptoms. Conversely, large studies have shown that the combination of NSVT and a structurally abnormal heart is a risk factor for sudden death. Treatment of NSVT with a structurally abnormal heart requires the use of an Automatic Implantable Cardioverter-Defibrillator (AICD). Beyond cardiac arrhythmias, dizziness has many causes. A proper history of the dizzy patient should include questions about syncope, hyperventilation, anxiety, visual disturbances due to incorrect eyeglasses or diplopia, and concussion. Physical examination should evaluate orthostatic hypotension, hypertension, and peripheral neuropathy. Anemia and hypoxia should be evaluated by laboratory studies.\nPUTTING IT ALL TOGETHER: A CASE OF PRIMARY HYPOGONADISM. E.J. Favus 1 , R.C. Brooks 2 , R. Granieri 1 ; 1 University of Pittsburgh, Pittsburgh, PA; 2 Pittsburgh VA Healthcare System, Pittsburgh, PA (Tracking ID #73840) LEARNING OBJECTIVES: 1. To recognize the clinical presentation of male hypogonadism. 2. To recognize the differential diagnosis of acquired primary hypogonadism. CASE INFORMATION: A 66 year-old male presented with bilateral gynecomastia, osteoporosis, and erectile dysfunction. Three years ago he was seen by a urologist for erectile dysfunction and hesitency. He was diagnosed with a low testosterone state (laboratory values unavailable), and treated with testosterone. He had improvement of his erectile dysfunction but worsening of his hesitency and was switched to sildenafil. No cause was found for urinary hesitancy, but he was treated with tamsulosin without relief. Two years ago he fell and broke his left fourth metatarsal. Dual x-ray absorptiometry scan revealed a T-score of À2.7, and he was treated with alendronate. He denied a history of glucocorticoid use. One year ago he noticed increased breast tissue bilaterally and a mass in the right breast. A bilateral mammogram was negative and a breast biopsy was negative for malignant cells. His medical history is significant for tinea capitis, treated with 2% ketoconazole shampoo. His medications are sildenafil, alendronate, and ketoconazole shampoo. He has no history of alcohol, tobacco, or drug use. Physical examination reveals a normal thyroid, moderate bilateral gynecomastia without masses, no kyphosis, and normal size and shape of the penis, testicles, and prostate. Laboratory tests show total testosterone 239 ng/dL (241±827ng/dL), free testosterone 5.28 ng/dL (5±21ng/dL), FSH 32.1 mIU/mL (adult male normal range 1.55±9.70 mIU/mL), LH 14.7 mIU.mL (adult male normal range 0±9.0 mIU/mL), prolactin 13.0 ng/mL (adult male normal range 3.7± 17.9 ng/mL), TSH 4.13 uIU/mL (0.35±5.5 uIU/mL), PSA 1.68 ng/mL (0±4ng/mL). Given his low-normal testosterone and his clinical symptoms, he was re-started on testosterone therapy. IMPLICATIONS/DISCUSSION: The clinical presentation of osteoporosis, erectile dysfunction and gynecomastia should provoke a search for a unifying diagnosis of hypogonadism. The medical history of this patient is suggestive of a low testosterone state. His serum testosterone level is in the very low-normal range, and the FSH and LH levels needed to maintain this low-normal testosterone level are three times the upper limit of normal. High levels of FSH and LH exclude the diagnosis of secondary and tertiary hypogonadism. The low-normal level of testosterone has not been enough for this patient to avoid developing clinical disease. Male hypogonadism is defined as a failure of the testes to produce a sufficient amount of testosterone. Hypogonadism can be primary (testicular failure), secondary (pituitary failure), tertiary (hypothalamic failure), or combined primary and secondary. Primary hypogonadism can be either acquired or developmental. Our patient's laboratory values suggest primary hypogonadism. Since he had normal sexual development and fathered two children, he likely has acquired primary hypogonadism. Acquired primary hypogonadism may be the result of amyloidosis, autoimmune destruction, cryptorchidism, leprosy, leukemia, mumps, polyarteritis nodosa, radiation therapy, trauma, surgery (castration), or drugs such as cancer chemotherapy, ethanol, ketoconazole, marijuana, or spironolactone. In this patient, long term administration of ketoconazole shampoo for the treatment of tinea capitis may have resulted in acquired primary hypogonadism.\nUSING THE HEAD AND NECK EXAMINATION TO DIAGNOSE FOCAL NEUROLOGIC DEFICITS. D. Fotino 1 , N. Parekh 1 ; 1 Tulane University, New Orleans, LA (Tracking ID #77089) LEARNING OBJECTIVES: USE THE EXAM SKILLS TO DIAGNOSE FOCAL NEUROLOGIC DEFICITS. CASE INFORMATION: A 41 year-old diabetic man presented with a sudden onset of slurred speech and left-sided weakness. On examination, he had severe dental caries, slurred speech, and a left facial droop. His tongue deviated to left on protrusion, and he had left upper and lower extremity weakness. There was tenderness over the right mastoid process. His WBC was 27,000 with 14% bands. A CT showed subdural fluid collection in the right frontoparietal region. Vancomycin, ceftazidime and metronidzaol were empirically started for a presumptive diagnosis of subdural empyema. Shortly after admission, he deteriorated to persistent and refractory status epilepticus. The CSF protein was 149; WBC 630 (70% neutrophils); the gram stain was negative. A repeat CT showed an area of hypodensity and a midline shift, consistent with subdural empyema. He underwent drainage of the abscess; the cultures grew out pseudomonas aeruginosa. Ciprofloxacin was added to his regimen to double cover the pseudomonas. His seizure activity relented soon thereafter. IMPLICATIONS/DISCUSSION: Sinusitis is the leading cause of subdural empyema, and can present with focal neurological signs if intra-cerebral extension occurs. Diabetic patients are also at risk for malignant external otitis, and empiric coverage for pseudomonas is warranted at even the lowest level of suspicion. Although this patient's symptoms suggested a cerebrovascular accident, the finding of mastoid tenderness directed the diagnosis towards the infected mastoid sinus and the subdural abscess. In diabetic patients, empiric pseudomonas coverage should be initiated early; double coverage should be instituted when a pseudomonas infection is confirmed, as this reduces the mortality of deep tissue infections by 50% when compared to single coverage. A thorough examination of the oro and nasopharyngeal cavities can direct and expedite diagnostic testing and prevent grave sequelae of treatable diseases.\nA ROCKY SITUATION. F. Francois 1 , J. Park 1 , C.T. Tenner 2 , S. Finkelstein 1 ; 1 New York University School of Medicine, Brooklyn, NY; 2 New York University School of Medicine, New York, NY (Tracking ID #74431) LEARNING OBJECTIVES: 1. Recognize the potential for bowel dysfunction in chronically ill patients 2. Consider the differential diagnosis of lower gastrointestinal bleeding in the elderly population 3. Review the management of stercoraceous masses. CASE INFORMATION: A 72-year-old HIV positive Hispanic male on highly active anti retroviral therapy (CD4 = 612 cells per cubic millimeter, viral load<50 copies per milliliter), was admitted for evaluation of hematochezia. The patient had a history of poorly controlled hypertension and had been hospitalized three weeks prior to this presentation with an acute change in mental status during which he was found to have a basal ganglia infarct. The family reported that the patient had been mainly bed bound since the stroke, and had intermittently complained of difficulty moving his bowels without any associated abdominal pain, nausea, or vomiting. On physical exam the patient was noted to have a right facial droop, right-sided hemiparesis, and normal active bowel sounds without abdominal distention, tenderness, or guarding. On rectal examination a freely mobile mass was palpated in the vault and bright red blood was seen on the glove. Laboratory evaluation was significant for a seven-point hematocrit drop from his baseline. On colonoscopy the patient was found to have 3 discontinuous areas of ulceration in the rectosigmoid region, with the largest measuring 6cm  2cm. The ulcers were associated with 3 large (4cm in diameter) hard fecal masses (fecalomas). The fecalomas were manually evacuated and the patient was started on a bowel regimen consisting of stool softeners and hydration. The patient's hematocrit remained stable with no further episodes of rectal bleeding and he was discharged to to the care of his family. IMPLICATIONS/DISCUSSION: Elderly patients may develop bowel dysfunction due to diabetes mellitus, hypothyroidism, renal failure, dementia, immobilization, cerebrovascular accidents, and medications. Once chronic constipation occurs the patients are at risk for fecal impaction and stercoral ulcers (a.k.a. Huntley syndrome). These ulcers result from pressure necrosis on the colonic mucosa by the rock-like fecal mass. Patients may present with rectal bleeding or colonic perforation with peritonitis. In the elderly population, the differential of hematochezia as a presenting sign in the setting of a change in bowel habits includes, malignancy, infectious colitis, and ischemic colitis. Initial management of a stercoral lesion involves manual evacuation of the stecoraceous mass followed by a bowel regimen with stool softeners along with adequate hydration. Patients with recurrent ulcer bleeding or perforation may require surgery as definitive therapy.\nSEA-BLUE HISTIOCYTOSIS: A NEWLY REPORTED COMPLICATION OF PARENTERAL NUTRITION. S. Frost 1 ; 1 Cleveland Clinic Foundation, Cleveland, OH (Tracking ID #73897) LEARNING OBJECTIVES: 1) Diagnose sea-blue histiocytosis syndrome as a complication of total parenteral nutrition (TPN). 2) Recognize that bone marrow sea-blue histiocytes are associated with many diseases in which the degradation of lipids and cellular lipid products is exaggerated. CASE INFORMATION: A 40 year-old man receiving chronic TPN with lipid emulsion after esophagogastrectomy following caustic ingestion was admitted to the hospital with fever for 5 days, jaundice, hepatitis, and pancytopenia. The patient did not appear ill. The physical exam was notable for a clean subclavian Hickman catheter site without erythema or tenderness, hepatomegaly, no heart murmur, and no peripheral stigmata of endocarditis. Laboratory revealed WBC = 3.8 K/uL, hemoglobin = 10 g/dL, platelet = 70 K/uL, AST = 223 U/L, ALT=160 U/L, alkaline phosphatase = 339 U/L, and bilirubin = 9.4mg/dL. Investigation for bacterial, fungal, mycobacterial, and viral infection was negative. Abdominal sonogram and CT scan revealed hepatomegaly without other abnormality. Echocardiogram revealed no vegetations. Upper extremity ultrasound was negative for catheter associated thrombosis. The Hickman catheter was removed and catheter culture specimens were sterile. Fever persisted despite therapy with piperacillin/tazobactam, vancomycin, fluconazole, and amphotericin B. Bone marrow biopsy revealed sea-blue histiocytes. TPN was held and improvement in liver function abnormalities and cell counts began on hospital day (HD) 6 and 7 respectively. Fever dissipated on HD 8, and antimicrobials were discontinued. TPN was restarted without lipid emulsion, and the patient has remained well with resolution of hepatitis and cytopenias. IMPLICATIONS/DISCUSSION: Sea-blue histiocytes (SBHs) are macrophages with lysosomal lipid accumulations that appear blue with Giesma staining. The sea blue histiocytosis syndrome is a typically benign idiopathic disorder characterized by hepatosplenomegaly, purpura, thrombocytopenia, and pulmonary infiltrates. SBHs have also been noted in association with hematologic disorders such as chronic myeloid leukemia, and metabolic illnesses such as Niemann Pick disease. SBHs in the bone marrow of patients with hepatosplenomegaly, jaundice, and pancytopenia receiving TPN containing lipid emulsions was first reported in 1996. Lowering the TPN lipid content has been shown to improve pancytopenia. Lipid induced hematopoetic cell membrane alterations triggering macrophage phagocytosis is one postulated mechanism accounting for TPN related sea-blue histiocytosis (TPN-SBH). This patient's illness is consistent with prior descriptions of TPN-SBH, except for fever, which has not been previously reported. However, the resolution of fever coincident with the improvement in pancytopenia and hepatitis after the discontinuation of parenteral lipids suggests an association with TPN-SBH.\nIT'S BLACK, IT'S HAIRY . . . BUT IS IT A PROBLEM? C.C. Fu 1 , G. Applebaum 1 ; 1 University of California, Los Angeles, Los Angeles, CA (Tracking ID #74949) LEARNING OBJECTIVES: 1. Recognize the presentation of black hairy tongue. 2. Review possible predisposing conditions. 3. Emphasize benign nature of this phenomenon. CASE INFORMATION: A 53-year-old Mexican man with no significant medical history presented with one month of easy bruising and gum bleeding. The patient's only medicines were occasional aspirin for pain and bayberry extract. Lab results were significant for 2.2 WBCs, Hb 8.2, and 4 platelets. Bone marrow biopsy revealed a hypocellular and fatty marrow consistent with aplastic anemia. The patient's three-week hospital stay was complicated. He had extraction of an infected tooth and was started on a peroxide-based swish and spit regimen. Treatment for aplastic anemia was begun with anti-thymocyte globulin and cyclosporine. The patient experienced prolonged neutropenic fevers for which he was treated with ceftriaxone, tobramycin, metronidazole, and vancomycin. During this period, the patient's tongue was noted to develop a dark gray,``hairy'' appearance, the surface of which could actually be brushed with the end of a cotton swab. IMPLICATIONS/DISCUSSION: Black hairy tongue is a rare condition caused by the elongation of the filiform papillae associated with oral bacterial overgrowth. Black hairy tongue is idiopathic and has been associated with various predisposing factors including alcoholism, systemic illness, immunosuppression, cancer therapy, coffee, antibiotic use, and use of peroxide-based mouthwash. Our patient was a likely candidate to develop black hairy tongue as he had been given broad-spectrum antibiotics and used peroxide based mouthwash during immunosuppressive therapy. Treatments for black hairy tongue have included topical retinoin gel, podophyllum, and attempts at repeated tongue brushing. However, in consideration of our patient's underlying immunosuppression and profound thrombocytopenia, we chose not to treat the patient's tongue. It is important not to complicate the treatment of an ill patient with the unnecessary treatment of a benign, albeit strange, condition. A 45 year old Mexican female with a history of diabetes mellitus was brought to the emergency room by her family for increasing abdominal pain and a four day history of nausea, vomiting and diarrhea. There was no recent history of fevers, chills, or dysuria. Initial examination revealed an ill-appearing woman, moaning in response to painful stimuli, but not following commands. Although she was afebrile and normotensive, her resting heart rate of 119 beats per minute established systemic illness. Her other examination was remarkable for right lower quadrant tenderness in a soft abdomen, but no peritoneal signs. There was evidence of costovertebral angle tenderness bilaterally. Initial laboratory results were consistent with diabetic ketoacidsosis (DKA). Leukocytosis and pyuria led to the additional admitting diagnosis of pyelonephritis. Declining mentation led to rapid transfer to the intensive care unit on the day of admission. Computed tomography (CT) of the abdomen showed bilateral emphysematous pyelonephritis, emphysematous cystitis, bilateral perinephric abscesses, and air surrounding the inferior vena cava (IVC). Broad spectrum antibiotics were initiated. Emergent urology consultation recommended medical management due to high perioperative mortality risk. Bilateral percutaneous drains were placed under CT guidance. Nevertheless, the patient's condition rapidly deteriorated with development of thrombocytopenia and septic shock, requiring intubation and pressor support. She died the following hospital day. Blood, urine and perinephric fluid cultures grew Klebsiella pneumoniae. IMPLICATIONS/DISCUSSION: Emphysematous pyelonephritis (EPN) is a rare condition that causes an acute necrotizing parenchymal and perirenal infection due to gas-forming uropathogens. Bilateral disease is exceedingly rare. EPN occurs predominantly in diabetic patients and in patients with urinary tract obstruction. E.coli, Klebsiella and Proteus are the most frequently identified organisms. Computed tomography is used for diagnosis and evaluation of the extent of disease. Antibiotics and percutaneous drains can lead to successful outcomes in patients with limited disease. However, nephrectomy is often considered the treatment of choice, especially in patients with extensive disease or multiple poor prognostic factors. Bilateral disease makes the surgical decision even more difficult. Understanding the indications for surgical versus medical management may mean the difference between life and death in patients with EPN.\nJ.A. Garcia 1 ; 1 Wayne State University, Troy, MI (Tracking ID #74175) LEARNING OBJECTIVES: Physicians should be able to recognize early adverse events of commonly prescribed drugs. CASE INFORMATION: 56 y/o male, previously heatlhy presented to ER with fever, left calf pain and swelling which started after a brief hospitalization for a febrile illness. Lower extremity dupplex revealed thrombosis involving the Left popliteal and lesser saphenous veins. He was started on unfractionated heparin. Labs: WBC 10.2, Hemoglobin 11.7 and Platelets, 386k. Infectious work up was negative and he was eventually switched to coumadin and discharged. On the 4th day, he presented with fever and worsening left calf pain. A Lower extremity dupplex showed progression of the thrombus involving the posterior tibial vein. Heparin was restarted. WBC count 16.1, Hemoglobin 9.7 and platelets 240k . The fourth day of this admission Hb dropped to 6.5 with plts of 150k. On the 9th day, patient developed chest pain. EKG showed ST elevations and he had increased cardiac biomarkers compatible with acute lateral Myocardial Infarction. Cardiac catheterization revealed clean coronary arteries. On the 12th day, he complained of constipation, abdominal distension and pain. Abdominal Series showed ileus. Amylase 136, Lipase 73, ALT 21, AST 29, Alkaline Phosphatase 131, Tbili 0.9. WBC 27.2 Hemoglobin 11.7 and Platelets 72k. Because of the dropping platelets and the multiple thrombotic events, heparin was held, antibodies drawn, and liperudine was started. Heparin induced platelet aggregating antibodies were positive with ELISA. Abdominal CT showed focal areas of low attenuation with peripancreatic fat stranding compatible with necrotizing pancreatitis. IMPLICATIONS/DISCUSSION: Heparin is a widely used medication. Uncommon presentation of HITT should be early recognized to prevent unwarranted management. LEARNING OBJECTIVES: 1. Recognize the common toxicities of ecstasy 2. Diagnose water intoxication and its complications. CASE INFORMATION: KD is a 20-year old woman brought into the UCSF ED because she developed altered mental status after taking ecstasy. Seven hours prior to admission, she took 1.5 tablets of ecstasy, while her companion took 2 tablets of the same preparation. They remained at home and``drank lots of water.'' One hour prior to admission, KD complained of severe headache and multiple episodes of vomiting. She then``stopped making sense.'' In the ED, KD was alert and oriented to person and place but appeared agitated and confused. She then had a generalized tonic-clonic seizure, became apneic, and was intubated. A NCHCT showed bilateral uncal herniation with diffuse effacement of the sulci. Significant laboratory abnormalities included leukocytosis, serum sodium of 124, rhabdomyolysis, and evidence of SIADH. A urine toxicology screen was positive only for amphetamines. An ECG showed sinus tachycardia with diffuse ST elevations, and a chest radiograph showed bilateral patchy infiltrates consistent with pulmonary edema. Repeat physical exam three hours after admission documented the absence of brainstem reflexes. She expired 24 hours later, and her organs were harvested for transplantation. IMPLICATIONS/DISCUSSION: Ecstasy, or methylenedioxymethamphetamine (MDMA), is a recreational designer drug popular both for its pleasurable psychological effects and for its safe reputation. Acute adverse effects can range from nausea, headache, and myalgias to insomnia, paranoia, and depression. An ecstasy tablet often contains contaminants and other drugs. In addition, the dose of MDMA itself can range from 0 to 200 mg per tablet, and the toxic or even fatal dose range overlaps that of recreational use. The pattern of major physical toxicity is usually hepatic, cardiovascular, hyperpyrexic, or cerebral, although DIC, rhabdomyolysis, and acute renal failure also contribute to morbidity. Cerebral toxicity is manifested as seizures or cerebral edema, thought to result from hyponatremia. In most cases, the low sodium level is hemodilutional; users often sweat profusely and then drink large amounts of free water to rehydrate and to prevent overheating. Occasionally, SIADH contributes to hyponatremia in this setting. In addition to the physical toxicity, ecstasy can also lead to increased risk-taking behavior. Despite the widespread use of ecstasy and the range of potential toxicity, fatalities attributable to the drug are uncommon. KD's demise likely began with ingestion of MDMA, which led to water intoxication and SIADH. Hyponatremia followed, precipitating cerebral edema, increased intracranial pressure, and then uncal herniation. Respiratory arrest occurred with compression of the brain stem. The immediate cause of death was hypoxic encephalopathy. Several case reports suggest young women, such as KD, and children appear to be at the highest risk of poor outcomes associated with hyponatremia. This unfortunate case emphasizes the often overlooked and unpredictable morbidity and mortality associated with casual use of ecstasy.\nGET YOUR COLONOSCOPY, GET YOUR CURE. D. Garrow 1 ; 1 New Hanover Regional Medical Center, Wilmington, NC (Tracking ID #74301) LEARNING OBJECTIVES: 1. Present a case of metastatic colon cancer. 2. Provide screening recommendations with colon cancer in a first-degree relative. 3. Discuss genetic factors implicated in colon cancer. CASE INFORMATION: The patient is a 57 year-old female who presented with dysequilbrium and hand tremor for one month. Head MRI revealed multiple intracranial lesions. ( fig. 1 ) Biopsy revealed poorly-differentiated adenocarcinoma. A colonoscopy was performed because of the above histology and a family history of colon cancer involving two brothers and a son. A near-obstructing mass was discovered in the right colon. Patient also had liver, spleen and multiple lymph node involvement via PET scan.\nIMPLICATIONS/DISCUSSION: The majority of studies have shown a two-to four-fold increase in the incidence of colon cancer in patients with similarly afflicted first-degree relatives. Screening for colon cancer with a positive history in a first-degree relative is advised ten years prior to the index case. Hereditary nonpolyposis colon cancer (HNPCC) is a consideration in this patient. The genetic basis for HNPCC is heterogeneous involving chromosomes 2, 3, and 7. With the multiple genetic focus of HNPCC, genetic testing is seldom employed. The family is clinically positive for HNPCC as there are more than two cases of colon cancer spanning at least two generations. HNPCC is caused by defective DNA mismatch repair genes. Afflicted patients develop polyps at a rate similar to the normal population. However, because of genetic defects, precursor adenoma lesions undergo a more rapid progression to carcinomas than usual adenomas. HNPCC typically presents during the fourth and fifth decades of life. Affected patients are predisposed to a higher frequency of proximal colonic lesions with more advanced pathology, as seen in this case.\nA. George 1 , R.D. Hobbs 1 ; 1 Oakwood Healthcare System, Dearborn, MI (Tracking ID #76605) LEARNING OBJECTIVES: To recognize that shingles, although most commonly thought of as a sensory problem with a characteristic rash, can cause diaphragmatic paralysis. CASE INFORMATION: A 71 year-old white male with stable hypertension, CHF and COPD, presented to the emergency room with severe dyspnea. He had a 12-year history of chronic elevation of the left hemidiaphragm and had been well except for recent right-sided neck pain. There was no rash. He was intubated. The following day he developed a classic shingles rash involving the right side of the neck. An EMG revealed a right-sided C3-C4 motor neuropathy. Phrenic nerve function did not return. He died six weeks later when he extubated himself at a nursing facility. Autopsy revealed that the chronic elevation of the left hemidiaphragm was due to a benign angiolipoma and unrelated to the viral infection. IMPLICATIONS/DISCUSSION: Sir William Broadbent first described muscular paralysis in association with zoster in 1866. In 1949, Halpern and Covner published the first description of zoster induced diagphragmatic paralysis. Since then, anecdotal cases have been reported although complete historical data is frequently missing and it has been difficult to elucidate the natural history of the condition. Based upon cumulative data from 10 well-documented cases, several tentative conclusions can be made. The average patient is 69 years of age (range 54±79). The time from rash to paralysis can vary widely from almost immediately to 210 days. In 7/10 cases phrenic nerve function did not recover. In the remaining cases, recovery occurred between 30 and 540 days later. The right side was affected in 7/10 cases although this is not statistically significant. The ratio between male and female patients is 7/3 but not statistically significant in this series. Pathologically, zoster induces a myelitis that involves the dorsal root ganglia. Why certain individual develop this rare complication is unknown and will require many further observations. At present, though, the point is clear±herpes zoster can cause diaphragmatic paralysis in the elderly. In such cases the paralysis may develop as a late complication with a poor chance of recovery. CASE INFORMATION: A 63-year-old woman was referred to our center for evaluation of tinnitus in her right ear for 9 months. She described the sensation as,`like the sounds of a steam engine pumping in my ear,' and that it coincided with her heart beat. She noted that applying pressure with fingers over an area in the right retroauricular area would reduce this abnormal noise. Work-up at an outside hospital was negative including, carotid ultrasound, CT scan of mastoid and temporal bone, audiometry and MRI of the head and internal auditory canal. Examination was significant only for a loud continuos bruit over the retro-mastoid area. Complete blood counts, serum chemistry, and electrocardiogram was normal. Cerebral angiogram showed an extensive dural arteriovenous fistula (AVF) affecting the distal transverse and sigmoid sinuses with supply through both internal and external carotid arteries as well as right vertebral artery. There was no evidence of cortical venous thrombosis. Thrombophilia work-up was normal. She underwent a combined transarterial embolization and stereotactic radiosurgery using gamma knife. Tinnitus resolved completely after intervention. She remains asymptomatic at the 3-month follow-up visit. IMPLICATIONS/DISCUSSION: Pulsatile tinnitus resulting in audible retro-auricular bruit could result either from systemic diseases, causing alteration in hemodynamic, or due to local disorders within or close to the petrous bone. The most common causes of pulsatile tinnitus includes benign intracranial hypertension, atherosclerotic disease, and glomus tumor. Dural AVF is a rare cause of pulsatile tinnitus (2%).However, 90% of patients with dural AVF present with pulsatile tinnitus. Dural AVF has been reported following trauma, infection, pregnancy related thrombophlebitis, surgery on the sigmoid and ethmoid sinuses. Cerebral angiogram remains the diagnostic modality of choice. If untreated, a third of dural AVF's develop intracranial bleeding or infarction. Treatment of dural AVF involves transarterial particulate embolization and radiosurgery using a gamma knife .High index of suspicion and thorough physical examination remains crucial in the early recognition of dural AVF. 2) Recognize principles of management of diabetes mellitus during Ramadan. CASE INFORMATION: A 40-year old female immigrant from Somalia with the history of poorly controlled type II diabetes for the past seven years was enrolled in our continuity clinic. She had been on numerous oral hypoglycemic agents without adequate benefit. Her blood glucose ranged from 200±380 mg/dl and glycosylated hemoglobin was 10.7%. She was started on insulin 70/30, 100 units in the morning and 70 units in the evening with addition of oral thiaglitazone agent. During the month of Ramadan her insulin dose was decreased to 50 units in the morning and evening and she was advised to take her oral medication in the evening as most of her meals were spread over night till dawn. While accompanying her daughter for routine checkup she complained of feeling of generalized``shakiness'' which was new to her. We obtained a random blood glucose level, which revealed a blood glucose of 37mg/dl. She was treated with supplemental glucose with complete resolution of her symptoms. Her insulin regimen was subsequently adjusted further and dietary education and hypoglycemia education was given. Frequent telephone calls were made to adjust her insulin doses and monitor her blood sugars. IMPLICATIONS/DISCUSSION: Fasting in the month of Ramadan represents a recurring annual event in the life of a Muslim patient. Patients with brittle type I diabetes and poorly controlled type II diabetes could develop complications during month long fasting with change in dietary habits. Physicians should monitor their diabetic patients closely and adjustment in the insulin regimen should be made to prevent hypoglycemia. Long acting oral hypoglycemics and premixed insulin should be avoided. Dietary principles should be reinforced. Education regarding home monitoring of blood glucose, hypoglycemia episodes is recommended. After Ramadan, patient's therapeutic regimen will need to be changed back with repeat overall education. Obtaining detailed history could help in ascertaining additional cultural factors which could interfere with management of chronic illnesses.\nADULT ONSET RECALCITRANT PRURITIS Ð TIME FOR A SERIOUS LOOK. P. Chahal 1 , A.K. Ghosh 1 ; 1 Mayo Clinic, Rochester, MN (Tracking ID #74142) LEARNING OBJECTIVES: 1) Recognize the differential diagnosis of worsening chronic pruritus.\n2) Recognize that patients with adult-onset recalcitrant eczema may have underlying noncutaneous lymphoproliferative disorder. CASE INFORMATION: A 38-year old male presented with history of two and a half years of insidious onset of severe itching involving his feet and gradually spreading and becoming generalized in few months. Subsequently, he developed a rash, which consisted of hard bumps', intensely pruritic in nature. He was seen by several physicians and had multiple skin biopsies demonstrating folliculitis and wound cultures growing Staphylococcus species. Numerous treatment attempts were unsuccessful. Examination revealed hundreds of equally spaced excoriated crusted pink papules measuring up to 1 cm diameters. Rest of his systemic examination was normal. Investigations revealed a normal complete blood count, hepatitis profile, and complement levels. HIV and fungal serology and PPD test were negative. Skin biopsies showed chronic dermatitis and the direct immunofluorescence was negative. Chest radiograph revealed multiple lung masses and subsequent CT scan of chest revealed extensive anterior mediastinal, bilateral hilar, right paratracheal, and subcarinal adenopathy and multiple bilateral pulmonary nodules. CT abdomen was normal. He underwent mediastinoscopy and lymph node biopsy, which revealed the diagnosis of Hodgkin's Lymphoma, Nodular Sclerosing type. He was started on combination chemotherapy with ABVD. IMPLICATIONS/DISCUSSION: Chronic unexplained eczema and pruritus of adult onset might be associated with underlying lymphoproliferative malignancy. Although pruritus has been associated with Hodgkin's disease, the reports of prurigo nodularis' association with Hodgkin's lymphoma are rare. Prurigo nodularis is also associated with other malignancies like transitional cell bladder cancer, leukemia and HIV. When an obvious cause like drugs or atopy year old Asian male presented with complaint of inability to move his arms and legs. His symptoms began one day prior to admission after walking a significant amount while shopping at a local mall. After leaving the mall, he started to experience numbness and tingling of his arms, legs and abdomen. Later that day, he started to experience generalized weakness and went to sleep early due to fatigue. When he awoke the morning of admission, he could not walk or move his body. He had to be assisted out of bed and brought to the emergency room. He had no prior medical problems and was not taking any medications, drugs or supplements. Upon further questioning, he revealed that his mother has had problems with low potassium levels. His review of symptoms prior to this episode was completely negative. Physical exam was unremarkable except for neurological exam. He was alert and oriented. His cranial nerves and sensory exam were intact. Motor strength was 0/5 in upper extremities and 1/5 in lower extremities. The patient was areflexive. Labs: Na 145, K 2.0, Cl 106, CO2 16, creatinine .7, CPK 519, Ca 7.9, Phos 1.3, Mg 1.5, TSH 0.0, T4 total 13.8, T3 total 236.4. His urinary potassium excretion was normal. EKG: low voltage t waves and u waves. Patient was diagnosed with hypokalemic periodic paralysis and treated with oral and intravenous potassium chloride. Within a few hours, his potassium improved to 5.0 and his strength was 5/5. The next day, he was discharged and told to follow up with endocrinology for his hyperthyroidism IMPLICATIONS/DISCUSSION: The diagnosis of hypokalemic periodic paralysis is made most easily when hypokalemia is documented during an attack of weakness and other secondary causes are excluded. Attacks begin during the first three decades of life and occur during the rest period following exercise, stress or high carbohydrate meal. The condition can develop in patients with a familial predisposition (autosomal dominant inheritance) or in those with thyrotoxicosis. The latter form is more common in Asian males. Although, the mechanism is not entirely understood, it is thought to be related to sudden shift of potassium into cells. Patients have normal potassium values between episodes and may have decreased magnesium and phosphorus during episodes. Treatment includes oral potassium chloride and correction of thyrotoxicosis. Prevention may include beta blockers, oral supplements, potassium sparing drugs and low carbohydrate meals. Recognition is important because the condition can lead to respiratory muscle paralysis and subsequent death. LEARNING OBJECTIVES: 1. Emphasize the importance of draining an infected galactocele and describe a less-invasive modality to achieve this. 2. Highlight follow-up measures in women with a recurrent galactocele. CASE INFORMATION: A 37 year-old lady presented with a non-tender lump in her left breast 4 weeks after birth of her fourth child. She was lactating without difficulty and denied redness of the overlying skin or fever. Following clinical examination, she had an ultrasound of the breast revealing a 3.7cm cystic mass with internal echoes. Ultrasound-guided aspiration was performed and 30cc of milky fluid aspirated with complete resolution of the mass. However, the galactocele recurred within 24 hours but remained asymptomatic otherwise. Three weeks later, the patient presented with evidence of mastitis, was started on antibiotics, and the galactocele re-aspirated. With persistent signs of infection, galactocele-fluid cultures revealing Streptococcus viridans, and re-accumulation of the milk in the galactocele, incision and drainage was contemplated. This was subverted with a less invasive means by inserting an 8 french multiple side-hole pigtail catheter into the galactocele enabling complete drainage and healing of the underlying infection. The catheter was removed in 10 days and antibiotics given for a total of 2 weeks. To prevent development of a milk fistula through the drain site, fluid reaccumulation was monitored with ultrasound and re-aspiration performed at 4 and 16 days after drain removal. The patient recovered with complete healing of the catheter tract. The galactocele persisted even after cessation of breast-feeding. A mammogram was then performed which revealed no findings of underlying malignancy. IMPLICATIONS/DISCUSSION: A galactocele most commonly presents as a breast mass soon after the cessation of lactation, and an ultrasound evaluation is usually confirmatory. These masses usually disappear in few weeks to months. However, the persistence of a galactocele, particularly after repeated aspirations, necessitates further work-up to rule out ductal obstruction from an underlying malignant process. Our patient had another infrequent complication Ð an infected galactocele. The concern of incision and drainage of a galactocele was that of a high likelihood of milk fistula. On the other hand, if not drained, infection was likely to persist and progress to a breast abscess. An alternative was therefore considered of introducing a pigtail catheter into the galactocele to enable complete drainage while instituting appropriate antibiotic coverage. Catheter drainage of breast abscess has been described in the literature, but this is the first report, to our knowledge, of catheter drainage of an infected galactocele. A 78 year-old lady reported a``whitehead like'', non-irritating lesion on her right nipple. Watchful waiting was recommended and over the course of the next 3 weeks, this lesion progressed into a rash involving the nipple and areola with a painful central abrasion and crusting. With the use of antibiotic cream, the rash improved in 3±4 days but mild flaking of the skin persisted. Three months later, the patient presented to the Breast Clinic for evaluation of a mild, but persistent nipple rash. She denied breast lumps, pain or nipple discharge. Breast cancer risks included nulliparity, and use of hormone replacement therapy for nearly 40 years. On examination, there was mild scaling of the skin over the right nipple with no associated induration, erythema or excoriation. On palpation, no dominant mass, nipple discharge, axillary or supraclavicular lymphadenopathy was identified. Mammograms revealed increasing microcalcifications in the sub-areolar region of the right breast. Stereotactic biopsy of these calcifications was performed and demonstrated high-grade ductal carcinoma in situ (DCIS). Skin biopsy of the scaly region revealed Paget disease of the nipple characterized by tumor cells with large nucleoli distributed singly in the superficial epidermal layers with clusters in the basal portion of the epidermis. These tumor cells were immunoreactive to anti-cytokeratin 7 and carcinoembryonic antigen (CEA) antibodies confirming the diagnosis of Paget disease. In compliance with the patient's wishes, right mastectomy was performed and on pathologic evaluation, residual DCIS was noted around the biopsy site along with Paget disease of the nipple. IMPLICATIONS/DISCUSSION: Paget disease, with an incidence ranging from 0.5 to 2.6%, presents as a scaly persistent rash, and often mimics eczema, or contact dermatitis of the nippleareolar complex. It may or may not be associated with a breast mass. Diagnosis is established by a skin biopsy, revealing characteristic Paget cells. Delay in diagnosis may result when the rash is mild in intensity and if unassociated with a breast mass. Clinical management of a patient with a nipple, or areolar rash mandates continued follow-up. If the rash fails to resolve, a high index of suspicion for Paget disease of the nipple will enable appropriate work-up and optimal outcome.\nBREAST NODULE AFTER PROPHYLACTIC MASTECTOMY. K. Ghosh 1 , K.R. Brandt 1 ; 1 Mayo Clinic, Rochester, MN (Tracking ID #75726) LEARNING OBJECTIVES: 1. Build awareness among physicians that prophylactic mastectomy does not completely eradicate the possibility of breast cancer. 2. Emphasize that any breast lump after prophylactic mastectomy warrants complete work-up to rule out breast cancer. CASE INFORMATION: A 56 year-old lady with fibrocystic changes in the breast had undergone several benign right breast biopsies for mammographically detected abnormalities, and finally decided to have bilateral prophylactic mastectomy to prevent the anxiety associated with these biopsies. Following the mastectomies, she had bilateral pedicle transverse-rectusabdominis-myocutaneous (TRAM) flap reconstruction of the breasts with a smooth postoperative recovery. She was very satisfied with the outcome of the procedure. Four months after the surgery, the patient incidentally felt a lump in the left upper breast and when it persisted for about a month, the patient sought further evaluation. Examination revealed a firm, non-tender, fixed mass with ill-defined margins in the left upper breast. Ultrasound evaluation of the palpable mass revealed a heterogenic echotexture with no evidence of malignancy. Diagnostic left mammogram revealed no evidence of malignancy. Magnetic resonance imaging of the breast with dynamic gadolinium enhancement was then recommended and showed benign appearing localized collection of fat lobules and postoperative changes. The patient continues to do well on follow-up. IMPLICATIONS/DISCUSSION: The management of women at high-risk of breast cancer includes the options of surveillance, chemoprevention, prophylactic mastectomy and/or prophylactic oophorectomy. Prophylactic mastectomy is an option for risk reduction of breast cancer, and in women with a strong family history, has been associated with a 90% relative risk reduction. The procedure does not however, eradicate the risk of breast cancer as residual breast tissue may be left behind depending on the surgical technique. Therefore, any breast lump after PM must be evaluated to rule out breast cancer. In general, breast lumps after PM are often benign and related to fat necrosis as was the case in our patient, but the possibility of malignancy must be borne in mind when evaluating these patients.\nA MAN NEWLY DIAGNOSED WITH HIV REFUSES TO INFORM HIS SPOUSE. C. Gibbs 1 , J. Tsui 1 ; 1 Emory University, Atlanta, GA (Tracking ID #74714) LEARNING OBJECTIVES: 1.) To appreciate a care giver's ethical and legal responsibilities when partner notification is refused by a newly diagnosed patient; 2.) To identify various partner notification strategies and weigh their advantages and disadvantages. CASE INFORMATION: A 36 year-old man presented to the emergency room complaining of fatigue, shortness of breath, severe dyspnea on exertion, and cough worsening over several weeks. The patient had no significant past medical history and denied tobacco, alcohol, or IV drug use. He denied any previous history of blood-product transfusions.He stated he had been in a monogamous marriage for the ten years and had had no other sexual partners. Further, the patient reported a negative HIV test three years ago. Chest X-ray revealed bilateral patchy opacifications without any focal consolidation. Laboratory results were significant for a WBC of 5.1, an LDH of 514, and an ABG of 7.46/39/55. The patient was admitted for communityacquired pneumonia and started on ceftriaxone and doxycycline; however, given his clinical picture, the patient's sputum was sent for silver stain. It returned positive for pneumocystis carinii. The patient's medications were changed to trimethoprim/sulfamethoxazole and prednisone, and he was counseled about the likelihood of immunocompromise. At this point, the patient agreed to HIV testing. He subsequently tested positive for HIV and had a CD4 count of 26. Patient education regarding HIV infection and AIDS was initiated. The patient acknowledged the possibility that his wife may have been infected and agreed that she should be notified. Initially, he wished to talk with her himself (self-referral). After a few days, it was discovered that the patient had not disclosed his HIV status to his wife. With much encouragement and further education, he agreed to a dual referral approach involving a healthcare provider being present when he notified his wife. However, when the appointed meeting day arrived, the patient once again refused. Patient education and support continued. As the patient's discharge approached, the medicine team feared that the patient's wife would never be informed of his seropositive status and her potential risk. These concerns were discussed with the patient and he was informed that if he did not notify his wife, the medicine team would act according to state and federal laws and notify her of her HIV exposure and need for testing. The patient informed his wife within 24 hours. IMPLICATIONS/DISCUSSION: This case demonstrates various approaches to partner notification and deals with the ethical and legal decisions physicians are forced to make when newly diagnosed HIV patients refuse to participate. Partner notification strategies can be classified into three basic categories: self-referral, dual referral, and provider referral. Each approach has its own advantages and disadvantages. Regardless of the strategy chosen, the patient's rights to autonomy, trust, confidentiality, and safety must be respected while balancing the physician's moral and legal responsibility to patients' partner(s), children, and community. Healthcare providers must recognize their responsibilities, know their federal and state laws, and act according to individual cases. A 50 year old African American man with no significant past medical history presented to the office with malaise, weakness, anorexia, watery diarrhea and mild headache of 2 days duration. He was treated conservatively for presumed viral gastroenteritis, until he presented to the emergency room 2 days later with increasingly severe frontal headache and fevers. Physical examination was remarkable for a temperature of 39.6 deg C and nuchal rigidity, but no focal neurological signs or impaired sensorium. Laboratory studies included a WBC count of 8800, and cerebrospinal fluid analysis which demonstrated a CSF WBC count of 150/mm3 with a differential of 11% neutrophils, 88% lymphocytes, 1% monocytes, and a CSF-protein level of 117 mg/dl. CSF gram stain was negative. The patient was admitted and placed on empiric ampicillin and cefrtriaxone. Blood cultures and CSF bacterial, fungal and protozoal studies remained negative. The patient slowly defervesced and his headache improved, and he was discharged to home. Notably, this case occurred at a time when public awareness of West Nile Virus infection in Allegheny County, Pennsylvania was just emerging; the virus had been isolated in several dead birds in the area, although there had been no reported cases of human infection. During the hospitalization, serum was therefore sent for West Nile Virus serologic testing. Following discharge, results of IgM and IgG antibody ELISA assays subsequently returned positive, and were later confirmed by plaque reduction neutralization test (PRNT). The patient gradually improved, although notably after developing a transient morbilliform rash on his chest, back and arms, observed during outpatient follow-up approximately one week after discharge. IMPLICATIONS/DISCUSSION: West Nile Virus infection is a mosquito-borne arboviral illness that is commonest in the United States in late summer and early fall. One of the early clues to an epidemic is the presence of dead birds in the surroundings. Infection is most often asymptomatic, and clinical illness is generally mild, presenting with constitutional symptoms much like those of other viral syndromes. Among patients with more serious CNS illness, isolated meningitis Ð such as occurred in our patient Ð is relatively less common than encephalitis or meningoencephalitis. Treatment is supportive, and advanced age is the biggest risk factor for severe neurologic disease. This case illustrates the need to consider West Nile Virus infection, however, in cases of aseptic meningitis occurring in endemic areas.Diagnosis is by serum or CSF IgM antibodies to West Nile Virus; first-line testing is by ELISA assay, and positive results are generally confirmed by the more specific PRNT.\nAN UNEXPECTED CAUSE OF FEVER, HEADACHE AND ELEVATED ERYTHROCYTE SEDIMENTATION RATE. A.N. Githaiga 1 , E. Anish 1 ; 1 University of Pittsburgh, Pittsburgh, PA (Tracking ID #76143) LEARNING OBJECTIVES: 1) To recognize sarcoid as a cause of central nervous system dysfunction in a patient without other systemic evidence of sarcoidosis. 2) To appreciate the value of performing a brain biopsy to establish a diagnosis of neurosarcoidosis when CSF analysis and neuroimaging are nondiagnostic. CASE INFORMATION: A hypertensive 50-year-old African American woman presented to the hospital with a one-month history of retro-orbital headache, recurrent fevers, chills, sweats and weight loss. Temperature on admission was 38 deg and her neurologic exam was non-focal. Initial labs were significant for WBC-11,800,PLT-610, 000 and ESR-148. A brain CT scan showed old lacunar infarcts. Lumbar puncture showed protein of 111mg/dl, white cell count of 3 and 79% lymphocytes. Viral, bacterial and fungal cultures were negative. Autoimmune serological markers were negative. A bone marrow biopsy was negative for malignancy and granulomatous disease. Serum ACE level was normal ( 29 Units/L.). Chest CT scan was normal, and abdominal CT scan revealed numerous small mesenteric and retroperitoneal lymph nodes that were not accessible for biopsy. The patient was started on empiric prednisone therapy for temporal arteritis at 60 mg per day despite negative bilateral temporal artery biopsies. She noted rapid improvement of her headache and resolution of her fevers and was discharged home. Over the next 8 weeks her ESR dropped to 22 and she remained asymptomatic. However, as the steroid dose was being tapered, the patient began to experience recurrence of headache and fevers, as well as gait and cognitive dysfunction. An MRI was performed that revealed multiple subcortical areas with increased signal. Repeat CSF analysis showed a white cell count of 28 with 89% lymphocytes and increased protein (79 mg/dl). Brain biopsy performed revealed focal perivascular lymphocytic infiltrates with non-necrotizing granulomas. Special stains for AFB and fungus were negative. A diagnosis of neurosarcoidosis was made. Her symptoms steadily improved with high dose solumedrol and subsequent oral prednisone. IMPLICATIONS/DISCUSSION: This case illustrates the importance of considering a diagnosis of neurosarcoidosis as a cause of fevers and CNS dysfunction in an individual who does not have other overt manifestations of sarcoid. In 5% of cases, neurosarcoid is the initial presentation of the disease. CNS presentations typically include cranial nerve palsies, meningitis, optic nerve dysfunction and neuroendocrine disorders (e.g. diabetes insipidus and central hypothyroidism). CSF findings are nonspecific and include elevated protein and mononuclear pleocytosis. Contrast enhanced MRI may reveal meningeal/cortical masses or hydrocephalus. As demonstrated in the case presented, neural tissue biopsy may be useful, revealing perivascular non-caseating granulomas, which while not diagnostic, will be highly suggestive in the appropriate clinical context. FOOLED BY FAT/PERPLEXED BY PROPOFOL. E. Gjersvik Cichowski 1 , H. Sakowski 1 , H. Hashish 1 , R. Baltaro 1 ; 1 Creighton University, Omaha, NE (Tracking ID #74955) LEARNING OBJECTIVES: 1) Recognize laboratory error in the measurement of serum bicarbonate.\n2) Utilize the Henderson-Hasselbach equation to indentify blood gas analysis errors.\n3) Recognize interfering substances as potential causes of laboratory errors. CASE INFORMATION: A 72 year-old male was admitted for respiratory distress and confusion, and found to have a right upper lobe lung mass and hypercalcemia. He was intubated on the second hospital day due to worsening of his respiratory status. Propofol was initiated for sedation and methylprednisolone and levofloxacin were given for a presumed postobstructive pneumonia. His initial arterial blood gas after intubation showed a pH 7.38 pCO2 38 pO2 143 on an Fio2 of .60. His measured HCO3 was 26 meq/l. Over the next 4 days, his measured bicarbonate progressively dropped to 8 meq/l despite no change in his arterial blood gas (pH 7.38 pCO2 36 pO2 103 on an FiO2 of .45). His anion gap was calculated at 19. Serum lactate was normal, and serum ketones were absent. Consultation with the pathology department revealed the patient's serum to be grossly lipemic. A review of the chart revealed the patient did receive lipid infusions with TPN 36 and 18 hours prior to this discovery. A lipid panel was obtained and revealed marked hypertriglyceridemia at 4426 mg/dl. The lipid infusions were discontinued, and the propofol was weaned off. The bicarbonate level dropped to a low of 3 meq/l approximately 7 hours after the medication was discontinued. Four hours later, the bicarbonate had corrected to 21 meq/l. The serum, however, remained grossly lipemic. The patient's condition continued to decline with the development of septic shock, multi-organ failure and ventricular arrythmias. Results of a previous bronchoscopy demonstrated small cell carcinoma. The patient's family requested no further aggressive treatment and he expired later that day. IMPLICATIONS/DISCUSSION: This patient developed marked derangement in his measured bicarbonate levels that did not correspond to his arterial blood gas analysis. A laboratory error was hypothesized as the cause. Due to the finding of lipemic serum, the hypertriglyceridemia was initially suspected as the interfering substance. Upon discontinuing the propofol, the serum bicarbonate level normalized, the serum, however remained lipemic. In a review of the literature, neither propofol nor hypertriglyceridemia have been reported as potential causes of this lab error. Further testing is needed to determine the role of propofol as an interfering substance in bicarbonate laboratory analysis.\nDYING TO BE THIN. R.E. Graham 1 ; 1 Lenox Hill Hospital, New York, NY (Tracking ID #75181) LEARNING OBJECTIVES: This case demonstrates the limitations of our current knowledge about herbal products, particularly in dose-related toxicities, and calls attention to the need for further studies. This case highlights the need for health care providers to be knowledgeable about the herbal products their patients may be using, and to be able to identify potential herbassociated adverse effects. I hope this case illustrates CAM's prevalence, benefits, and potential dangers but most importantly highlights the importance of obtaining a complete medical history; including asking our patients if they are using and/or incorporating alternative therapies into their general healthcare. CASE INFORMATION: 26 year old thin female presented to the ED at about 5:15 PM complaining of a frontal headache, nausea, and feeling weak after fainting. The patient stated to the triage nurse that she had eaten Mexican food at about 3:30 PM, went home and took``a fat burner pill'' at about 4PM. She has been on fat burner pills and herbal teas for about one year. Suddenly she began to feel nauseous, developed a worsening headache, then fainted which prompted her to seek medical attention. Initially, the patient was taken to the back of the ED. While awaiting physician examination, the patient syncopized and began to have a seizure. The patient was wheeled into trauma and found to be in Ventricular fibrillation with a potassium of 2.4. She was defibrillated and then became hypotensive and progressed into asystole, she was resuscitated and then intubated. She began posturing without corneal reflexes. After stabilization the patient was taken to CT; which revealed±Extensive subarachnoid hemorrhage with the presence of intraventricular hemorrhage. Saddly, over the next 48 hours she was placed on life support and an organ donation referral was made. Soon after she expired. IMPLICATIONS/DISCUSSION: Complementary and Alternative medicine (CAM) is one of the fastest growing sectors in health care today. In 1997 , Eisenberg & et al JAMA, 1998 :280:1569±1575, revealed that 42.1% of Americans (83million) used CAM over the past year. In the same article Eisenberg, showed that only 38% of patients disclose to their physicians their use of alternative therapies when not asked directly. As we continue to see an increase in the rate of obesity in the United States, our patients will incorporate both prescription and nonprescription products in hopes of obtaining their desired weight loss. This current vignette exemplifies the need for the health care provider to first, ask the unasked question, secondly, to have a basic understanding and knowledge regarding CAM, and finally to provide an important role in educating, advising and discussing the potential risks and benefits of CAM, and the need to closely monitor any use of them. CXR showed R mediastinal mass. CT showed diffuse LAN (mediastinal, peritoneal, retroperitoneal) and numerous liver metastases. HIV and hepatitis panel were negative. FNA of neck revealed atypical lymphoid cells. Excisional biopsy showed anaplastic carcinoma, no melanin but HMB-45 and melan A (melanocyte markers) were immunostain positive. IMPLICATIONS/DISCUSSION: Distinguishing between localized and generalized LAN helps guide the appropriate differential diagnosis. Diffuse LAN suggests HIV, mycobacterial infection, infectious mononucleosis, SLE, medications (i.e., phenytoin), metastatic carcinoma and lymphoma. The history and physical exam become crucial in the diagnostic work up of generalized LAN. The history should include signs and symptoms suggestive of infection or malignancy, exposures (cats±cat scratch disease; raw meat±toxoplasmosis; tick bite±lyme disease; unprotected sex or injection drug use-HIV), constitutional symptoms and medications. A complete physical exam should guide the appropriate diagnostic work-up. Patients with generalized LAN should have a CBC and CXR. If these are normal, consider PPD, HIV test, RPR, ANA, and heterophile test, although these tend to be low yield if not specifically indicated. An FNA for cytology is generally the first step due to ease and safety, especially for granulomatous versus malignancy and lymphoma versus carcinoma, yet an FNA fails to provide enough material to examine architectural detail or to perform immunostains. This is especially relevant with younger patients where hematologic malignancies or uncommon solid tumors are a consideration. Metastatic melanoma without a known skin primary accounts for around 3% of melanomas. Prior to melanocyte-specific immunostains, these cases were considered carcinoma of unkown primary. Cases of metastatic melanoma lacking primary skin lesion include primaries of mucosal surfaces (sinuses, oral mucosa, vulva, vagina and anorectum), occular and primary skin lesion which have regressed. Thus, the lack of skin lesion does not rule out metastatic melanoma.\nNo effective systemic therapy exists at this time for metastatic melanoma. Biotherapeutic approaches combining immunomodulators such as IL-2, INF, and vaccines with conventional cytotoxic chemotherapy are currenlty under active investigation.\nA COURAGOUS CALL: SALINE FOR HEART FAILURE? M. Guidry 1 , J. Wiese 1 , J. Wiese 1 ; 1 Tulane University, New Orleans, LA (Tracking ID #77054) LEARNING OBJECTIVES: 1. Recognize the perils of over-diuresis in bi-ventricular heart failure. 2. Use disciplined methods of diagnosis to prevent errors in pattern recognition diagnosis. 3. Use the physical examination to predict cardiovascular function. CASE INFORMATION: An 80 year-old woman presented with two days of shortness of breath and orthopnea. Her blood pressure was 132/65 with a heart rate of 116. She had bibasilar crackles and 3+ pitting bipedal edema. There was an S3, S4, and an inferiorly displaced PMI. Her room-air ABG at presentation was pH: 7.44; pCO2:35; pO2:58. She was diuresed with a total of 320 mg of furosemide over one day. Her symptoms improved. On the third hospital day, she had a sudden worsening of her shortness of breath. Her neck veins were flat and there were no crackles. A repeat ABG was pH: 7.28; pC02: 57; p02: 81; HCO3:42 Her BUN tocreatinine ratio increased from 23/1.4 at presentation to 41/2.3 on the third hospital day. Diuresis was stopped, and hydration with normal saline was initiated. Her shortness of breath improved, with normalization of her acid base values. IMPLICATIONS/DISCUSSION: Dyspnea can result from hypoxia or hypercarbia. This patient initially presented with dyspnea due to congestive heart failure; she was appropriately managed with preload reduction via diuresis. Continuing diuresis was excessive however. In the setting of bi-ventricular heart failure, the over-diuresis resulted in an inadequate right ventricular preload, and a subsequent drop in her pulmonary artery pressure. With the alveolar pressure unchanged and the pulmonary artery pressure lowered, the percentage of dead space increased. This resulted in a ventilation to perfusion mismatch resulting in hypercarbia. The physical examination was the tell-tale sign, revealing that her volume status was low, instead of high. The ABG confirmed the diagnosis; the rehydration increased the pulmonary artery pressure, resolving the mismatch, and eliminating the dyspnea. In caring for patients with biventricular heart failure who are sensitive to preload reduction, it is important to remember that hypercarbia from dead space ventilation can result from aggressive lowering of the pulmonary artery pressure.\nCOLCHICINE±INDUCED MYOPATHY. K. Gupta 1 , T.A. Townley 1 ; 1 Creighton University, Omaha, NE (Tracking ID #76577) LEARNING OBJECTIVES: 1) Review the differential diagnosis of proximal muscle weakness in an elderly male 2) Review symptoms and signs of colchicine-myopathy 3) Review the risks factors for colchicine myopathy. CASE INFORMATION: INTRODUCTION: Colchicine has been used for 200 + years, but there are few reports of myopathy associated with its use. We present such a case. CASE: Patient is a 70-year-old male who was hospitalized for abdominal pain/diarrhea. He had fallen on his hip at home 1 week prior to his admission and became increasingly weak with considerable reductions in his ADL such as inability to get in and out of bed, stand from sitting and walk. His PMH includes COPD on home O2, CHF, HTN, DM, CRI (baseline Cr 2.2), obesity and gout. Medications include Oxycodone 5/325mg prn, metoprolol 50mg qd, colchicine 0.6mg qd, allopurinol 100mg qd, furosemide 80mg qd, glipizide 30mg qd, isosorbide dinitrate 30mg qd, fosinopril 40mg qd, ASA 325mg qd, KCl 20mEq qd, Insulin 70/30, MVI qd. His initial physical exam was normal except for reduced reflexes in upper limbs 2+/4 and lower limbs 1+/4 with no sensory deficits. Lab work revealed acute renal insufficiency on CRI (BUN 69/Cr 2.6), in addition LFT's were twice normal. CK was 1059 with MB fraction <5%. CXR and abdominal series were clear and EKG was unchanged. His abdominal pain resolved, and ARI improved with hydration and discontinuation of ACE-I. Initially his weakness was felt to be secondary to intercurrent illness superimposed on chronic illness. However weakness failed to improve and a repeat CK was 1825 with ESR of 36. His weakness localized to proximal muscles of upper and lower extremities, with strength 3/5. Distal muscle strength was 5/5. Colchicine was suspected as the cause of his myopathy and discontinued. He noticed a significant improvement in his proximal muscle of 4/5 on the following day. EMG showed myositis with predominantly proximal muscle irritability. Repeat labs 5 days after discontinuing colchicine, showed BUN 44/ Cr 1.4, and CK 958. There was a noticeable improvement in strength, with ability to stand from sitting, ambulate across room and get into and out of bed alone. IMPLICATIONS/DISCUSSION: DISCUSSION: Patients taking colchicine over an extended period of time have been reported to develop a subacute, often severe myopathy in conjunction with a relatively mild polyneuropathy. Chronic renal failure is a risk factor for this syndrome. Our patient had been on colchicine for more than a year. In this case an untreated myopathy may have resulted in nursing home placement versus a trial of steroids for polymyositis. In our patient, who had pre-existing renal insufficiency worsened by the dehydration, the clinical presentation and the improvement on discontinuation of colchicine strongly supports the diagnosis of Colchicine induced myopathy.\nMANAGEMENT OF RAMSAY±HUNT SYNDROME. S. Gupta 1 , O. Melamed 2 ; 1 UCLA School of Medicine, Los Angeles, CA; 2 Olive View±UCLA Medical Center, Sylmar, CA (Tracking ID #75744) LEARNING OBJECTIVES: 1. Clinical presentation and pathophysiology of Ramsay±Hunt Syndrome 2. Lack of outcome benefit from head imaging, IV therapy, and surgery for RHS 3. Improved outcome with early treatment. CASE INFORMATION: A 30-year-old female presented to the urgent care clinic for evaluation of left ear pain, vertigo, left facial paralysis and small vesicles over the left pinna. The patient's past medical history was significant for chickenpox at the age of eight. On physical examination the patient had left ear swelling and erythema with multiple 0.5±1.0 mm vesicles around the pinna and marked erythema involving the external auditory canal. There was a positive Bell's phenomenon (the eyeball rolling up and outward on attempting to close the affected eye). A facial paralysis involved the left levator labii and left frontalis muscles. A left sensorineural deficit was observed with Weber test lateralizing to the right and a positive (normal) Rinne test bilaterally. The diagnosis of Ramsay±Hunt Syndrome was made. The patient was discharged from the clinic on a regimen of Acyclovir and Prednisone. She was instructed to avoid pregnancy, contact with pregnant women or neonates. IMPLICATIONS/DISCUSSION: Clinical presentation of unilateral facial paralysis and a vesicular rash on the ear (zoster oticus), often accompanied with hearing loss, tinnitus, nausea, vomiting, vertigo and nystagmus was first described by J Ramsay Hunt in 1907. Vestibulocochlear symptoms were explained by the anatomical proximity of the eighth nerve and the geniculate ganglion within the temporal bone. The pathophysiology of RHS involves the reactivation of varicella-zoster virus (VZV) within the geniculate ganglion and secondary edema in the facial nerve resulting in facial nerve palsy. VZV is considered a major etiologic agent of Bell's palsy. Use of contrast enhanced MRI in diagnosis of RHS does not appear to provide much information as enhancement of the geniculate ganglion, meatal fundus, and facial nerve is observed independent of etiology. Most patients with Bell's palsy tend to recover to complete or near normal function even without treatment. One prospective study found that 85% of patients showed signs of remission within the first 3 weeks, with 71% recovering complete function, 13% left with insignificant sequelae, and 16% left with permanently diminished function. Although most patients with RHS improve, many patients are left with functional and cosmetic deficits. Treatment of RHS with oral acyclovir and prednisone (AS therapy) may improve outcome. Two separate studies showed complete recovery in 75% of patients with RHS treated with AS. There has been no evidence of improvement in outcome with intravenous administration of either acyclovir or prednisone, though many patients are still admitted to the hospital for IV therapy. There is also insufficient evidence for efficacy of surgical facial nerve decompression. Evidence for improved outcomes with earlier administration of therapy have been observed. In one study 75% of patients beginning AS treatment within 3 days of onset of facial paralysis had complete recovery, compared with 30% of patients beginning therapy after 7 days. Earlier therapy decreased nerve degeneration and improved recovery of hearing.\nSEIZURES ON POSTPARTUM DAY SEVEN. K. Gustafson 1 , R. Khurana 1 ; 1 University of Alberta, Edmonton, Alberta. (Tracking ID #76917) LEARNING OBJECTIVES: 1) To recognize that preeclampsia and eclampsia may occur in the postpartum period. 2) Diagnose eclampsia even when it presents without the typical signs of preeclampsia. We describe a case of postpartum eclampsia that was initially misdiagnosed. CASE INFORMATION: A 28 year old woman G2P2 was transferred to our tertiary case hospital on postpartum day 7 because of seizures. Her pregnancy had been uncomplicated and she had a vaginal delivery at term for a healthy 3515g female infant. Her past medical history was unremarkable and her blood pressure had remained normal throughout the pregnancy. She presented to a local hospital on postpartum day 7 complaining of severe headache, nausea and vomiting. Her blood pressure was 176/100. She was given analgesia, but then had two generalized seizures. She was given magnesium sulfate 5 g intravenously and transferred. When assessed, her blood pressure was 150/90. She had no focal neurologic findings, right upper quadrant tenderness, edema or hyperreflexia. Her liver enzymes and platelet count were normal. Urinalysis was negative for protein. Urate was slightly elevated. CT scan of the head was normal. EEG was normal. She was seen by the services of Neurology, Internal Medicine and the Intensive Care Unit. She was diagnosed with seizures of unknown origin and admitted for observation. The magnesium sulfate was discontinued. Her blood pressure varied over the next two days from 124/81 to 200/103. On postpartum day 10, she had another generalized seizure. MRI of her brain was consistent with hypertensive encephalopathy. She was given a diagnosis of postpartum eclampsia and treated with intravenous magnesium sulfate and antihypertensive therapy. She improved over the next few days and all therapy was discontinued. She remains well with no further seizure activity. IMPLICATIONS/DISCUSSION: Preeclampsia/eclampsia may worsen or even initially present in the postpartum period. Postpartum eclampsia represents 25% of all cases of eclampsia. The majority of these events occur in the first 24±48 hours postpartum, but have been reported to occur as late as 4 weeks postpartum. Eclampsia does not always occur in patients who have the classic triad of preeclampsia (hypertension, proteinuria and edema). About 20% of patients with eclampsia have no proteinuria. Computed tomography and EEG are often unremarkable, but should be performed to rule out other causes of seizure. When MRI abnormalities are present, hyperintense signals in cortical and subcortical areas on T2 weighted images may be seen. These are usually in the posterior parietal or occipital lobes. Management of eclampsia includes delivery of the fetus (if diagnosed antepartum), magnesium sulfate to prevent recurrent seizures and control of severe hypertension. A high index of suspicion is necessary to appropriately diagnose the woman with postpartum eclampsia and prevent further morbidity. LEARNING OBJECTIVES: To recognize focal muscle infarction as an unusual, and sometimes bilateral, cause of severe limb pain in patients with diabetes. CASE INFORMATION: A 55 year-old diabetic woman developed disabling pain in both upper legs, with thickening and tenderness of the soft tissues of the right lateral thigh. There was no evidence of infection. She had neuropathy but sensation in the thighs was normal. There was a history of disc disease but an MRI examination of the spine was unimpressive. She had arterial disease but ankle/brachial indices were only in the claudication range. There was a history of venous thrombosis but she was taking warfarin and duplex studies showed no clots. Plain films and a bone scan were negative. MRI examination showed diffuse edematous infiltration of the subcutaneous fat of both thighs, with increased signal in the right vastus lateralis muscle. These findings were consistent with infarction of the vastus lateralis, and conservative treatment was recommended. IMPLICATIONS/DISCUSSION: Diabetic muscle infarction causes pain, swelling, and tenderness, most commonly in the thigh, but sometimes in the calf. The opposite limb may be involved, either sequentially, or concurrently, as in this case. The MRI appearance is characteristic and allows the diagnosis to be made without the risks of biopsy. Treatment is supportive. This rare but important entity must be considered when diabetic patients suffer severe limb pain.\nA CASE OF SPONTANEOUS BILATERAL CAROTID CAVERNOUS FISTULAS. S. Habib 1 , P.K. Han 1 ; 1 University of Pittsburgh, Pittsburgh, PA (Tracking ID #76354) LEARNING OBJECTIVES: 1) To recognize the clinical symptoms and signs of carotid cavernous fistula. 2) To describe an unusual presentation of carotid cavernous fistula. CASE INFORMATION: An 85-year-old woman presented with a 3±4 week history of progressive difficulty with eye opening, poor vision, and tinnitus. She denied headaches, ocular or orbital pain, diplopia, worsening of symptoms with activity, dizziness, focal weakness, or other recent illnesses. She had a history of recent falls, but no head trauma. Her past medical history was significant for hypertension, primary hyperparathyroidism, normocytic anemia, Paget's disease of bone, and a fifty pack-year smoking history. She was on ferrous sulfate but no other medications Physical examination revealed a blood pressure of 168/74 without orthostatic changes, mild conjunctival pallor, and bilateral complete ptosis. There was chemosis of the right eye and opthalmoplegia, with bilateral palsies of cranial nerves III, IV, and IV. Both pupils were mid-dilated and non-reactive to light. No other neurological deficits were noted. Diagnostic evaluation included a normal CT scan of the brain, while brain MRI demonstrated changes suggestive of bilateral carotid cavernous fistulas. Cerebral angiography was performed, which confirmed the presence of bilateral type A carotid cavernous fistulas. The patient then underwent trans-carotid endovascular embolization, which was unsuccessful. Unfortunately the patient developed right-sided hemiparesis a few days later, and subsequently declined further intervention. IMPLICATIONS/DISCUSSION: Carotid cavernous fistula is an unusual problem caused by an abnormal communication between the internal and/external carotid artery and the cavernous sinus. It is classically characterized by the triad of pulsating exophthalmos, ocular chemosis and an orbital bruit. Other features include orbital pain or tightness, pulsatile tinnitis, visual loss due to ischemia of optic nerve and retina, retinal vein congestion, and glaucoma. Ophthalmoplegia often results from pressure upon local cranial nerves; CN VI is affected most often, and CN III and IV are involved less frequently. Our patient did not present with the classic clinical triad, but with bilateral complete ophthalmoplegia and pulsatile tinnitis. Trauma is the most common cause of carotid cavernous fistula although 20 percent of cases are non-traumatic. Spontaneous bilateral carotid cavernous fistula, as observed in our patient, is a very rare entity. Risk factors for carotid cavernous fistulas include older women, mothers during childbirth, hypertension, atherosclerosis and collagen vascular diseases. A four-type classification of carotid cavernous fistulas based on the anatomy of the lesion has been proposed. Our patient had type A fistulas, presumably due to a rupture of an intra-cavernous carotid artery aneurysm. Early recognition and intervention is important to prevent permanent visual loss and ophthalmoplegia. Endovascular interventions (embolization and balloon occlusion) represent a successful treatment option although stroke is a well-recognized complication in 5±15 % of cases. LEARNING OBJECTIVES: 1, To recognize that autoimmune hepatitis is a rare complication following allogeneic stem cell transplantation. 2, To understand the pathophysiology of auto immune hepatitis folllowing stem cell transplantation. CASE INFORMATION: A 43 year-old white female developed Ph-chromosome positive chronic myeloid leukemia. She received cytoreductive therapy, followed by a 6/6 HLA matched allogeneic peripheral stem cell transplant (SCT). Tacrolimus and prednisone were continued for 18 months. One month after tacrolimus dose reduction, her serum AST and ALT levels were 723 and 965 respectively, but bilirubin and alkaline phosphatase were normal. An abdominal sonogram was normal. After the tacrolimus dose was increased to its baseline level, serum AST and ALT levels decreased to 45 and 60. Tacrolimus was then gradually reduced and eventually discontinued, which caused a rise in AST and ALT levels. She had no risk factors for viral hepatitis. Physical examination was normal except for Cushingoid appearance. CBC with differential were normal. Biochemical tests showed total bilirubin 0.7, AST 88, ALT 128, ALP 96, GGT 74, albumin 3.7, and total protein 9.8. Other biochemical profile including TSH, iron studies, ceruloplasmin, a 1 anti-trypsin and a feto protein were all within normal limits. ANA and anti thyroglobulin antibodies were positive whereas anti LKM, anti mitochondrial, anti smooth muscle antibodies were all negative. She had negative serology for EBV, CMV, HZV and hepatitis A, B, and C viruses. The histology was most consistent with autoimmune hepatitis. In situ hybridization study for Y chromosome established that immune reactive cells were exclusively of donor origin. There was no evidence of recurrent chronic myelogenous leukemia. Serum transaminase levels normalized with prednisone 20 mg a day but relapsed on dose reduction. Azathioprine was therefore added. Her transaminase levels have been normal for 28 months. IMPLICATIONS/DISCUSSION: This report documents a case of liver dysfunction, following SCT, which was indistinguishable from`definite' autoimmune hepatitis (baseline diagnostic score > 15) as defined by the modified criteria of International Autoimmune Hepatitis Group. To our knowledge, the present case represents the first report of this complication after allogeneic peripheral SCT. One intriguing feature of this case is that the target liver cells and immune reactive cells are of different origins. The donor origin of immune reactive cells was clearly established in our female patient by in-situ hybridization labeling of the Y chromosome. As the term`autoimmune hepatitis' does not accurately reflect the disease process, we propose the term`alloimmune hepatitis' for this syndrome. A 50 year old white male was admitted to our hospital in July with three days of fever, chills, headache, and neck stiffness. He worked as an air-conditioner repairman and spent a considerable amount of time shirtless, outdoors in East Texas. Physical examination was significant for fever of 102.4 degrees Fahrenheit, meningismus, and Kernig's sign. The patient was awake, alert, and oriented to person, place, and time. Initial CSF analysis revealed WBC 91 with 92% neutrophils and 8% monocytes; protein 81, glucose 68; gram stain revealed few WBCs, and was negative for bacteria; latex agglutination screen was negative for H. influenzae, S. pneumoniae, N. meningitis A, B, C and group B streptococcus. CBC revealed a WBC count of 10.6 with 70% neutrophils, 20% lymphocytes, and 10% monocytes. Within two days of admission, the patient became progressively more lethargic and delirious and was oriented only to person, month, and year. Repeat CSF analysis revealed WBC of 223 with 15% neutrophils, 59% lymphocytes, and 26% monocytes; glucose of 66 and protein of 81. CSF bacterial cultures remained negative. CSF studies for syphilis, tuberculosis, and herpes simplex virus were negative. CSF IgM was positive for West Nile virus. IMPLICATIONS/DISCUSSION: Until 1999, West Nile virus was found only in Africa, Asia, and the Middle East. Since that time, West Nile virus has infected over 3852 individuals in the U.S. in at least 39 states. West Nile virus is a single-stranded RNA Flavivirus virus that belongs to the Japanese encephalitis complex. Birds are the natural hosts of West Nile virus and mosquitoes, particularly of the culex species, act as vectors. Infection in humans typically occurs in late summer through early fall when mosquitoes are most predominant. Severe neurologic disease accompanying West Nile virus infection is rare. One out of every 150 infected patients develops meningitis or encephalitis. Age greater than 50, is the major risk factor for developing life-threatening neurologic disease. West Nile viral encephalitis is similar to other viral encephalitities and is characterized by fever, headache and an altered level of consciousness. CSF studies typically show a lymphocytic pleocytosis with elevated protein.\nEarly viral encephalitis, however, may demonstrate CSF with a predominantly neutrophilic CSF pleocytosis, as in our patient. Lymphopenia is a common feature of West Nile virus encephalitis and thus it may aid in distinguishing this disease from other forms of viral encephalitis. Definitive diagnosis is obtained through confirmation of blood or CSF IgM to West Nile virus. Treatment is supportive. The main intervention is prevention by promoting the use of mosquito repellant and protective clothing. year old white male presented to our hospital with complaints of fever, diarrhea, and fatigue. His admission was preceded by two days of watery, non-bloody stools, fever to 102, fatigue, malaise, and generalized weakness. The patient denied any sick contacts or travel, although he noted living in a mosquito-infested area of East Texas. On admission, the patient's temperature was 102.2, pulse 69, blood pressure 117/61 and respiratory rate 24. The patient appeared fatigued and exhibited a diffuse, erythematous rash and injected conjunctivae. The neurologic examination revealed normal tone and 5/5 strength throughout. Sensation was intact and reflexes were 2+ and symmetrical in all extremities. The rest of the physical examination was unremarkable. Within days, the patient developed severe generalized weakness with areflexia and ultimately, respiratory failure requiring mechanical ventilation. Analysis of CSF revealed a WBC count of 78 with 55% lymphocytes, 30% neutrophils, and 5% monocytes; glucose 154, and protein 116. Electrophysiologic studies were performed and were consistent with a diffuse axonal polyneuropathy. Serologic studies were positive for the West Nile virus. IMPLICATIONS/DISCUSSION: Before 1999, West Nile virus, a single-stranded RNA virus belonging to the Japanese encephalitis complex, was found only outside the western hemisphere. Since then, West Nile virus has infected over 3852 individuals in the U.S. in at least 39 states. Many clinicians are aware that West Nile virus is associated with encephalitis and meningitis, however, the association between West Nile virus infection and acute flaccid paralysis is less well known. CDC surveillance during the most recent outbreak of West Nile Virus in Louisiana revealed that almost 20% of patients described as having severe neurologic disease presented with acute flaccid paralysis. Early reports of patients with West Nile virus acute flaccid paralysis often confused the condition with Guillain-Barre syndrome. Clinicians should be aware that unlike most patients with Guillain-Barre syndrome, patients with West Nile associated acute flaccid paralysis typically have fever, CSF pleocytosis, and electrodiagnostic studies consistent with a predominantly axonal neuropathy. Most patients with Guillain-Barre syndrome are afebrile, have an elevated CSF protein, few if any WBCs in the CSF, and electrodiagnostic studies consistent with a demyelinating neuropathy. The presence of a maculopapular or morbilliform rash, such as our patient had, may also suggest the presence of West Nile virus infection. LEARNING OBJECTIVES: 1. Develop a differential diagnosis for thrombocytopenia in a well-appearing adult. 2. Learn to diagnose and treat ITP in adults using the best available evidence. CASE INFORMATION: A well-appearing 72 year-old Chinese-American man being treated with glipizide for type II diabetes presented to clinic with a petechial rash on his trunk, arms, and legs. A CBC revealed isolated thrombocytopenia (platelet count 23,000). Glipizide was discontinued but thrombocytopenia persisted. His platelet count normalized with a prednisone taper but in the ensuing months, despite splenectomy, he continued to require 5 mg of daily prednisone to maintain a normal platelet count. His diabetes became poorly controlled while on prednisone, and he had one episode of pneumonia requiring antibiotics. The decision was made to stop prednisone. After the initial petechiae he has had no further evidence of bleeding, but his platelet count remains between 10,000 and 40,000. IMPLICATIONS/DISCUSSION: Medications and ITP are common causes of isolated thrombocytopenia in well-appearing adults. Glipizide is among the many medications with published case reports of thrombocytopenia as a side effect. ITP is a clinical diagnosis made in the setting of isolated thrombocytopenia with a normal peripheral smear and no other identifiable cause of low platelets. Experts recommend a bone marrow biopsy in patients over age 60 to rule out myelodysplasia. Anti-platelet antibodies are not specific for ITP. Case series suggest that in adults spontaneous remission occurs less than 10% of the time. Splenectomy is curative in 50±65% of all cases, with better results in patients less than 40 years old. Lifethreatening bleeding rarely occurs with a platelet count of greater than 10,000. Prednisone is not curative and is most helpful as a supportive measure in patients with platelets under 10,000 or other high bleeding risks (ie: surgery). Asymptomatic patients with platelet counts above 10,000 and no other risks for bleeding can most likely be safely managed without steroids. Develop an approach for the evaluation of dyspepsia through review of the current evidence and recommendations 3. Review diagnostic testing and treatment for H. pylori. CASE INFORMATION: 46 y/o woman presented with epigastric discomfort and nausea 1±2 months after she ran out of her cimetidine. Symptoms were similar to ones she experienced 2 years prior when she may have had a``stomach bug'' for which she thought she was given antibiotics. The patient did endorse drinking 2 beers/day but denied dysphagia, anorexia, weight loss or melena. Physical exam was benign. Symptoms initially improved with reinstituting cimetidine and decreasing alcohol intake. Initial labs demonstrated a normal CBC, LFTs, amylase and lipase, but was impressive for an H. pylori titer 1:87 U/mL. Despite continued cimetidine, the patient's symptoms recurred within months and lansoprazole was initiated. An H. pylori breath test was not pursued due to the patient's reluctance to discontinue medications prior to the test. Subsequent endoscopy revealed 2 small, linear, cleanbased ulcers biopsy positive for H. pylori. Symptoms resolved after treatment with amoxicillin, clarithromycin and lansoprozole. IMPLICATIONS/DISCUSSION: Dyspepsia, or chronic and/or recurrent pain or discomfort of the upper abdomen, is a common complaint accounting for 5% of all PCP visits. H. pylori is implicated in several of the etiologies of dyspepsia, thus the management of dyspepsia must involve strategies to appropriately diagnose and treat H. pylori. Testing for H. pylori is recommended only if treatment is intended; however, guidelines regarding the indications for treatment of H. pylori put forth by the American College of Gastroenterology (1998) and the Maastricht 2±2000 (European) are conflicting. Both agree on the need to treat in cases of active ulcer disease or gastric/MALT lymphoma, but the Europeans are more aggressive in their recommendations advising treatment in entities such as non-ulcer dyspepsia or concurrent NSAID use. A recent meta-analysis (Laine, et. al. 2001) helps to clarify the issue of non-ulcer dyspepsia Ð which accounts for the majority of dyspepsia Ð suggesting that eradication of H. pylori does not improve symptoms in non-ulcer dyspepsia. Yet the uncertainty regarding the utility of eradication of H. pylori makes the appropriate work up of uninvestigated dyspepsia somewhat unclear. We will review several algorithms for the evaluation of dyspepsia and discuss the current data supporting the most often recommended``test and treat'' approach with regards to H. pylori and dyspepsia. In addition, we will discuss the range of diagnostic testing available for H. pylori and the appropriate use of these tests during the evaluation of dyspepsia. The topics of treatment and treatment failures will also be addressed. spells of altered mental status and syncope. The patient's behavior ranged from combative and agitated to lethargic. These variations in behavior usually occurred early in the morning, lasted one to three hours, and were associated with diaphoresis, marked confusion, and generalized weakness. The patient often had no recollection of these events and returned to baseline between episodes. This pattern increased in frequency and severity prior to admission. The spells were associated with blood sugar measurements in the low forties. The patient was hospitalized for further work up and underwent a monitored fast. Serial lab values were consistent with insulinoma. Imaging studies to locate a tumor included body CT, ultrasound, endoscopic ultrasound, and nuclear imaging, which were non-diagnostic. Initially the patient's glucose was maintained at normal levels with intravenous 10% dextrose and frequent snacking. Diazoxide therapy was initiated which allowed the intravenous dextrose to be discontinued; however, even with frequent snacks the patient still had occasional low sugars. With failure of medical management the patient underwent exploratory laparotomy with intraoperative ultrasound which did not detect a distinct mass. A distal pancreatectomy was performed, which demonstrated hyperplasia of islet cells. Post-operatively the patient was able to maintain normal blood sugars without diazoxide. IMPLICATIONS/DISCUSSION: Nesidioblastosis is a rare cause of hyperinsulinemic hypoglycemia that is differentiated by an insulinoma only by pathology. In this case, the patient's hypoglycemia was associated with syncope and altered mental status. LEARNING OBJECTIVES: 1) to review neutropenia as a presentation of SLE 2) to discuss strategies to avoid misdiagnoses and how to manage lupus neutropenia. CASE INFORMATION: A 53 y/o Egyptian woman presented with two weeks of fevers reaching 102±103 degrees F. She denied cough, headache, dysuria, diarrhea, abdominal pain, joint pain, oral lesions, rash, weight loss, sexual promiscuity, recent travel, or drug use. She last visited Egypt four years ago and worked as an apartment manager. She took occasional Tylenol, no other medication. On examination she had a temperature of 103 degrees F and appropriate tachycardia. No lymphadenopathy, breast masses, abdominal masses/pain, lung crackles, cardiac murmurs, or neurological findings were found. She had a WBC 1.7, 64% PMNs and mild anemia Hgb 11.1. LFT's, TSH, Fe/Ferritin, and chemistry normal. An extensive infectious workup was negative including blood and fungal cultures, AFB cultures, urinalysis, CXR, visceral leishmaniasis, malaria, brucellosis, EBV, CMV, HIV, RPR, PPD. CT of the chest, abdomen, pelvis, and head found only mild splenomegaly. Transesophogeal echo revealed no valvular vegetations. The patient continued with daily fevers to 102±103 degrees F, anemia worsened to a Hgb 8.4, WBC decreased over ten days to a nadir of 0.7 (ANC 60). GCSF was started for neutropenia and empiric Ceftazidime and Tobramycin were begun. Bone marrow biopsy showed normal cell elements and myeloid to erythroid ratio 1:1. Her mammogram was normal. At this point ANA returned +1:320 as well as Coombs positive. Antids DNA and Anti-Smith was sent. On second day of GCSF patient developed polyarthritis in hands, wrists, elbows, knees, ankles as well as faint malar rash. Follow up CXR revealed small left pleural effusion. GCSF was stopped and patient was started on Prednisone with subsequent resolution of fevers, rash, and arthralgias. Anti-ds DNA returned +1:80. IMPLICATIONS/DISCUSSION: Lupus can present in a variety of ways. In this case, the initial presentation was neutropenic fever. Only after receiving GCSF did the diagnosis become apparent as it precipitated a lupus flare. The diagnostic criteria for Lupus developed by the ACR are 96% sensitive and specific. Non-specific symptoms such as fever, fatigue, and weight loss are common presenting complaints and are often attributed to causes other than lupus leading to misdiagnoses. Leukopenia occurs in more than 50% of lupus patients with either granulocytopenia or lymphopenia. GCSF has been used in the treatment of lupus induced neutropenia resulting in a rise in neutrophils within 48 hours, however the elevation is transient with return to pretreatment levels within days after withdrawal of therapy. In addition treatment with GCSF has been associated with flares in lupus that resolve when the drug is stopped. A 63 year old male with hypertension, chronic renal insufficiency, and peripheral vascular disease presented to the Emergency Department with acute onset of bilateral lower extremity numbness and weakness. Six weeks prior, he had received an arteriovenous fistula in preparation for hemodialysis and was feeling well until the morning of admission, when, upon rising from bed, he fell to the ground because of numbness and weakness of his legs. Review of systems was positive for urinary incontinence,``sharp'' back pain, and shortness of breath which had developed at the same time as the paresthesia. Upon reviewing his medical records, it was discovered that his prostate specific antigen had been elevated at 24.9, but the patient had not followed up with the urologist for further evaluation. Physical exam showed a well-developed male, alert and oriented, in moderate respiratory distress. His respiratory rate was 24, blood pressure 142/90, heart rate 82, and temperature 36 C. Other significant physical findings included bilateral crackles, a pericardial friction rub, absent sensation to light touch at an L3 level, 2/5 bilateral lower extremity strength, absent ankle and patellar reflexes, absent pedal pulses (previously documented), diminished but equal femoral pulses, and an enlarged prostate. Labs showed BUN of 71mg/dl and creatinine of 9.3 mg/dl. Chest radiograph demonstrated mild pulmonary edema. Thorax and abdomen CT without contrast showed a normal aorta. A vascular catheter was placed, and emergent hemodialysis started. The neurology and neurosurgery services were consulted, and plans were made to get an emergent MRI of the lumbosacral spine to rule out cord compression after the patient was stabilized. An hour after dialysis had begun, the patient lost consciousness, and ACLS for pulseless electrical activity was initiated. After 25 minutes of unsuccessful resuscitation, the patient was declared deceased. An autopsy was performed which showed hemopericardium from type A aortic dissection which descended to the bifurcation of the aorta. IMPLICATIONS/DISCUSSION: In this case, aortic dissection caused peripheral arterial ischemia which presented as paresthesia and weakness. Stroke is the most common neurologic presentation of aortic dissection, although 10% of patients present with ischemic peripheral neuropathies. Although pain describing dissection has classically been taught to be``ripping or tearing'', most patients classify the pain as``sharp.'' Given the high mortality associated with aortic dissection, a high index of suspicion should be maintained for patients presenting with paresthesia and back pain. year-old white male with unremarkable medical history presented with acholic stools, dark urine, and constitutional symptoms. Physical examination revealed a diffuse erythematous, maculo-papular rash of trunk and legs, profound jaundice, liver span of 14cm with a firm edge, no splenomegally and no stigmata of chronic liver disease. His initial outpatient laboratory tests revealed: alkaline phosphatase 413 U/L, total bilirubin 3 mg/dl, SGOT 510 U/L, and SGPT 515 U/L. Viral hepatitis serologies, an abdominal ultrasound and CT, and an endoscopic retrograde cholangiopancreatography were unremarkable. Three weeks later, the patient underwent a percutaneous liver biopsy at which time his laboratory values were: WBC 1.9  103/ 3 , Hgb 12.6 g/dl, Platelets 62,000, alkaline phosphatase 706 U/L, total bilirubin 26.9 U/L (direct 21.8 U/L), SGOT 190 U/L, and SGPT173 U/L. The liver biopsy showed a high-grade malignant leukemic infiltrate. Bone marrow biopsy confirmed the diagnosis of B cell/ Burkitt's type acute lymphoblastic leukemia with flow cytometry showing CD19+/CD10+/CD20-/CD34-/CD13+/HLA DR+ cells. IMPLICATIONS/DISCUSSION: Acute lymphoblastic leukemia is primarily a disease of children; adults only account for approximately 20% of cases and Burkitt's B cell ALL, in turn, makes up only 5% of the cases of ALL. Unfortunately, over 60% of adults with ALL are not cured of their disease. The clinical features of ALL are often nonspecific and primarily constitutional as well as manifestations of cytopenias secondary to the bone marrow infiltration. Also, ALL has a predilection for the CNS and symptoms of meningeal leukemia or hyperleukocytosis are not uncommon at presentation. Cholestatic jaundice, however, is a much less common presenting feature with a limited number of case reports in the literature. As demonstrated in this case, one should entertain the diagnosis of acute lymphoblastic leukemia in those presenting with acute cholestatic jaundice. He also had intermittent episodes of gum bleeding, anorexia and shortness of breath. His medications included Atenolol. On physical exam, his vital signs were stable and he was noted to have pale conjunctiva, minimal gum bleeding and a non-radiating systolic murmur. His white blood cell count was 1,600 with a normal differential. He had a hematocrit of 14.9 and a platelet count of 7,000, and lactate dehydrogenase of 794. His remaining lab findings were normal. His peripheral smear showed decrease red blood cells and platelets with normal morphology and no immature white blood cells. Two bone marrow biopsies were both dry taps and confirmed a diagnosis of Acute Myelogenous Leukemia (AML) of the Megakaryocyte subtype (FAB classification M7). His course was complicated early on by neutropenic fever and broadspectrum antibiotics were initiated. Chemotherapy was started after a 48-hour period feverfree period with Idarubicin for 3 days and Cytosine-arabinoside for seven days. He tolerated the treatment well, but remained pancytopenic. Two days after completing induction, he became febrile and complained of severe abdominal pain and diarrhea. Broad-spectrum antibiotics for both bacterial and fungal pathogens were started. Imaging of his abdomen showed bowel wall thickening consistent with neutropenic colitis (typhlitis). Treatment initially consisted of broad-spectrum antibiotic coverage and supportive care. His condition progressively worsened, and both he and family did not want any aggressive intervention. The patient passed away shortly thereafter. IMPLICATIONS/DISCUSSION: Acute leukemia accounts for about 10% of human cancers and is the leading cause of death in adults younger than 35 years of age. In our case, the patient had poor prognostic factors of age greater than 60 years old and poor functional status. The treatment of AML consists of supportive care and chemotherapy with induction therapy conducted with an anthracycline and Cytosine-arabinoside (except for subtype M3). Complications from leukemia include those related to immunosuppression and pancytopenia. Typhlitis represents inflammation and/or necrosis of the cecum, appendix, and/or ileum and is a frequent complication of the treatment of AML. Patients usually present with abdominal pain, diarrhea and fever. The etiology of typhlitis is unknown, but profound neutropenia and mucosal injury from cytotoxic drugs play important roles in the pathogenesis. The mortality rate from typhlitis in leukemic patients can be high and the treatment consists of broad antibiotic coverage, nasogastric suction and supportive care. Surgical intervention is indicated if there is bowel perforation or persistent bleeding. CASE INFORMATION: A 74-year-old female, who was previously independent and ambulating well, presented with a four day history of progressively worsening bilateral knee pain and swelling. Her past medical history is significant for degenerative joint disease with chronic pain in her knees. Her medications included Rofecoxib, Oxycontin, Oxycodone, and Calcium with vitamin D. The patient had received an injection of Hylan G-F 20 in both knees four days prior to presentation for chronic knee pain. A similar injection had been performed one year previously with relief of pain. The next day she developed fever, chills and the knees became warm, erythematous, and swollen, with worsening pain, 10/10 in severity. Upon presentation, the patient was afebrile and both knees were swollen and tender. The patient was unable to bear weight, range of motion of the knees was limited to about 30 degrees bilaterally, and there was tenderness in the lateral joint line bilaterally. Laboratory results showed a normal white blood cell count, ESR 70, and C Reactive Protein 20.6. Venous doppler ultrasound was negative for DVT. Knee x-rays revealed bilateral effusions and evidence of severe osteoarthritis. Bilateral knee arthrocentesis showed (left/right) white blood cell count, 11300/10500 with a normal differential, red blood cells 475/38000, and no crystals. Gram stain and cultures were negative. The patient was admitted to the hospital for pain management and transferred to sub-acute rehabilitation for 3 weeks where she responded well to corticosteroid injections and physical therapy. IMPLICATIONS/DISCUSSION: Visculosupplementation involves using intra-articular injections of high elastoviscous solutions of hyaluronan and hylans in an attempt to restore the natural mechanical properties of synovial fluid. Complications of intra-articular injections with visculosupplementation occur in 2±4% of injections. Local reactions include joint pain, effusion, and rarely, warmth. Acute pseudogout, granulomatous inflammation, and severe systemic reactions have also been reported. Rest, analgesics, corticosteroid injections, cold packs, and therapeutic arthrocentesis may help relieve symptoms. The presence of fever, debilitating pain, and joint effusions in our patient suggested both a systemic and local reaction to the supplementation. This impacted her functional status as she became bedridden and she required a prolonged rehabilitation to regain her independent ambulatory status. LEARNING OBJECTIVES: 1. Recognize myxedema coma as rare, but important diagnosis in differential for obtunded patient 2. Recognize emergent need for treatment of myxedema 3. Recognize mortality due to the condition and its therapy. CASE INFORMATION: 80 year old Caucasian female with history of congestive heart failure, chronic obstructive pulmonary disease, hypothyroidism was found by neighbors to be in obtunded state. In emergency room, her temperature was 95.9 F, blood pressure 108/56, pulse 50, respiratory rate 11, glasgow coma scale score 3. Her skin was cool to touch, with induration and 1+ non-pitting edema of the lower extremities. She had peri-orbital swelling. Labs: Na 142, K 4.3, Glc 142, creatinine 2.9, Hgb 9.3, CPK 286, CPK MB 5, troponin I .1, TSH 309, T4 total < 1, T4 free .1. ABG (after intubation, 100% FIO2) pH 7.26 pCO2 52 pO2 624. EKG:sinus bradycardia at 50 beats/minute, low voltage. CT head was normal. Blood cultures were negative. After labs were drawn, patient was started on nasogastric levothyroxine but continued to deteriorate. The next day, she was switched to intravenous therapy and started on steroids. Her mental status improved markedly and she was extubated. However, a few days later, she was found in asystole and expired. IMPLICATIONS/DISCUSSION: Myxedema coma is a rare, deadly condition. It should be suspected in patients who present with hypothermia and altered mental status. They may also have hypotension, hypoventilation, hyponatremia, hypoglycemia, and cardiovascular abnormalities. It may be caused by severe longstanding hypothyroidism or precipitated by infection, infarction, cold weather, narcotics/sedatives. Elderly females are most commonly affected. Therapy should be based on clinical suspicion and started right after drawing TSH, free T4 and cortisol levels. Glucocorticoid therapy prevents adrenal crisis in cases with associated adrenal insufficiency. Type of thyroid therapy is controversial. Some prefer T3 because of its rapid onset and greater biologic activity. Others prefer T4, which should be given intravenously to avoid malabsorption. Careful dosing based on age and weight may prevent cardiac complications of thyroid therapy. The mortality rate is 30±40% despite adequate therapy. Even though intravenous therapy was delayed in this patient, she showed significant improvement prior to her demise. Thus, her death may have been due to cardiac complications of therapy rather than the delay in intravenous therapy. However, definitive cause of death was not determined. Since therapy is controversial and associated with complications, an additional lesson is to focus on prevention via recognition and treatment of hypothyroidism. In this tragic case, the patient had been lost to follow up.\nINTESTINAL T-CELL LYMPHOMA PRESENTING AS SMALL BOWEL PERFORATION. P. Hulick 1 , H. Tun 1 ; 1 Mayo Clinic, Jacksonville, FL (Tracking ID #74266) LEARNING OBJECTIVES: 1. Recognize the association of primary gastrointestinal T-cell lymphoma and celiac disease 2. Recognize the poor prognosis, especially with +CD56 marker 3. Treatment must include systemic chemotherapy. CASE INFORMATION: A 73-year-old white female presented to the emergency department following an acute exacerbation of abdominal pain that had progressed over the past two months. The pain was originally diffuse in nature but became sharp in character with the acute presentation. The patient denied radiation to the back while movement exacerbated the pain. The patient reported decreased appetite with a fifteen-pound weight loss over the preceding six months, nausea but no vomiting, and intermittent fevers to a maximum of 105F over the preceding two months. The patient denied other constitutional symptoms. On admission, the patient was afebrile with normal vital signs. Physical exam was notable for diffuse abdominal discomfort on palpation, decreased bowel sounds of normal pitch, and the absence of generalized lymphadenopathy Laboratory studies were Hgb 11.1g/dL, MCV 85 fL, and platelet count of 385,000. Electrolytes, creatinine and liver profile were unremarkable. LDH was 185 u/L. Computed tomography (CT) scan of the abdomen/pelvis revealed a pneumoperitoneum without identification of a specific site of perforation. Exploratory laparotomy revealed a perforated small bowel mass (7.0  4.0  1.0 cm) with infiltrating neoplastic lymphocytes positive for CD 3, 43, and 56 staining. Flow cytometry showed a T-cell phenotype with anomalous expression of CD 103 (T-cell mucosal homing antigen receptor). Staging evaluation did not reveal evidence of metastatic disease. The pathology findings in our case are consistent with enteropathy associated T-cell lymphoma. Our patient presented with the rarer T-cell CD56 positive variant which has a poorer overall prognosis and aggressive course. IMPLICATIONS/DISCUSSION: Primary gastrointestinal lymphomas account for 30±40% of extranodal lymphomas, but they are usually of B-cell lineage. Celiac disease, a malabsorption disorder related to gluten intolerance, is thought to be a risk factor for developing primary intestinal T-cell lymphoma. Intestinal T-cell lymphoma continues to have a poor prognosis. CD56 (neuronal cell-adhesion molecule) positive tumors have been associated with a more aggressive course. Additional poor predictors include nutritional status at presentation and presence of a bowel perforation. Survival data for cases presenting with bowel perforation has been estimated at 28% and 0% six months and one year post diagnosis. Treatment consists of surgical resection of the tumor and adjuvant chemotherapy which consists of cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) which our patient received. Systemic chemotherapy is necessary, regardless of stage, because of the malignant clonal population of T-cells that exists in the normal gastrointestinal mucosa. Relapse rate is thus high, even in patients with initial response to chemotherapy. year-old woman with recurrent ovarian cancer was hospitalized and conservatively managed for a partial small bowel obstruction. After a second unsuccessful attempt to anti-coagulate her right su-bclavian central venous catheter, the patient developed acute left-sided weakness associated with slurred speech. She denied chest pain but stated she was increasing short of breath overnight. Vital signs included a heart rate of 117 beats per minute and a respiratory rate of 26 breaths per minute. Physical exam was remarkable only for a left facial droop, left arm and leg weakness, and dysarthria. An emergent CT scan revealed an acute, non-hemorrhagic right-sided cerebral infarct. ECG, duplex scan of the carotid arteries and chest radiograph were normal. Cardiac isoenzymes were negative for myocardial infarction. An arterial blood gas analysis revealed severe hypoxia and an alveolar-arterial oxygen gradient of 42. A ventilation-perfusion scan showed high probability for pulmonary embolism and a transesophageal echocardiogram revealed a patent foramen ovale. IMPLICATIONS/DISCUSSION: Indwelling catheters can serve as a nidus for thrombus formation. Emboli may produce pulmonary sequela with concomitant increased right sided circulatory pressures. This results in paradoxical emboli entering the arterial circulation though a right to left shunt, such as a patent foramen ovale, with resultant cerebral ischemia. Patients who demonstrate symptoms of both a cerebral infarct and a pulmonary embolism should be evaluated with a CT scan, carotid duplex, ventilation-perfusion scan and transesophageal echocardiogram to identify the source and location of the embolus. Treatment involves immediate evaluation for emergent anticoagulation if not contraindicated. Operative closure of a patent foramen is not indicated if the elevation of right sided pressure is related to an acute event. year old Asian female without significant medical history presented with ten days of rash, fever, joint pains and stiffness. Rash, which was erythematous and non-pruritic, began on her thighs and spread to her back and chest. Joint pains spread from her hands and wrists to her shoulders, ankles and knees. Her course of illness included high fevers and a sore throat. Physical exam revealed temperature of 103.9 F and erythematous, salmon colored maculo-papules on her trunk and proximal extremities. Palms and soles were spared. The rash was exacerbated by fever spikes, which usually occurred in the afternoon. Koebner's phenomenon occurred when rubbing caused the rash to appear on previously uninvolved skin. Metacarpophalangial joints, wrists and left shoulder were tender, but without erythema, warmth or effusions. There was no lymphadenopathy or splenomegaly. Labs: WBC 22,000 (85% neutrophils, 5% bands). Blood cultures and tests for HIV, enterovirus, cytomegalovirus, Ebstein-Barr virus, hepatitis, parvovirus were negative. Anti-streptolysin O titers were negative. LFT, ANA, RF, chest xray, CT chest/abdomen/pelvis, echocardiogram were normal. Skin biopsy showed perivascular infiltrate, no vascultis. Patient was started on nonsteroidal anti-inflammatory medications and gradually responded to therapy. IMPLICATIONS/DISCUSSION: Adult Still's disease, although uncommon, should be considered in adults with fever, joint pains and rash. The diagnosis requires atleast 5 criteria, including atleast 2 major ones. The major criteria are temperature of > 39 C for atleast 1 week, arthralgias for atleast 2 weeks, typical rash (salmon, maculopapular, evanescent, koebners phenomenon), WBC > 10,000 (> 80% neutrophils). Minor criteria are sore throat, lymphadenopathy and/or splenomegaly, elevated transaminases or LDH, negative ANA and RF. Diagnosis also requires exclusion of infection, malignancy and other rheumatic diseases. Treatment is aimed at joint symptoms because extent and chronicity of arthritis is best predictor of long term consequences. Nonsteroidals, corticosteroids and immunomodulating drugs are therapeutic options. Choice of therapy depends upon organ involvement and severity of disease. LEARNING OBJECTIVES: 1) To recognize intussusception as a cause of intestinal obstruction, 2) To recognize that the treatment of adult intussusception differs from pediatric cases, 3) To recognize that adult intussusception is often a presentation of underlying pathology. CASE INFORMATION: A 53 year old male with a past history of small cell lymphoma treated three years prior with CHOP chemotherapy and radiation therapy presented to the emergency department with several days of diffuse, unremitting periumbilical abdominal pain. He denied melena or hematochezia, and, until recent constipation, his bowel habits were unchanged. His intake of food was reduced due to bloating, but he denied nausea or vomiting. He did not experience fatigue, night sweats, fevers, or weight loss. On examination, his abdomen was soft and non-distended. He had normal bowel sounds. Despite diffuse nonspecific tenderness, he did not exhibit guarding or rebound. The spleen and liver were normal size, and no masses were palpated. A rectal exam was normal, and stool hemoccult was negative. Because of his medical history, a CT scan of the abdomen was performed which revealed ileocecal intussusception and resulting obstruction. A barium enema confirmed an intussusception but was unable to reduce it. He was taken to the operating room where the surgeons found intra-abdominal lymphadenopathy and ischemia of the proximal colon requiring hemicolectomy. Pathology of the resected tissue revealed follicular B-cell lymphoma within the terminal ileum and cecum. IMPLICATIONS/DISCUSSION: Intussusception is a process of gastrointestinal invagination in which a proximal segment telescopes into a distal portion. While common in children as the second leading cause for acute abdomen, adult intussusception occurs in only 5±10% of all cases. Presentation may be either acute or of a chronic indolent nature. It is rarely idiopathic in adults as it is in children, and underlying pathology is readily identifiable in up to 90% of all cases. Common causes include polyps, Meckel's diverticulum, endometriosis, and inflammatory bowel disease, but 54%±69% of identified pathology are malignancies including lymphomas. The process of intussusception causes gastrointestinal circulatory obstruction which can lead to ischemia and bowel infarct. Avoiding delay in surgical intervention preserves tissue. While barium enema is both diagnostic and therapeutic in children, it is less efficacious in adults, and its role in adult therapy is actually controversial. Because of the likelihood of underlying pathology, primary resection is often the treatment of choice. Whether or not to attempt reduction before resection is arguable; some authors have suggested that time spent on barium enemas compromises the integrity of bowel tissue. While uncommon, intussusception should be readily recognized when it occurs. Treatment of the disease requires timely surgical intervention to preserve bowel tissue. It is paramount to recognize that treatment in adults differs from pediatric cases where barium enemas are often successful. Discovery of adult intussusception must arouse clinical suspicion of underlying pathology which should be addressed. He was taken to the local emergency department, which was unable to run ethylene glycol level assays. At the outside hospital, he had an anion gap of 25, but an osmolar gap was not measured. He was placed on an ethanol drip and transferred to a tertiary care center. A stat ethylene glycol level returned elevated at 432 mg/dL, and the osmolar gap was 140 mOsm/kg. At this time, although stuporous, he was arousable and conversed appropriately. Unfortunately, urine was not sent for analysis, but serum creatinine was 1.2 mg/dL. Instead of initiating hemodialysis, the patient was placed on intravenous fluids with dextrose and saline and given fomepizole every 12 hours. Treatment continued for the next 60 hours until his ethylene glycol level fell within a non-toxic range. Clinically, his mental status returned to baseline, and his serum creatinine improved to 0.8 mg/dL. The patient underwent psychiatric evaluation and after medical clearance was transferred to an inpatient psychiatric facility for treatment of his depression. IMPLICATIONS/DISCUSSION: Ethylene glycol is a sweet liquid found in antifreeze. The taste has led to accidental intoxication in young children, but ingestion also occurs during suicide attempts. It is metabolized into oxalate, which is excreted by the kidneys, through several enzymatic steps beginning with alcohol dehydrogenase. Early toxicity is manifested by mental status changes ranging from stupor to coma. Renal damage from direct toxicity to the kidneys develops later although the rate of occurrence depends on the severity of intoxication. Presumptive diagnosis, based on a history of ingesting solutions likely to contain ethylene glycol, such as antifreeze, is the first critical step in diagnosis. While ethylene glycol can be measured directly, the laboratory equipment may not be available readily in all emergency departments. However, diagnosis can be confirmed indirectly with basic laboratory tests. Ethylene glycol itself causes an increased osmolar gap, and its metabolite glycolic acid creates the anion gap metabolic acidosis. The end product of metabolism, oxalate, can precipitate in urine, and urinalysis may reveal calcium oxalate crystals. Treatment must occur expediently to preserve renal and neurological function. This should involve adequate hydration and inhibition of alcohol dehydrogenase to prevent breakdown of ethylene glycol into its toxic metabolites. Ethylene glycol is dialyzable, and severe intoxication may require hemodialysis. However, in most cases, fomepizole, a more efficacious inhibitor of alcohol dehydrogenase than ethanol, can be used alone without need for dialysis. If both methods are used the dose of fomepizole needs to be increased since it is a dialyzable agent. Fomepizole can preserve renal function without invasive vascular access required for hemodialysis. Recovery without permanent sequelae is possible but requires timely recognition of ethylene glycol intoxication and familiarity with the treatment methods. A 24 year old man with severe hemophilia A and AIDS was admitted to the medical service with new neurological deficits. Eight weeks prior to admission he was started on highly active retroviral therapy (HAART). Six weeks prior to admission he developed left facial droop and left upper extremity weakness. The patient's family described behavioral changes including mood disturbances and aggressive behavior. On the day of admission the patient experienced two witnessed generalized seizures. On physical examination he appeared chronically ill. Neurological examination revealed a left cranial nerve VII upper motor neuron palsy and left upper extremity weakness. There was no papilledema. An MRI of the brain revealed multifocal hyperintense signals in the right frontal, left frontal, and right parietal areas predominately affecting the white matter without enhancement or mass effect. A subsequent spinal tap revealed a normal opening pressure with 1 white blood cell, 0 red blood cells, total protein of 74, and glucose of 54. Evaluation of the cerebrospinal fluid for tuberculosis, bacteria, fungus, toxoplasmosis, syphilis, herpes, CMV, and cryptococcus were negative. JC virus DNA was detected by PCR from the CSF. Based on the clinical presentation, radiographic findings and positive JC virus DNA in the CSF, the diagnosis of progressive multifocal leukoencepholapthy (PML) was made. IMPLICATIONS/DISCUSSION: PML is a disorder characterized by multiple rapidly progressive focal neurological deficits. Hemiparesis, visual field defects, cognitive impairment, and cranial nerve deficits are most common. Intracranial pressure usually remains normal. Ninety-five percent of all patients with PML have an underlying immunosuppressive disorder such as HIV. Conversely, 1±3% of AIDS patients will develop PML. CT or MRI of the brain reveals multiple bilateral, usually asymmetric areas of demyelination. The lesions are usually localized to the periventricular area and subcortical white matter. There is no contrast enhancement or mass effect. The diagnosis is made based on the clinical picture of multiple neurologic deficits, characteristic radiographic findings, and isolation of the JC virus from the CSF. Detection of JC virus from the CSF has a sensitivity of 74±95% and a specificity of 92±100%. There is currently no proven effective therapy for PML. HAART therapy has been shown to slow the progression of the disease. However, most patients deteriorate rapidly within 6 months. LEARNING OBJECTIVES: 1. Recognize the importance of opthomologic exam in the differential diagnosis of aseptic meningitis 2. Understand the role of steroid therapy in Vogt-Koyanagi-Harada Syndrome. CASE INFORMATION: A 24-year-old previously healthy man was admitted to the medical service with progressively worsening generalized headaches, decreased visual acuity, and hearing loss for two weeks. The patient reported malaise, but denied nausea, vomiting and fevers. He had never traveled outside the United States and did not use drugs or alcohol. The patient was afebrile. HEENT examination revealed bilateral papilledema and decreased visual acuity and hearing. Neck examination was remarkable for nuchal rigidity. The neurological exam was otherwise normal. CT scan of the head and orbits confirmed the papilledema but did not reveal any mass effect. A subsequent spinal tap revealed normal opening pressure, 727 white blood cells with 70% lymphocytes, a protein = 81, and glucose = 53. The patient was begun on broad-spectrum antibiotics and anti-tuberculous therapy without significant improvement. RPR, HIV, ESR, and cryptococcal antigen were negative. Ophthalmologic consultation was obtained. Slit lamp examination revealed cells in the anterior chamber consistent with uveitis, choroidal inflammation, and depigmentation. The patient was diagnosed with Vogt-Koyanagi-Harada (VKH) syndrome based on the ophthalmologic findings, aseptic meningitis and hearing loss. The patient was started on high dose steroids with relatively rapid resolution of the headaches and visual and hearing loss. IMPLICATIONS/DISCUSSION: VKH is a uveomeningoencephaltic syndrome and appears to be an autoimmune reaction to melanocytes thus making it more common in Asians, Middle-Easterners, and Hispanics than whites. Melanocytes are located in the skin, uvea, retinal choriod, membrane of the inner ear, and leptomeninges, accounting for the particular pattern of involvement seen in this syndrome. The ocular findings may include cataracts, glucoma, and globe atrophy. Meningeal involvement may lead to encephalopathy, seizures, myelopathy, or other focal signs. CSF findings include a lymphocytic pleocytosis, elevated total protein, and normal opening pressure. Other manifestations of VKH include dysacousia, alopecia, poliosis, and vitiligo. Although the optimal steroid dosage for treating VKH has not yet been defined it is generally accepted to begin therapy with high-dose systemic steroids followed by oral steroids for at least 6 months. Patients typically have a rapid decrease in symptoms. In about half of the patients, disease recurs within six months of discontinuation of steroids.\nNONSTEROIDAL ANTI-INFLAMMATORY DRUG ASSOCIATE D LOWER GASTROINTESTINAL TOXICITY. J.T. Jacob 1 , A.K. Jaffer 1 ; 1 Cleveland Clinic Foundation, Cleveland, OH (Tracking ID #74333) LEARNING OBJECTIVES: 1) Recognize the lower gastrointestinal (GI) adverse effects of non-steroidal anti-inflammatory drugs (NSAIDs). 2) Examine the differential diagnosis of multiple gastrointestinal ulcers. 3) Implement appropriate testing and treatment of NSAIDinduced lower GI pathology. CASE INFORMATION: An 80-year-old female with coronary artery disease, hypertension, dyslipidemia and osteoarthritis was admitted to the hospital with chest pain and two episodes of syncope. Each episode of syncope was preceded by use of sublingual nitroglycerin. Review of systems was positive for chronic fatigue, but negative for weight loss, nausea, vomiting, abdominal pain or GI blood loss. Medications were metoprolol, omeprazole, hydrochlorothiazide, indomethacin, and gemfibrozil. Exam revealed a BP = 113/60 without orthostasis, a Pulse = 95 and 97% O2 sat. The exam of the abdomen was normal except for guaiac positive stool on rectal exam. A CBC showed a hemoglobin of 8.9 g/dl with a normal MCV and RDW. An iron panel, B12, folate, LDH, bilirubin, and haptoglobin were also normal. A dipyridamole thallium stress test was negative. The syncope was felt to be related to transient hypotension from nitroglycerin in the setting of anemia. She was therefore transfused blood and underwent upper and lower endoscopies. Multiple ulcers were noted in the stomach, terminal ileum, ileocecal valve, transverse colon and descending colon. Biopsies demonstrated acute nonspecific inflammation. Gastrin levels were normal. Indomethacin was discontinued, acetaminophen was initiated and omeprazole continued. She was discharged home in stable condition with a two-month follow up endoscopy. IMPLICATIONS/DISCUSSION: Adverse gastrointestinal (GI) effects of NSAIDs are not limited to the stomach and duodenum. Although these sites are most commonly affected, adverse effects related to the large bowel and the distal small intestine are increasingly being recognized in practice. NSAIDs can cause a variety of pathology in the lower GI tract including erosions, ulcers, diaphragms, strictures, small bowel enteropathy, diverticular bleeding and colitis resembling or exacerbating inflammatory bowel disease (IBD). The pathophysiology of these changes has not been elucidated. Other etiologies of multiple GI ulcers include: viral and bacterial infections, vasculitides, IBD and Zollinger-Ellison syndrome. Treatment consists of discontinuation and avoidance of all NSAIDs. Gastrin levels should be checked to rule out a gastrinoma. A repeat endoscopy in 6±8 weeks is needed to confirm healing otherwise other etiologies need to be considered. An increase in reporting of these adverse effects will lead to a better understanding of NSAID induced lower gastrointestinal toxicity amongst physicians. -old male with a history of DM1, inflammatory bowel disease (IBD) and PG was admitted for ileostomy closure. The patient discontinued his insulin pump the night prior to surgery. The preoperative glucose was 500 and the surgery was canceled. He was well until three weeks prior when his shin lesions started to get worse after minor trauma. Review of systems was otherwise negative. On exam, he appeared lethargic, T = 38.3 8C, Pulse = 145, and BP = 110/50 mmHg. The patient had four necrotic shin and ankle ulcers with exposed bone and tendon. Labs revealed a serum bicarbonate of 9 mg/dl, potassium of 4.6 mg/dl, glucose of 527 mg/dl, anion gap of 26 and elevated serum ketones. A blood gas revealed a pH of 7.24, pCO2 of 17 mmHg, pO2 of 123 mm Hg and HCO3 of 7 mg/dl. Culture of the ulcers grew gram positive and negative bacteria. The patient was treated with intravenous (IV) Insulin, fluids and antibiotics. After the DKA resolved, therapy for PG was begun using infliximab, topical cromolyn, clobetasol and whirlpool therapy. During infliximab infusion the patient developed transient hypotensive and shortness of breath. He was treated with diphenhydamine and hydrocortisone with resolution of his symptoms. He was switched to oral cyclosporine and discharged home. IMPLICATIONS/DISCUSSION: There are two accepted alternatives in the preoperative management of DM1. Patients can hold short-acting insulin on the morning of surgery and take one-half to two-thirds of their normal long-acting insulin dose or omit their morning insulin regimen entirely and be administered regular insulin on arrival to the hospital based on their glucose level. Patients with insulin pumps should continue their pumps at a basal rate until the morning of surgery. PG is an idiopathic dermatosis associated with epidermal and/or dermal inflammation. It is most commonly associated with systemic diseases such as IBD, rheumatoid arthritis and lymphoproliferative disorders. These lesions typically involve the lower extremities and are painful. Treatment is directed at the underlying systemic disease. Systemic steroids were avoided in this patient's case due to concerns for recurrent hyperglycemia. Alternatives include infliximab, dapsone, thalidomide, azathioprine, cyclophosphamide, 6-mercatopurine, tacrolimus and cyclosporine. Adjunctive therapies include antimicrobials, topical cromolyn, clobetasol and whirlpool therapy. female with Crohn's disease on long term TPN presented with shallow breathing, paresthesias involving the extremities and progressive motor weakness over a period of 2 days. Two weeks prior to presentation she was diagnosed with linerelated candidemia; the port was removed and she was treated with intravenous fluconazole. Exam revealed a cachectic female who was afebrile, BP = 110/80, HR = 90, RR=22, O2 sat = 97%. Neurological exam revealed a right facial droop, dysarthria, motor strength was 2/5 in the upper extremities (UE) and 0/5 in the lower extremities (LE) bilaterally. All reflexes were absent. Brain and spine MRI were unremarkable. The CSF cell count and glucose were normal while the protein was elevated. Blood, urine and CSF cultures were negative. The initial vital capacity (VC) was decreased to 0.89 L and the negative inspiratory force (NIF) was unobtainable. EMG revealed features of generalized sensorimotor polyneuropathy with demyelination and axonal loss. Recurrent asymptomatic episodes of non-sustained ventricular tachyarrhythmia were observed on telemetry but cardiac evaluation revealed no structural heart disease. The patient was diagnosed with GBS with cranial nerve involvement, a GBS variant called Miller-Fisher syndrome. Plasmapheresis was instituted and after a couple exchanges improvement was noted in her motor strength, VC and NIF. She was discharged after completing five session of plasmapheresis. 6-weeks post-discharge, her neurological examination was remarkable only for mild residual diplegia. IMPLICATIONS/DISCUSSION: GBS is an acute ascending inflammatory demyelinating polyradiculoneuropathy. The management of GBS requires early recognition because the respiratory muscles can be involved. Elective intubation should be considered when the forced VC falls below 15 mL/kg or when the patient is unable to generate more than 20 cm H2O of maximal NIF. Initial symptoms include parasthesias with rapid progressive muscle weakness and areflexia. The Miller-Fisher syndrome is a variant of GBS in which the cranial nerves are affected. Autonomic dysfunction with cardiac dysrythmias occurs in one half to two thirds of patients. The disease generally has a good prognosis with close cardiopulmonary monitoring. Plasmapheresis and IVIG have been shown to alter the natural history of GBS if treatment is instituted quickly. year old male presented for further workup after suffering a seizure episode on two separate occasions. 10 days prior to his presentation at the university hospital, the patient experienced a witnessed tonic-clonic event and was taken to a local hospital. The patient was admitted to that hospital and found to have a normal CT, MRI, and EEG. While preparing for discharge home, he had another tonic-clonic event which was witnessed by hospital personnel, and he was transferred to the university hospital for further evaluation. He complained of feelings of mild generalized weakness over the past few months as well as some tingling and numbness in his hands and feet. He denied any recent trauma or seizure history. He also denied any chills, fevers, nausea, vomiting, or weight loss. Past medical history was noncontributory. He denied the use of illicit or prescription drugs, tobacco, ethanol, or herbals. On examination, his vital signs were pulse 101, blood pressure 146/90, and temp 35.4C. Physical exam was unremarkable including normal neurological exam. Labs on admission were significant for K of 3.1 meq/L (nl 3.5±5.0), Cl of 96 meq/L (nl 100±110), CO2 33 meq/l (nl 20±32), Ca 9.0 mg/dl (nl 8.5±10.5), and Mg 1.0 meq/l (nl 1.5± 2.4). Records received from the outside hospital showed low Mg and K levels during the time of both wintessed seizures. Patient was repleted with IV Mg and K and placed on phenytoin. Repeat MRI and EEG were negative. The patient continued to have low serum magnesium levels in spite of aggressive IV and PO repletion. 24 hour urine studies revealed a significantly elevated urine magnesium excretion of 25 MEq/24hr (nl 6±10 MEq/24hr), as well as a decreased urine calcium excretion of 94 mg/24hr (nl100±300 mg/24hr). Urine diuretic screen was negative. Renin and aldosterone were both elevated at 15.8 ng/ml/hr (nl 0.2±1.6 ng/ml/hr) and 38.4 ng/dl (nl 1.6±16) respectively. The patient was diagnosed with Gitelman's Syndrome and discharged home on magnesium oxide, amiloride, aldactone and potassium supplements. He suffered no further episodes of seizure activity and his weakness improved. IMPLICATIONS/DISCUSSION: Seizures presenting with normal MRIs and EEGs can present a diagnostic dilemma for internists. This case emphasizes the importance of considering metabolic disorders as a cause of seizures especially in the setting of normal MRIs and EEGs. Gitelman's Syndrome, an autosomal recessive disorder which usually presents in early adulthood, is a rare variant of Bartter's syndrome resulting from defects in renal tubular function. Unlike Bartter's, Gitelman's patients present with hypomagnesemia and hypocalciuria, along with complaints of severe fatigue, cramps, muscle spasms, and polyuria. with progressive failure to thrive. Three exploratory laparotomies over that time for acute abdomen revealed no pathologic diagnosis. PMH includes asthma, G1P1. P.E. reveals a cachectic, female, 4ft. 11in., 36.1 kg. Evidence of hypotelorism, bilateral ptosis, decreased lateral gaze with nystagmus at end gaze. Abdominal exam reveals distended and tympanic epigastrum. Neurological exam reveals normal visual acuity, decreased C.N. 8 bilaterally, strength of proximal muscle groups 4/5 with atrophy, while distal muscle groups 5/5 bilaterally. DTR's brisk through out. Laboratory studies included CBC, basic metabolic panel, thyroid function tests, Vitamin B12, folate, and lactic acid which were normal except hemoglobin 9.9g/dl, low iron saturation 11%, ferritin 51. She underwent EGD which was normal including duodenal biopsies, aspirates and electronmicroscopy of duodenal biopsies. Her esophageal manometry revealed hypotensive LES, gastric emptying scan showed prolonged liquid gastric emptying with T 1/2 = 136 minutes (mean is 90 minutes). SBFT showed slow emptying of barium. In consultation with neurology, work up included urine porphyrin studies that were negative, while MRI of brain showed diffuse leukoencephalopathy. LP was recommended but nondiagnostic. Fatty acid levels were normal. Ophthalmology evaluation revealed retinal pigmented degeneration while audiometry revealed bilateral moderate sensorineural hearing loss. Muscle biopsy would be indicated if genetic testing were not available. Pt. had chromosomal karyotyping, as well as mitochondrial DNA and thymidine phosphorylase levels, which are absent in specific mitochondrial disorders. These confirmed the diagnosis of a mitochondrial disorder, subtype called mitochondrial neurogastrointestinal leukoencephalopathy(MNGIE). IMPLICATIONS/DISCUSSION: Mitochondrial disorders certainly accounts for <0.1% of causes of nausea and vomiting. On review of the patient's history and physical exam she also had salient findings of proximal muscle weakness, hearing and eye abnormalities.\nGastrointestinal involvement is an unusual presentation for mitochondrial disorders with only 21 reported cases. More typical findings of mitochondrial myopathies are progressive ptosis, ophthalmoplegia and proximal muscle weakness in young patients. All these findings can be found on an internists thorough physical exam, as this case illustrates.`I CAN'T TIE MY SHOES. '' ADENOCARCINOMA OF THE LUNG AND PARANEOPLASTIC ANTIBODY ENCEPHALOPATHY: FOLLOW UP 2 YEARS LATER. K.S. Jorn 1 ; 1 Mayo Community Internal Medicine, Jacksonville, FL (Tracking ID #74452) LEARNING OBJECTIVES: 1. To describe symptoms that may be seen in primary care that should prompt evaluation for paraneoplastic limbic encephalopathy 2. To describe the clinical course of and paraneoplastic antibody levels in this patient over time 3. To present the concept that paraneoplastic antibody levels may not be cost-effective for surveillance after cancer is treated, but additonal study is needed. CASE INFORMATION: A 47 yo nurse presented for primary care in May 2000 after recovering from a massive pulmonary embolus in February 2000. Previous medical history was significant only for tobacco abuse and history of depression and anxiety, stable with medical therapy. Evaluation for causes of the embolus revealed only a 1cm coin lesion in her right lung, stable on follow-up CT through June 2001. In Fall-Winter of 2000, the patient, co-workers, and her supervisors noted a decline in the patient's concentrating ability and documentation efficiency. An accusation of diversion of narcotics triggered severe depression and anxiety in December 2000. The patient was receiving medical and psychological therapy for depression and anxiety when apraxic symptoms such as inability to tie her shoes, loss of direction sense, fecal and urinary incontinence, and absence-seizure type episodes were noted in the spring of 2001. She was felt to have symptoms of limbic encephalopathy. A paraneoplastic antibody panel revealed an elevated P/Q calcium channel antibody. Thoracotomy with removal of the coin lesion July 2001 revealed it to be bronchoalveolar cell carcinoma. Patient's cognitive function has gradually improved to near-normal levels. Though the elevated P/Q antibody led to her cancer diagnosis, the antibody levels have not paralleled her clinical course. IMPLICATIONS/DISCUSSION: Neurologic paraneoplastic antibody syndromes are more typically associated with small cell lung carcinoma and often involve brain stem, cerebellar, or neuromuscular junction dysfunction. There is little literature to guide the follow-up use of paraneoplastic antibody testing after cancer has been treated. This unusual case is characterized by cognitive dysfunction in the presence of lung adenocarcinoma and suggests that follow-up paraneoplastic antibody testing does not add much to clinical assessment after treatment of cancer. Additional studies are needed to determine if there is prognostic information to be gained by surveillance with paraneoplastic antibody testing.\nEVALUATION AND SIGNIFICANCE OF PERSISTENT ASYMPTOMATIC ELEVATED BONE ALKALINE PHOSPHATASE. K.S. Jorn 1 ; 1 Mayo Community Internal Medicine, Jacksonville, FL (Tracking ID #74454) LEARNING OBJECTIVES: 1. To review a series of cases in which asymptomatic elevated bone alkaline phosphatase was discovered, evaluated, and followed. 2. To propose an algorithm for evaluation of asymptomatic elevated bone alkaline phosphatase. 3. To suggest that additional study is needed to determine if this algorithm is cost-effective. CASE INFORMATION: Asymptomatic elevated liver enzymes are commonly encountered in primary care and are well documented in medical literature. Asymptomatic elevated alkaline phosphatase (bone subtype) also appears to be common in primary care but there is a lack of guidance in the literature for further evaluation of the abnormality once it is discovered. The cases presented provide examples of persistently elevated bone alkaline phosphatase and the outcome of the evaluation and followup over three to five years. Diagnoses revealed during evaluation of asymptomatic elevated bone alkaline phosphatase include metastatic lung cancer, male osteoporosis, and an apparently benign elevation of the enzyme. An algorithm for evaluation of an asymptomatic elevated bone alkaline phosphatase is proposed. IMPLICATIONS/DISCUSSION: The author hopes that this limited case series will help to focus additional attention on this relatively common clinical problem, ultimately providing more robust guidance to the clinician in its evaluation and management. -old female with a history of fibromyalgia and penicillin allergy presented several weeks after rectal abscess drainage with malaise, generalized myalgias and subjective low grade fever. On the first visit to her physician, the patient was sent home with the presumptive diagnosis of fibromyalgia exacerbation. Having continued to experience symptoms that she described as different from her usual``fibromyalgia pain'', she returned to the office to seek additional medical care. She was afebrile and her physical exam was notable for a diastolic murmur and generalized muscle tenderness. Pertinent labs included H/H of 11/ 39, WBC 14 and ESR 66. Blood cultures were drawn and returned 24±48 hours later, being positive for Enterococcus faecalis in 3 out of 3 bottles. TEE demonstrated an aortic valve vegetation with regurgitation. The diagnosis of IE was made and she was treated initially with vancomycin and gentamicin. She was successfully desensitized to penicillin and her treatment course with IV penicillin and gentamicin continued for 6 weeks. The patient had gradual resolution of her presenting symptoms during her treatment course. IMPLICATIONS/DISCUSSION: Patients with fibromyalgia or chronic pain syndrome can be challenging as they present frequently with multiple nonspecific complaints. It can be tempting to attribute all of their symptoms to these underlying disorders. However, physicians should objectively evaluate the patient and entertain other diagnoses when these patients present with new or worsening symptoms. In our case, this finally led to the diagnosis of IE.\nThe symptoms and signs of IE often are constitutional and, when localized, result from a complication of IE. Consequently, if physicians are to avoid overlooking the diagnosis of IE, a high index of suspicion must be maintained. This is especially true in cases of IE that have a subacute course. Subacute endocarditis is characterized by an insidious onset and by nonspecific signs and symptoms of chronic illness, such as low grade fever, fatigability, and malaise. Immunologic phenomena occur when the disease is protracted. Musculoskeletal symptoms, as was described in this patient, are common. Although fever is the most common sign in patients with IE, it is typically low grade. Heart murmurs are noted in 80 to 85 percent of patients with native value endocarditis (NVE) and are emblematic of the lesion predisposing to IE. Enterococci, which comprise normal gut flora, account for 5 to 15 percent of cases of NVE. There are no specific clinical manifestations nor is there a single lab test that establishes the diagnosis of IE unequivocally. A combination of major and minor clinical findings, outlined in the Duke criteria, makes the diagnosis highly probable. In the absence of surgical indication, optimal therapy for enterococcal endocarditis requires synergistic bactericidal interaction of antimicrobial agents targeted against the bacterial cell wall (penicillin, ampicillin, or vancomycin) and an aminoglycoside. Because of her overall clinical and laboratory presentation, the pituitary-adrenal axis was evaluated. The AM Cortisol was level 1.4 and ACTH was 5. Brain MRI and CT abdomen were unremarkable. The diagnosis of secondary adrenal insufficiency, potentially secondary to Megace 1 , was made. Patient was started on hydrocortisone 20 mg PO qAM, and 10 mg PO qPM. She had marked improvement in her symptoms. Megace 1 was discontinued and hydrocortisone was ultimately tapered. IMPLICATIONS/DISCUSSION: Findings in adrenal failure are nonspecific, and, without a high index of suspicion, the diagnosis of this potentially lethal but readily treatable disease is missed easily. Symptoms due to cortisol deficiency include anorexia, nausea, vomiting, weight loss, weakness, and fatigue. Although orthostatic hypotension is more marked in primary than secondary adrenal insufficiency because of aldosterone deficiency, it does occur in the latter. Hyperpigmentation (due to increase ACTH), hyperkalemia and volume depletion (due to aldosterone deficiency) occur only in primary adrenal failure. Hyponatremia is seen in both. In patients in whom adrenal insufficiency is merely to be ruled out, plasma cortisol is measured between 8 and 9 AM. Concentrations of <3 Ag/dl are indicative of adrenal insufficiency and obviate the need for other tests. Concentrations greater than 19 Ag/dl rule out the disorder. The short cosyntropin stimulation test is the most commonly used test for the diagnosis of primary adrenal insufficiency. Secondary adrenal insufficiency is characterized by low blood cortisol and ACTH levels, a low baseline rate of steroid excretion, and abnormal ACTH and metyrapone responses. Megace 1 is a progestational agent with activity in advanced breast and endometrial cancer and AIDS-related cachexia. Long-standing Megace 1 therapy can result in marked suppression of serum cortisol levels and decreased ACTH .The depressing effect of megestrol acetate on the pituitary-adrenal axis does not cause clinically evident problems in the overwhelming majority of cancer patients. However physicians should be aware of the possibility of adrenal insufficiency while taking or upon withdrawal of this drug. This is especially important in severely ill patients, in whom the subtle signs and symptoms of adrenal insufficiency can be attributed to their primary disease. LEARNING OBJECTIVES: 1) To recognize some causes of gait disorder in the elderly 2) To recognize the importance of a detailed history and 3) To recognize the role of genetic testing in appropriate elderly patients with gait disorders. CASE INFORMATION: 64-year-old female patient with past medical history of diabetes, mild osteoarthritis, and hypercholesterolemia developed gait problems over the last year and a half. She started losing balance around that time and her gait had become slow. She denied headaches, seizures, swallowing problems, memory disturbances or any sensory involvement. Her family history is significant for one sister and all male members in her father's family who developed gait disorders in their sixties. On examination, slurring of speech was present. Extraocular movements were intact. Strength in all extremities was normal and there was no pronator drift. Sensory examination was intact. Reflexes were symmetric and brisk. Gait was wide-based and she could not walk on her heels or toes. Her handwriting was small and she had difficulty with rapid alternating movements. An MRI showed mild cerebellar atrophy. Tests for Lyme disease, rheumatoid factor and anti-nuclear antibody were negative. Vitamin B12 level and sedimentation rate were normal. Genetic testing revealed that she possessed the CAG/ CAA repeat expansion mutation associated with spinocerebellar ataxia 17. Genetic counseling has been offered to her family members. IMPLICATIONS/DISCUSSION: There are many potential causes for gait disorders in the elderly. The most common are related to the presence of a sensory-motor polyneuropathy such as seen with diabetes, or strokes related to cerebrovascular disease. Also common are disorders secondary to compressive myelopathy. In this patient the presence of slurred speech made myelopathy and neuropathy unlikely and the absence of structural lesions on MRI suggested a progressive degenerative disorder. Spino-cerebellar ataxia(SCA) type 17 is a rare autosomal dominant neurodegenerative disease caused by an abnormal CAG expansion in the TATAbinding protein (TBP) gene. SCA 17 was identified in 4 Japanese pedigrees and first reported in 2001. Most individuals present between the ages of 20 and 30 with gait ataxia and dementia, progressing over several decades to include bradykinesia, dysmetria, dysdiadokokinesis, hyperreflexia, and paucity of movement. Neuroimaging shows cortical and cerebellar atrophy. Even the small number of SCA 17 patients identified so far suggests that there would be substantial phenotypic variability, probably secondary to incomplete penetrance. This case emphasizes the details of the differential diagnosis of gait disorders in the elderly and the importance of the family history even in the elderly patient. This has become especially important with the greater availability of genetic testing that will provide the basis for genetic counseling. 2) The importance of a complete history in pinpointing a rare diagnosis; CASE INFORMATION: A 37 yo white female with no significant PMH presented to the ER with a chief complaint of constant, progressive weakness over the past ten days. The patient initially noted swelling, paresthesias, and muscle tenderness in her legs, followed by progressive weakness from the ankles to the hips. She described difficulty in standing from a seated position, climbing stairs, and ambulating. She denied any alleviating or exacerbating factors or similar past symptoms. She had taken only acetaminophen as needed and had no known drug allergies. She denied any ingestions, exposures, or recent travel. On ROS, she denied fever, changes in appetite, weight, urine output or color, any GI upset, recent illness, or rashes. She was a smoker, but denied any alcohol or drug use. Exam revealed a thin, well-developed female laying in bed in no apparent distress. Her temperature was 98.28F, pulse was 100, BP was 139/ 82, RR was 20, weight was 115 lbs., and height was 5 H 7 HH . HEENT exam revealed poor dentition, otherwise normal. Cardiac, lung, neck, and abdominal exams were normal. Musculoskeletal exam showed strength to be 5/5 in the upper extremities, 3/5 in the proximal lower extremities, 4/5 in distal lower extremities. A positive Trousseau's sign (carpopedal spasm) was noted upon inflation of BP cuff. DTRs were slightly brisk in all 4 extremities but no spasticity was noted. Otherwise, neurologic exam was normal. Skin exam showed no rashes. Laboratory evaluation revealed significant hypokalemia of 1.7 mmol/L (normal 3.5±5.0), plasma chloride of 94, plasma CO2 of 31, serum calcium of 7.4, serum magnesium of 1.3, and serum phosphorus of 3.3. Other electrolytes, BUN/Cr, and glucose were normal. Serum CPK was significantly elevated at 5,098 U/L (normal 24±170). Urine sodium was 177 mmol/L, urine potassium was 8 mmol/L, and 24 hour urine potassium was 14 mmol/L. MCV was 104 fL, folate level 3.3 ng/mL, and albumin 3.7 g/dL. Toxicology and diuretic screens were negative. The profound hypokalemia, hypokaliuria, macrocytic anemia, and hypoalbuminemia were consistent with the suspicion of a 18 dietary disorder; i.e, low intake vs. eating disorder. The patient consistently denied an eating disorder or distorted body image. Upon obtaining further dietary history, the patient's daily consumption of 5±6 L of Pepsi 1 stood out as an apparent etiology. The patient stated that she had been drinking caffeinated colas heavily for several years. Upon cessation of cola consumption and replacement with a total of over 820 meq of KCL, the patient's weakness improved. IMPLICATIONS/DISCUSSION: Colas and other caffeinated beverages have been reported to be causes of significant hypokalemia, likely due to caffeine toxicity and/or phosphatepotassium binding. Although a rare diagnosis, this case of cola induced myopathy was made by using fairly simple, bedside physician-patient discussion.\nPAINFUL RASH. K. Kamjoo 1 ; 1 University of California, Los Angeles, Sylmar, CA (Tracking ID #76685) LEARNING OBJECTIVES: 1-To recognize the clinical presentation of sarcoidosis and Lo È fgren's syndrome. 2-To understand that sarcoid is a systemic disease with multi-organ involvement and 3-To discuss treatment modalities for sarcoid and sarcoid arthritis. CASE INFORMATION: A 24 year old male without significant past medical history presented to Emergency Department (ED) with complaint of painful rash over both legs for 2±3 weeks. Patient is an Argentinean exchange student who traveled to the US approx. six months prior to this presentation. Patient was seen at an outside clinic for the same complaint and was treated with Keflex and Ibuprofen without improvement. Patient reported that few days after appearance of rash; both of his ankles became swollen and painful. Although patient reported that walking was very painful, he denied any weakness or limitation of movement. In addition to the rash, patient admitted that he has been having night sweats and subjective fevers for the past couple of weeks. Patient also reported a slight dry cough. Initial vitals in ED were: temp of 38.2, blood pressure of 132/70, pulse of 95, and respiratory rate of 16. On physical exam patient appeared alert but in mild distress due to leg pain. Heart exam was regular sinus without any murmurs and lungs were clear to auscultation bilaterally. Abdomen was benign. Examination of skin revealed large nodules that were raised, red and tender to touch. Nodules were warm to touch and distributed randomly all over both legs. Both ankles had non-pitting edema but they had full range of motion. Ankles were tender to touch and passive range of motion was painful. Initial lab results showed a serum calcium level of 9.4 and a sedimentation rate of 47. Chest x-ray examination revealed bilateral hilar lymphadenopathy and EKG showed a normal sinus rhythm without any conduction abnormalities. Patient was subsequently admitted to hospital and placed in respiratory isolation. Patient had negative coccidiomycosis titers and ruled out for pulmonary Tuberculosis. HIV test was also negative. Patient remained clinically stable but continued to have a low-grade fever. CT scan of chest showed bilateral hilar and sub-carinal lymphadenopathy as well as multiple 1 cm nodules within lung fields. Subsequently, patient had bronchoscopy with biopsy and pathology report confirmed presence of non-caseating granulomatous lesions. Patient was started on 40 mg of prednisone per day and made a remarkable recovery. IMPLICATIONS/DISCUSSION: Sarcoidosis is a multi-system disorder of unknown cause characterized by a non-caseating granulomatous reaction in affected organs. Lo È fgren's syndrome is a variant of sarcoidosis in which acute sarcoidosis often presents with constitutional symptoms, polyarthritis, and erythema nodosum. The diagnosis of sarcoidosis should be based on a tissue biopsy, but a patient with typical Lo È fgren's syndrome may not need biopsy proof. Non-steroidal anti-inflammatory agents usually effectively alleviate sarcoid arthritis and joint symptoms associated with erythema nodosum. In severe acute arthritis and in chronic arthritis, corticosteroids may be required to control the symptoms. with a productive cough of white sputum and intermittent fevers, chills and night sweats for 1 month. She also complained of dizziness, weakness, a decreased appetite and a 5 pound weight loss over 4 weeks. She had been treated with antibiotics for 14 days which provided no relief. She denied visual changes, head pain, neck stiffness, chest pain, shortness of breath, dyspnea on exertion, nausea, vomiting, dysphagia, abdominal pain, diarrhea, urgency, dysuria, arthralgias or myalgias. Her medications included hydrochlorothiazide (HCTZ) and lisinopril. On physical exam, she appeared cachectic with dry mucous membranes and decreased skin turgor. Temperature was 38.4. Lung exam was clear bilaterally. Cardiac exam revealed a II/VI systolic murmur. There was no lymphadenopathy or skin rash. Abdominal exam revealed no masses or tenderness. She was hydrated and her HCTZ was held. She had multiple episodes of fever of up to 39.1. At the end of 7 days, her diagnostic testing revealed the following: normal CBC with differential; normal LFT's; 6 blood and 2 urine cultures were negative; Legionella antigen was negative; non-reactive PPD and a TSH of 2.6. Chest x-ray revealed biapical pleural thickening. ESR was 85. ANA, Rheumatoid factor (RF) and Anti-ds DNA were negative. Transthoracic echocardiogram (TTE) was negative for vegetation. Chest and abdominal CT scans revealed no significant findings. Bone marrow biopsy, aspirate and culture were negative. Bilateral temporal artery biopsies revealed severe temporal arteritis (TA). She was discharged on prednisone and her symptoms resolved after 1 week of therapy. IMPLICATIONS/DISCUSSION: FUO is defined as a temperature greater than 38.3 on several occasions for more than 3 weeks or after 1 week of thorough diagnostic testing performed in the out-or inpatient setting. Earlier studies reported that infections represented 41% of all cases. Collagen vascular diseases represented 30% and tumors represented 13%. Miscellaneous causes represented 2% and in 13% of cases the etiology was not identified. More recent studies reveal that infections are less common than multisystem disease and this trend was even more pronounced in patients over 70 years of age. A cost-effect approach in the elderly consists of confirmation of fever and a thorough history and physical exam. Laboratory analysis should consist of a CBC with differential, ESR, TSH, chemistry and liver panel, urine analysis and multiple blood and urine cultures. HIV, EBV, CMV, ANA and RF should be considered in certain cases. Stop all nonessential drugs. Imagining should include a TTE, chest and abdominal CT scans. A temporal artery biopsy should be considered if the ESR is more than 40 mm/hr, even in the absence of symptoms. Gallium scan for hidden abscesses and liver and bone marrow biopsies should be considered only in certain cases. Explorative laparotomy is a final resort. LEARNING OBJECTIVES: Introduction: Typhoid fever is a common disease in the developing countries.\nStep-wise rising fever, bacteremia, diarrhea associated with abdominal pain and hepatosplenomegaly are the usual manifestations. We report a case of typhoid fever with sore throat as an integral part of the clinical picture. CASE INFORMATION: A 21-year-old immigrant male from Ecuador presented with a seven-day history of sore throat associated with a foreign body sensation and dysphagia for solid foods. Three days prior to presentation, in addition to his sorethroat, the patient complained of vomiting, diarrhea, mild abdominal discomfort, myalgias, fatigue and subjective fevers. On examination, his temperature was 94.68F with a pulse rate of 66 beats/minute. The oropharynx was markedly erythematous. The monospot test and rapid streptococcal screen were non-reactive. A diagnosis of acute viral syndrome with pharyngitis was made and he was treated symptomatically with Ibuprofen. He returned to the emergency department two days later with worsening sore throat, fever, new onset of low back pain, and mild abdominal discomfort with nausea. Physical examination revealed a temperature of 103.28F, pulse rate of 60 beats/minute, mild peri-umbilical tenderness and splenomegaly. The patient was hospitalized and started on intravenous fluids and oral Ciprofloxacin 750 mg twice daily. His fever subsided and he showed significant symptomatic improvement on antibiotic therapy. On the fourth day of hospitalization, blood cultures yielded Salmonella Typhi. He was discharged home on day five with two weeks of ciprofloxacin. IMPLICATIONS/DISCUSSION: Conclusion: Once considered an integral part of the symptomatology of typhoid fever, sore throat is infrequently reported, perhaps due to differences in causative strains, less emphasis on documentation of all symptoms and early and effective treatment in the antibiotic era. Physicians should be aware that typhoid fever could present as pharyngitis and should be considered especially in immigrants, to aid in early diagnosis and treatment. \nA 27 year old African American female with a past history of the human papilloma virus, gonorrhea, and chlamydia presented with two weeks of diffuse joint pains. The pain started in her knees and progressed sequentially to include her elbows, shoulders, back, and hips. Her associated symptoms were nausea, fever, and a sallow complexion. She denied any history of intravenous drug use, but did endorse a history of multiple sexual partners in the past. Physical exam was remarkable for a temperature of 38.3 C, scleral icterus, and jaundiced skin; However, there was no cardiac murmur or hepatosplenomegaly. Musculoskeletal exam revealed no joint effusions, warmth, erythma, or tenderness. A pelvic exam was normal with negative gonorrhea and chlamydia testing. Labs demonstrated a normal CBC with differential, and an erythrocyte sedimentation rate was 11; An antinuclear antibody was less than 1:40; Rhuematoid factor, hetrophile antibody, cytomegalovirus (CMV) IgM, and rapid plasma reagin tests were all negative; AST and ALT were elevated at 605 and 1303, respectively; Bilirubin was 3.3, alkaline phosphatase 204, and albumin 3.3. At this point, work up was initiated for acute hepatitis. While hepatitis A and C serologies were negative, hepatitis B surface antigen and anti-hepatitis B core antigen were positive. HIV testing was negative. A diagnosis of acute hepatitis B was made. It was presumed that the aquisition of hepatitis B in this patient was from sexual contact. Over the next 10 days her AST and ALT peaked at 4500 and 5500 respectively, bilirubin at 10, albumin decreased to 2.0, and INR was 1.9. Treatment was supportive in nature including fluids and pain control. At time of discharge her AST and ALT were trending down toward normal levels and her symptoms were resolving. She was educated about transmission of hepatitis B with attention to safe sex practices. IMPLICATIONS/DISCUSSION: Migratory polyarthralgia has a challenging and broad differential diagnosis. Infectious causes are many and include gonorrhea, syphilis, CMV, Epstein-Barr virus, Lyme disease, and bacterial endocarditis. Viral hepatits is a possible but uncommon infectious cause. Inflammatory causes are a major category and include rhuematoid arthritis, systemic lupus erythematosis, scleroderma, and polymyositis. Postinfectious causes include rheumatic fever and Reiter's syndrome. Crystalline diseases namely gout and psuedogout can also cause polyarthralgias. Joint aspiration and analysis is the key to diagnosis, however our patient had no joint effusions. With the advent of vaccination, acute hepatitis B virus (HBV) infection is now much less common in the United States. However, it remains a major global problem, particularly in developing countries. Signs and symptoms include malaise, fatigue, myalgias, anorexia, nausea, vomiting, abdominal pain, fever, and jaundice. Migratory polyarthralgia, particularly with a benign joint examination, constitutes an atypical presentation of acute hepatitis B. The incubation period for HBV is 1 to 6 months. While 30% of patients who acquire HBV present with acute symptoms, the majority of patients remain asymptomatic with anicteric hepatitis. Ninety percent of cases of acute hepatitis B clear infection and resolve symptoms within 6 months. They remain asymptomatic carriers of HBV. This is demonstrated by disappearance of the heptitis B surface antigen and appearance of the hepatitis B surface antibody. Less than 1% of patients develop acute fulminant hepatitis. The potential outcome of this subgroup is poor and therefore close follow up of synthetic function is warranted during the initial period. Approximately 5 to 10% progress to eventually develop symptomatic chronic HBV infection. Sequelae can include cirrhosis, hepatic decompensation, and hepatocellular carcinoma. Diagnosis of acute HBV is confirmed with serology, and quantification can be done with the polymerase chain reaction. Treatment is mainly supportive in nature, and patients should be educated about disease transmission. Antiviral therapy is not currently recommended or approved for the treatment of acute HBV.\nA. Kapoor 1 , J. Hefner 1 ; 1 University of Pittsburgh, Pittsburgh, PA (Tracking ID #76642) LEARNING OBJECTIVES: 1.) Recognize an unusual cause of persistent hypokalemia; 2.) Diagnose an atypical presentation of Cushing syndrome; 3.) Recognize the clinical presentation of an ectopic ACTH-producing tumor. CASE INFORMATION: A 77-year-old male with hypertension and Type II diabetes mellitus presented with a 2 month history of fatigue and weakness. He subsequently developed a gait dysfunction and had frequent falls. Outpatient potassium (K) was 1.9 and he was admitted for management of hypokalemia. Physical exam revealed a BP of 180/90, facial plethora and bilateral diffuse rales at the bases of the lungs. Proximal muscle strength was 4/5 in the upper and 3/5 in the lower extremities. Distal strength was 5/5. No moon facies, buffalo hump or abdominal striae were found. His K remained low despite aggressive replacement and secondary causes were investigated. Results of diagnostic testing included a urinary Na 17, K 52, pH 6 (4.8±8), serum Na 140, Cl 95, HCO3 40, BUN 28, Cr 1.4, renin 0.6 (<5 mcg/ml) and aldosterone 5.7 (2±9 mcg/ml). Urinary metanephrines were normal. Cortisol at 8 am was 100 (7±20 mcg/ml) and ACTH 380 (9±52 pg/ml). Post high-dose (8mg) dexamethasone suppression test (DST) cortisol was 118 and ACTH was 126 (2±8 pg/ml). FSH, LH, prolactin and IGF-1 were normal. Brain MRI revealed a normal sized pituitary. Chest CT showed pleural effusions, no masses or lymphadenopathy. Abdominal CT revealed a 3 cm renal mass and bilateral adrenal hypertrophy. Biopsy of the renal mass showed poorly differentiated small cell carcinoma (SCC). Treatment with ketoconazole failed to suppress his cortisol. The patient decompensated and expired. Autopsy revealed numerous nodules in the liver, the right renal mass with focal hemorrhage and no lung masses. Pathology revealed SCC of renal origin with metastasis to the liver. IMPLICATIONS/DISCUSSION: Cushing syndrome is an unusual cause of hypokalemia. Other causes include primary or secondary hyperaldosteronism, Type 2 renal tubular acidosis, Bartter syndrome, Liddle syndrome, diuretic use and VIPoma. Patients with ectopic Cushing secondary to carcinomas often lack the clinical manifestations associated with classic Cushing syndrome±the centripetal fat distribution, moon facies and striae. The metabolic abnormalities, however, are more common in ectopic ACTH syndrome namely hypokalemia, metabolic alkalosis, excessively high cortisol and elevated ACTH levels. Differentiation of ectopic from pituitary dependent Cushing is made by non-suppression of cortisol with the 8 mg DST. There have been reported cases of ectopic ACTH with lung SCC, bronchial carcinoid, medullary carcinoma of the thyroid, gastrointestinal and prostatic adenocarcinoma and pheochromocytoma. However, there are no reported cases of SCC of renal origin causing ectopic ACTH production as was present in our patient.\nA CATASTROPHIC COMPLICATION OF GIANT CELL ARTERITIS. P. Kapoor 1 , S. Parikh 1 , P. Radhakrishnan 1 , M. Yungbluth 1 , D. John 1 ; 1 St. Francis Hospital, Evanston, IL (Tracking ID #75985) LEARNING OBJECTIVES: 1. Awareness of the association of aortic aneurysm (AA) with giant cell arteritis (GCA) will aid prompt recognition and therapy. 2. To highlight the significance of using Chest XRay (CXR) as a screening modality in patients of GCA for timely detection of thoracic AA.\nA 75-year-old male presented with acute, severe right upper quadrant (RUQ) pain, radiating to the right shoulder and associated with nausea and vomiting. He denied any fever, chest pain, shortness of breath, change in bowel habits or similar episodes in the past. He had a history of coronary artery disease, well-controlled hypertension and biopsy-proven GCA, diagnosed 15 months ago. Physical examination: afebrile;pulses: 86/min, regular and symmetric; BP: 170/108 mm;tenderness in the RUQ with palpable gall bladder 4 cm below the right costal margin; an ejection systolic murmur in the aortic area. The rest of the physical examination was essentially unremarkable. Laboratory data: WBC-10000, Hb-13.8 and platelets-251,000. The CT scan of abdomen was suggestive of acute cholecystitis. A dissection of the superior mesenteric artery (SMA) was also noted. CXR showed aneurysm of ascending aorta. CT scan of thorax showed a Stanford Type A aortic dissection (AD) with aneursymal dilation of 7 cm. Communication of the true and false lumen suggested chronicity (see figure) . After stabilization of BP, he underwent an emergency laparoscopic cholecystectomy which led to a resolution of his abdominal pain. Subsequently, he was referred for an elective repair of his asymptomatic, chronic aortic dissection. IMPLICATIONS/DISCUSSION: Horton's syndrome or GCA is the commonest primary systemic vasculitis in the United States, associated with diverse manifestations and outcomes. The true incidence of aortic involvement is unknown, but is estimated to be 3±15 %. Patients with giant cell aortitis may be asymptomatic or may present with aortic arch syndrome, dilation of aorta, aortic aneurysm, AD or aortic incompetence. Although, in this patient AD may well be attributed to atherosclerosis,the history of GCA and segmental involvement of ascending aorta and SMA implicate GCA as a predisposing condition.A high incidence of thoracic aortic aneurysm has led Evans et al to recommend annual CXR, including lateral views in patients of GCA. Since the diagnosis of GCA is commonly made by the internists, awareness of it's possible complications helps us manage these patients better. A 37 year old Mandarin speaking woman with no known PMH presented with chest palpitations for 6 months. Her history was obtained in a crowded, noisy ER through a telephone translator, with her husband as a middle translator. She states having symptoms for over 6 months with acute decompensation over the last 2 months involving constant fast heart rate, swelling in her face, abdomen, and extremities for 2 months, dyspnea when walking 2±3 blocks, a 20 lb unintentional weight loss, poor concentration, and constipation. She went to a doctor 1 month ago and was told she has a thyroid problem, but has not been taking the prescribed medications. More than 10 years ago in China, she had problems with her thyroid and was given Western medications for 6 months with improvements. Otherwise, review of systems was unremarkable. On exam, her pulse was irregular, atrial fibrillation, at 125±145, BP 98/64. She was anicteric with swollen eyelids, no proptosis. She had an enlarged thyroid, JVD, a hyperdynamic precordium, no heart murmurs, a distended abdomen, and non-pitting edema in all extremities. Lab studies showed a mild transaminitis, negative hepatitis serologies, and positive thyroid stimulating antibodies. She was diagnosed with Graves disease. Echocardiography showed severe tricuspid regurgitation and right heart failure without evidence of left sided dysfunction, pulmonary artery hypertension or septal defects. Her atrial fibrillation was treated with beta blockers and anticoagulation, and she was scheduled for repeat echocardiogram in 2 months to evaluate for valve disease that may be contributing to her right sided failure. She would like more children, so she opted for medical therapy with propythiouracil to treat her hyperthyroidism and would consider radioactive iodide or surgical therapy after her thyroid disease is under control. IMPLICATIONS/DISCUSSION: 1. Obtaining a complete history in non-English speaking patients can be difficult. Despite seeing a private doctor, this patient came to the ER without her primary doctor's support, without her medications, and without the ability to communicate with hospital doctors. Studies have shown that intepreter services increase cost-effectiveness and quality of care as well as patient understanding and participation in healthcare, despite potential increases in the time and resources to provide such services. 2. Recurrent hyperthyroidism, most commonly due to Grave's disease, is treated similarly to the initial episode. Anti-thyroid medications are the drug of choice in pregnant women and are not known to effect reproductivity. Radioactive iodide is contraindicated in pregnancy, although has been shown to have no effect on reproductivity in the long term. Surgery is effective, although has no advantages and has been associated with increased fetal losses if done during pregnancies. A 74 year old male was brought by his friend to the emergency room with a four week history of gradually progressive shortness of breath that began 2±3 days after dual-chamber pacemaker placement for Mobitz II heart block and symptomatic bradycardia. The procedure was without any immediate complications. CXR post-precodure was negative for pneumothorax. He currently denied chest pain, cough, fevers, chills, sweats, lightheadedness, or dizziness. PMH was significant for, in addition to Mobitz II heart block, coronary artery disease, severe aortic stenosis and hypertension. Medications included felodipine 10 mg PO qd, simvastatin 10 mg PO qd, and lisinopril 20 mg PO qd. He had a remote history of tobacco use and was a former steel mill worker. On physical examination, VS: HR 105, RR 26, BP 150/90, pulse ox 92% (on room air). Pertinent findings included no JVD, a II/VI SEM at the right upper sternal border radiating to carotid arteries, laterally displaced PMI, decreased breath sounds on the left 2/3 up the lung fields, no organomegaly and 1+ pretibial edema. CXR showed a free flowing large left pleural effusion. The patient was admitted to the hospital and a thoracentesis was performed from which serosanguinous fluid was obtained. CT of the chest demonstrated a perforated right ventricle from the pacer wire. Surgical repair was successful but the patient had a very complicated postoperative course and expired. IMPLICATIONS/DISCUSSION: Pacemaker insertion is a relatively common and safe procedure and can be lifesaving. Ventricular rupture is a rare, albeit potentially serious, complication. The elderly, in whom most pacemakers are inserted, are at increased risk of complications. The Pacemaker Selection in the Elderly (PASE) study, a prospective multicenter trial involving 407 patients age 65 years and older, documented a 6.1% complication rate; 4.4% of patients required a repeat procedure. The most common complications were lead dislodgement (2.2%), pneumothorax (2.0%and observed only in those with subclavian venous access) and cardiac perforations (0.98%). Most perforations occur intraoperatively and rarely cause symptoms. However, as in our patient, serious complications, including death, have been reported postoperatively. Complications of pacemaker insertion may masquerade as other less serious entities with symptoms of dyspnea, chest pain, fever and weakness. It is paramount that internists be alert to the potential serious complications of cardiac rupture and pneumothorax in the patient presenting with these symptoms postprocedure. Symptomatic patients require surgical intervention. A 53 year-old Puerto Rican male presents with a chief complaint of two years of increasing dyspnea on exertion. Past medical history was notable for multiple episodes of pneumonia. Although the patient had been successfully treated as an outpatient during these episodes with oral antibiotics, his chronic baseline dyspnea and decreasing exercise tolerance continued to worsen. For one of these exacerbations, the patient was admitted to the hospital, where physical exam revealed nystagmus, poor visual acuity, oculocutaneous albinism, and a bleeding diathesis. Radiological studies revealed bilateral lower lobe infiltrates and diffuse pulmonary honeycombing fibrosis. The patient improved following a ten-day course of IV cefotaxime, gentamycin, and chest physical therapy. IMPLICATIONS/DISCUSSION: The combination of oculocutaneous albinism, a bleeding diathesis, and pulmonary fibrosis in this Puerto Rican patient raised the suspicion for Hermansky-Pudlak Syndrome. HPS is an inherited disease which results in a platelet storage pool defect and lysosomal accumulation of a substance called ceroid lipfuscin. HPS is an autosomal recessive syndrome found primarily in individuals from the northwestern quadrant of Puerto Rico. Patients have legal blindness, nystagmus, iris transillumination, foveal hypoplasia, decreased number of melanocytes, pulmonary fibrosis, renal disease and granulomatous enteropathic disease. Lab studies in patients with HPS may show an increased bleeding time with a therapeutic response to desmopressin, decreased bone density, and genetic testing showing a mutation on the HPS1 gene on chromosome 10q23. The most accurate diagnosis, however, is made by a pathognomic platelet morphology diagnosed only by electron microscopy. Management consists of recognizing the disease, having a lower threshold for admitting individuals for inpatient management, and administration of immunosuppressants and steroids. Current trials of a test drug called pirfenidone are being conducted and offer patients some hope for a treatment. Back to our patient: A careful history taking revealed that the patient presented above had all of the above manifestations, even decades prior to his official diagnosis being made. Management of this patient's disease ultimately consisted of various trials of steroids, interferon, and methotrexate. While the inpatient stay focused primarily on his pulmonary pathology, outpatient management consisted of patient education to minimize complications of the disease and carefully orchestrated care with numerous specialists. week history of progressive pain and swelling in his left index finger after an attempt to straighten an iron pipe with this left hand. Past medical history was significant for a similar episode involving his right index finger after trauma that resolved with symptomatic treatment. Patient had a 50pack year history of smoking. The rest of his review of systems was unremarkable. Physical examination was unremarkable except for a swollen and tender left index finger with a gangrenous tip. The left ulnar pulse was absent. His radial pulses were intact in both extremities. Laboratory data including ANA and sedimentation rate were within normal limits. An arteriogram of the left upper extremity revealed thrombosis of the distal ulnar artery and occlusion of digital arteries of the second digit that were thought to be secondary to embolic events from the ulnar artery thrombus. Duplex ultrasound also revealed a thrombosed pseudoaneurysm in the left ulnar artery at the hypothenar eminence. Diagnosis of Hypothenar Hammer Syndrome (HHS) was made. IMPLICATIONS/DISCUSSION: HHS is a rare clinical entity that results from repetitive palmar trauma leading to damage of the ulnar artery as it passes over the hammate bone. The syndrome usually manifests as unilateral digital ischemia caused by embolic digital artery occlusion from the thrombosed palmar ulnar artery. It is predominantly seen in males around the age of 40. In a small study of 21 patients, 12 out of 13 who had arteriography of both upper extremities had bilateral palmar ulnar disease (1). Diagnosis is made on clinical features and confirmed by angiography that shows a palmar ulnar artery abnormality and digital artery occlusion. Treatment may include surgery involving excision of abnormal palmar ulnar artery segment. LEARNING OBJECTIVES: To recognize and manage pustular psoriasis and to differentiate it from other causes of erythroderma. CASE INFORMATION: DP is a 58-year-old African American male who presented with an exfoliative erythroderma and pustules in the palmar-plantar areas that had coalesced to form`l akes of pus.'' The patient noted a similar but milder eruption 3 months earlier in which he was treated at another facility with oral prednisone with some improvement. He reported that the pustules had started on his hands, progressing rapidly over several days to affect the entire body. Erosions later appeared in the mouth. He complained of an intense pruritus and burning sensation along with a fetid odor. At the time of presentation he was noted to be febrile with a leukocytosis. A skin biopsy showed spongiform pustules in the upper epidermis with numerous PMNs present. Inpatient management included a combination of actretin, IV vancomycin, and topical mid-potency steroids, with rapid improvement in the patient's condition. IMPLICATIONS/DISCUSSION: A presentation of erythroderma should prompt the physician to consider pustular psoriasis, pemphigus foliaceus, pityriasis rubra pilaris, underlying leukemia/lymphoma, severe atopic dermatitis, ichthyoses, drug induced erythroderma, diffuse seborrheic dermatitis, scabies, and staphylococcal scalded skin. Pustular psoriasis can be differentiated by sudden onset of acral pustular lesions, fever, intense pruritis, and occasionally, hypocalcemia and mucous membrane lesions. Patients will often have a personal or family history of plaque psoriasis and/or psoriatic arthritis. Treatment of choice is acitretin (a second generation retinoid). Topical steroids are often helpful, but systemic steroids should be avoided.\nWITH KLIPPEL-TRENAUNAY-WEBER SYNDROME. M. Kennedy 1 , C.V. Mueller 1 ; 1 University of Cincinnati, Cincinnati, OH (Tracking ID #77195) LEARNING OBJECTIVES: 1) Learn about the vascular malformations seen in Klippel-Trenaunay-Weber Syndrome. CASE INFORMATION: A 25 yo Caucasian male originally presented to an outside hospital with bright red blood per rectum. He denied associated abdominal pain, nausea, vomiting, fever or prior history of rectal bleeding. Initial systolic blood pressures were in the 80-90's with a heart rate in the 130's. Work up at the outside hospital included flexible sigmoidoscopy that revealed nonbleeding internal hemorrhoids, a negative tagged RBC scan and an unremarkable CT of the abdomen. Upon transfer to our facility, he was hemodynamically stable, but found to have a hemoglobin of 7.5 with a baseline of 13.5. The morning after admission, the patient developed massive hematochezia associated with diaphoresis and tachycardia. He was urgently sent for tagged RBC scan that showed distal ileal bleeding. Subsequent mesenteric angiography did not identify any active bleeding or vascular malformation. The patient again decompensated and was taken for exploratory laparotomy that revealed bleeding ileal and colonic varices. He underwent ileocecectomy without complications and had no further episodes of bleeding. He required a total of 20 units of PRBC's during his hospitalization. IMPLICATIONS/DISCUSSION: KTWS is classically described as a venous monoangiodysplasia, but is actually associated with a variety of vascular malformations including: subcutaneous hemangiomas, cavernous hemangiomas, AV fistulas, deep vein malformations and/or aplasias, varices and a variety of lymphatic abnormalities. Vascular malformations may involve visceral organs such as the bowel, liver, kidneys, and lungs and can, uncommonly, cause complications in those organs. Treatment of KTWS is supportive and/or cosmetic unless orthopedic or bleeding complications arise, at which point surgery is generally indicated.\nLEARNING OBJECTIVES: 1) Consider the potential for a fatal overdose of Metformin when treating diabetic patients who are at risk of commiting suicide. 2) Recognize the constellation of signs that are consistent with a Metformin overdose.\n3) Recognize the need to quickly and aggressively treat a Metformin overdose and its medical consequences. CASE INFORMATION: A 42 year-old woman with a history of diabetes, hypertension and schizoaffective disorder was found obtunded in her apartment and admitted to the MICU. She had recently been hospitalized for suicidal ideation. Empty pill bottles of a benzodiazepene, an atypical antipsychotic, a SSRI and Metformin were found. Six hours after admission, the patient developed lactic acidosisand a severe sepsis-like syndrome consistent with Metformin overdose. She subsequently became critically-ill, and had a clinical course which included severe ARDS and anuric renal failure. Support was withdrawn at the request of her family following a prolonged course of aggressive treatment. IMPLICATIONS/DISCUSSION: Metformin is commonly used in primary care and its potential for lethal side effects is well known in the setting of medical illness. This case demonstrates how Metformin also has potential for great harm in the setting of an overdose. Physicians should be aware of the risks of prescribing Metformin to diabetic patients with depression or a history of impulsive behavior. A 49 year old female with recent diagnosis of pulmonary embolism due to oral contraceptive use presented with acute onset of left sided abdominal pain associated with nausea but no vomiting, diarrhea or constipation or changes in urinary habits. Patient's medications were warfarin, ibuprofen prn, home O2 and amitryptiline. Had 25 pack year smoking history. On examination, she seemed to be in moderate distress with severe tenderness on left side of midline from epigastrium to suprapubic region, worse with any movement associated with mild fever. Vital signs were stable otherwise. Stools hemoccult negative. Lab studies revealed a wbc count of 12,700, Hb of 10.7, noted to be reduced from previous admission. Creatinine 0.7 mg/dl, lipase 94 mg/dl, PT 23.1, INR 3.73 with normal liver function tests and normal UA. Contrast enhanced CT revealed acute hematoma of left rectus sheath, 20cm  6cm with active bleeding. Patient's Hb further dropped so eventually transfused, coumadin stopped and surgery consulted. They suggested conservative treatment with analgesia and rest and later on, IVC filter placement. Repeat CT Scan showed stable hematoma. Patient clinically better with stable Hb. So, again started on low dose coumadin and sent home with caution not to smoke and avoid trauma as on further questioning, eventually, patient provided history of fall. IMPLICATIONS/DISCUSSION: Left sided abdominal pain and fever can be mistaken for number of causes of acute diseases of abdomen. But, due to advent of CT, an unusual diagnosis is relatively easy to make. But, since rectus sheath hematoma is one of the reported complications of anticoagulation therapy, this diagnosis should always be kept in mind. Once diagnosis is established and if this hematoma is not causing severe symptoms, the condition can be managed nonoperatively with bed rest and analgesics. Ideally, anticoagulation should be discontinued. It is rarely necessary to reverse the coagulation defect if operation is not undertaken. As in our case, patient was clinically improved so it was decided to manage her conservatively and avoid surgery. She was restarted on anticoagulation because risk of pulmonary embolism is greater than hematoma. It was made sure to counsel her about any trauma as it increases risk of hematomas in future. Patient continued to do well on further follow ups.\nM. Khalil 1 , R. Granieri 1 ; 1 University of Pittsburgh, Pittsburgh, PA (Tracking ID #73745) LEARNING OBJECTIVES: 1. To recognize ascites as a complication of eosinophilic gastroenteritis (EG) 2. To diagnose and treat EG. CASE INFORMATION: A 50 year-old female with no significant PMH presented with a 2 month history of epigastric pain that was associated with nausea and decreased appetite. She reported the pain to be constant and exacerbated with food, but mildly relieved with a bowel movement. She denied any vomiting, melena, hematochezia, fever, chills, recent travel, change in diet or alcohol abuse. Two weeks prior to admission, she developed non-bloody, watery diarrhea, increased abdominal girth, and 12 lb. weight gain, despite the poor appetite. Outpatient evaluation with abdominal ultrasound was unremarkable and an upper GI series revealed dysmotility of the lower esophagus with tertiary contractions. Treatment with rabeprazole provided minimal relief. Physical examination upon admission was remarkable for a mildly distended and diffusely tender abdomen with positive shifting dullness but no rebound, guarding or organomegaly. The remainder of physical exam was normal including heme negative stool. Lab work was significant for WBC of 14,000 with 30% eosinophils. Liver function tests were normal. Serum albumin was 2.4 and ascitic fluid analysis showed: pH 7.31, albumin 1.8, negative cytology, and WBC of 4700 with 96% eosinophils. Stool culture and sensitivity, ova and parasites were negative. Further workup with abdominal ultrasound with Doppler showed patent vessel and normal direction of flow. Upper endoscopy revealed erythema of the antrum. Colonoscopy was normal. CT scan of the abdomen and pelvis showed diffusè`L akes of pus'' small bowel thickening without fat stranding and moderate ascites. Trans-vaginal ultrasound was negative for ovarian mass. Bone marrow biopsy was normal. The patient refused surgical bowel biopsy and diagnosis of EG was established on the basis of exclusion. She was started on oral dose of prednisone for 6 weeks and discharged to home. Follow up visit and blood work showed 0% eosinophils, resolution of ascites, resolution ofabdominal pain, and good appetite. IMPLICATIONS/DISCUSSION: EG is an uncommon disorder characterized by gastrointestinal symptoms, blood eosinophilia, and eosinophilic infiltration of the gastrointestinal wall. The signs and symptoms of EG are related to the layer and extent of the bowel involved with eosinophilic infiltration. Patients with subserosal infiltration may develop eosinophilc ascites. The diagnosis of EG is confirmed by a full thickness biopsy of the bowel in the absence of intestinal parasitic infection. Treatment of EG is empiric because no prospective, randomized clinical trials are available to guide treatment. Prednisone (20 to 40 mg/day) is recommended for two weeks and should be tapered over the next two weeks. Improvement usually occurs within two weeks of therapy. Despite the rarity of EG, it should be suspected in a patient with any GI symptoms associated with peripheral eosinophilia because this treatable disease can disguise itself as the irritable bowel syndrome. LEARNING OBJECTIVES: 1) Recognize that pulmonary hypertension is not uncommon patients with SLE; 2) Recognize that primary pulmonary hypertension in SLE patients often presents earlier than pulmonary hypertension from a secondary cause. CASE INFORMATION: A 21-year-old woman was transferred to our hospital for management of her pulmonary hypertension (PHT) and systemic lupus erythematosus (SLE). She initially presented a year ago with progressive cough and pleuritic-type chest pain. 8 months prior to admission, she underwent an echocardiogram which revealed a pulmonary artery pressure (PAP) of 27 mm Hg. Four months later, she was admitted to a community hospital for congestive heart failure and was found to have positive anti-Smith, antiphospholipidand anti-nuclear antibodies, and was diagnosed with SLE. Her symptoms gradually progressed to dyspnea on exertion, paroxysmal nocturnal dyspnea and Raynaud's phenomenon. She was subsequently readmitted to that hospital 2 weeks prior to transfer to us for worsening of her symptoms. There, she had a generalized seizure and was initiated on plasmapheresis treatment. Examination revealed a thin, anxious lady in mild dyspnea and an obvious malar rash. Cardiac auscultation revealed a loud and palpable P2, and a II/VI systolic murmur at the tricuspid area. She had a non-tender hepatomegaly and mild bilateral pedal edema. Echocardiogram revealed a PAP of 76 mm Hg, with evidence of right heart enlargement and mild tricuspid regurgitation. A chest CT scan with angiography and a ventilation-perfusion scan showed no signs of a pulmonary embolism. A right heart catheterization was done as well showing moderate PHT and a moderate response to epoprostenol challenge. Workup for lupus cerebritis was negative. She was initiated on prednisone and Cytoxan for SLE. Plasmapheresis was discontinued. Diltiazem, Valsartan and Warfarin were initiated to control her pulmonary hypertension. She tolerated the medications well and was discharged with outpatient follow-up, with the consideration of using epoprostenol in the future should her condition worsen. IMPLICATIONS/DISCUSSION: Prevalence of PHT in SLE ranges from 5% to 14%. A third of SLE patients have positive antiphospholipid antibodies, although the significance of this in the development of PHT is unknown. In one study of SLE patients, 5.9% were found to have pulmonary hypertension, of which 48% of them had no identifiable cause, the rest found to have a secondary cause. For the subgroup with secondary hypertension, 50% of them were due to valvular abnormalities. The primary pulmonary hypertension tended to present far earlier after diagnosis of the SLE compared to secondary PHT, 8.8 versus 43.2 months. Raynaud's phenomenon in secondary hypertension is associated with a more severe pulmonary artery pressure. These patients seem to respond to epoprostenol therapy although there have been documented cases of fatal thrombocytopenia. year-old female with rheumatoid arthritis, hypertension, and seizure disorder presented with two days of hematuria. She denied fevers, dysuria, urinary frequency, headaches, visual changes, or cough. Her last seizure was two weeks prior to presentation. Medications included gabapentin, phenytoin, methotrexate, and amlodipine. There was no fever and her physical examination was otherwise negative for lymphadenopathy, hepatosplenomegaly or petechiae/purpura. Initial workup revealed a platelet count of 7,000, H/H of 7.3/24, WBC of 8.2, LDH of 900, and Cr of 3.5. Peripheral smear demonstrated schistocytosis. The patient was diagnosed with TTP-HUS and began daily plasmapheresis and prednisone, during which her platelet count improved to 130,000 and her LDH normalized. She was discharged to continue outpatient pheresis but was readmitted one week later with fever (1038 F) and a platelet count of 78,000. She was begun on plasmapheresis with cryoprecipitate-poor plasma, vincristine, and steroids. She responded well; her pheresis was decreased to every other day and prednisone was tapered. She was discharged with a platelet count of 398,000 to continue outpatient pheresis and vincristine, which were discontinued after one month. One month later, the patient had a platelet count of 70,000, LDH of 1,186, and schistocytes on peripheral smear. Plasmapheresis and prednisone were restarted, and a splenectomy was performed. Discharge platelet count was 199,000. Two weeks later, her platelet count was 57,000. Pheresis and vincristine were restarted with good response. IMPLICATIONS/DISCUSSION: TTP and HUS are diseases with abnormalities in multiple systems. Classically, TTP is described as having more severe neurologic disease, whereas HUS has more of a renal component. Clinically, however, patients are described as having TTP-HUS because most present with the same symptoms, have identical pathologic changes, and receive the same treatment. The classic pentad of thrombocytopenia, microangiopathic hemolytic anemia, neurologic abnormalities, renal abnormalities and fever is present in few patients. The diagnosis is made primarily by the presence of thrombocytopenia and microangiopathic hemolytic anemia. The etiology is a deficiency of von Willebrand factor (vWF) cleaving protease, which results in the accumulation of unusually large vWF multimers, causing platelet aggregation. Causes of TTP-HUS include E. coli 0157:H7 and medications such as ticlopidine, cyclosporine, and quinine. Most adult cases, however, are idiopathic. Treatment is plasmapheresis, the mechanism of which is not completely understood. 10 to 20% of patients have an incomplete response; steroids, vincristine or IVIG can be added in these cases, but are not alternatives to pheresis. Some studies show improvement with splenectomy, especially in conjunction with pheresis and steroids. 30 to 50% of patients have exacerbations of thrombocytopenia and hemolysis when pheresis is tapered. Relapse is defined as recurrence after 30 symptom-free days without treatment. Both are treated with reinstitution of daily pheresis. It is essential that physicians involved in the care of the patient with TTP-HUS recognize that many patients may have worsening disease with tapering of pheresis and to be aware of alternate life-saving treatments if this occurs. year-old female presented with 4 weeks of malaise, a dry cough, and recent high fevers. She also had night sweats for the 6 months since she started hormone replacement therapy. She had no pets and no recent travel history. Though exposed to tuberculosis as a child, she had never been tested or treated for it. She had begun minocycline just over a month earlier for rosacea and was on loratidine for seasonal allergies. She did not drink alcohol and quit smoking 5 years prior. Physical exam was notable for high fever, tachypnea, pulse oximetry of 96% on room air, inspiratory crackles at the right base and dullness at the left base. Labwork: WBC 15.2 with 84% neutrophils; blood eosinophilia increased over several days to 15%. ESR was 70. Chest radiograph showed small bilateral effusions, no cardiomegaly, and bilateral, peripheral, apical alveolar infiltrates. PPD was negative. Her fevers persisted and the left pleural effusion increased on intial therapy for community-acquired pneumonia. The effusion was transudative, with 1755 WBCs, 12% neutrophils and 10% eosinophils. A bronchoalveolar lavage (BAL) and transbronchial biopsies were performed; BAL fluid contained 63% eosinophils and biopsies revealed focal aggregates of eosinophils around the airways, consistent with eosinophilic pneumonia. Oral prednisone was started and both symptoms and eosinophilia resolved 48 hours after her first steroid dose. IMPLICATIONS/DISCUSSION: EP is characterized by eosinophilic pulmonary infiltrates. It presents as an acute febrile illness with dyspnea, night sweats, weight loss, dry cough, crackles or wheezing on lung exam, increasing blood eosinophilia and bilateral, peripheral patchy infiltrates on chest radiograph. The ESR is often elevated. Etiologies include common medications, including minocycline, parasitic infections, HIV, and fungal infections. A high index of suspicion, confirmed by BAL fluid eosinophilia and diffuse eosinophilic infiltration of the lung on biopsy, leads to the diagnosis. The response to steroids is dramatic, as was seen in our patient, with symptoms, eosinophilia, and lung infiltrates decreasing within 48 hours. Treatment is continued for 3 months to prevent relapses, which respond to re-intitiation of steroids.\nA POTENTIAL BLOOD DONOR. J. Kimberly 1 , R. Watkins 2 ; 1 Wake Forest University Baptist Medical Center, Winston-Salem, NC; 2 Wake Forest University, Winston-Salem, NC (Tracking ID #76222) LEARNING OBJECTIVES: 1) Be able to counsel patients with evidence of infection with HTLV-I and 2) appreciate associated diseases. CASE INFORMATION: A 43 year old African American female with a history of hypertension, diabetes, and treated syphillis presents to clinic reporting that she fears she may have contracted HIV disease. She recently donated blood and subsequently received a letter stating she could no longer donate blood``because of an infection like AIDS.'' The patient presented the letter which notified her of the presence of HTLV-I/II antibodies with Western Blot confirming HTLV-I infection. ELISA for HIV 1 and 2 was performed and was negative. The patient was initially fearful and had many questions regarding HTLV-I infection: How did she get infected and was it sexually transmissible or contagious via other routes? Would she develop immunosuppression similar to that seen with HIV disease? Were there any other possible adverse effects on her health? IMPLICATIONS/DISCUSSION: HTLV-I is a retrovirus endemic in parts of Japan with other clusters of high prevalence including the southeastern US. The virus is transmitted by three known routes: 1) from mother to child, especially through breast feeding, 2) through sexual activity, and 3) through blood, either from contaminated needles or transfusion. In areas where HTLV-I is endemic, many inflammatory and autoimmune diseases have been attributed to the virus though causation has only been shown for two diseases. HTLV-I is clearly linked to adult T-cell leukemia/lymphoma (ATL), classically an aggressive, usually deadly, hematogenous malignancy characterized by rapid progression of skin lesions, pneumonitis, hypercalcemia, and lymphocytosis. The second disease with a clear association with HTLV-I infection is tropic spastic parapesis or HTLV-I-associated myelopathy (HAM) which, unlike ATL, is characterized by an insidious onset with symptoms often including stiffness or weakness in one or both legs, back pain, urinary incontinence, and mild sensory changes. Onethird of patients are bedridden by ten years. Both ATL and HAM occur in 2-5% of HTLVinfected patients. ATL is slightly more common in infected males and usually occurs after 20-30 years of infection whereas HAM is more prevalent among females and can occur just a few years after infection. More than 95% of patients with ATL and HAM have evidence of HTLV-I infection. The patient under discussion was educated about the nature of HTLV-1 and how it differed from HIV, and counselled to practice``safer sex'', avoid donating blood, and, should she become pregnant, avoid breast feeding. She was also informed of the risk, albeit a fairly low one, of ATL and HAM. 2) Identify the diagnostic criteria for eosinophilic gastroenteritis.\n3) Understand the management and prognosis of eosinophilic gastroenteritis. CASE INFORMATION: A 38 year-old woman presented to the ER with vomiting and diarrhea for four weeks. She experienced 6-8 watery, non-bloody stools per day; vomiting after consuming any food or liquid; and a``pulling sensation'' across the lower abdomen. One week after the onset of symptoms the patient was admitted to the hospital, rehydrated, and discharged with a diagnosis of acute gastroenteritis. The symptoms worsened soon after discharge, and the patient was readmitted to the hospital. Evaluation included routine stool cultures and ova and parasites. The patient was treated empirically with levofloxacin and metronidazole and was discharged home. Stool cultures were later found to be negative. The patient's symptoms quickly worsened and she presented again to the ER. Past medical history was remarkable for asthma and lactose intolerance. On physical examination, BP = 115/62, P = 110, and T = 99.18F. Abdominal examination revealed a moderately distended abdomen with shifting dullness and mild tenderness to palpation in the upper abdomen. The remainder of the examination was unremarkable. The white blood cell count was 15,600 with 55.3% eosinophils; the hemoglobin was 8.9. CT of the chest, abdomen, and pelvis revealed a thickened terminal ileum and cecum, ascites, and bilateral small pleural effusions. Paracentesis was performed. The peritoneal fluid revealed 1,920 white blood cells with 3 neutrophils, 12 lymphocytes, and 85 eosinophils. Ascites fluid albumin was 3.3; the serum-ascites albumin gradient was 1.2. Upper and lower endoscopy revealed only melanosis coli. Biopsy of the duodenal mucosa revealed lymphocytic and eosinophilic infiltration. The patient was treated with prednisone. Her symptoms improved and she was discharged home. IMPLICATIONS/DISCUSSION: Eosinophilic Gastroenteritis (EG) is a rare disease whose etiology is unknown, though it may be a Type I hypersensitivity reaction. Patients with EG present a range of symptoms from nausea, vomiting, and diarrhea, to ascites and bowel obstruction. The symptom complex is determined by the degree of eosinophilic infiltration of the gastrointestinal tract. The diagnosis of eosinophilic gastroenteritis requires the presence of gastrointestinal symptoms, eosinophilic infiltration of one or more areas of the gastrointestinal tract on biopsy, absence of eosinophilic involvement of multiple organs outside the gastrointestinal tract, and absence of parasitic infection. Eosinophilic gastroenteritis without ascites is treated with cromolyn; the presence of ascites requires glucocorticoid treatment. More than 90% of patients respond quickly to treatment. The relapse rate is high with cessation of treatment (>33%). Those who relapse often require maintenance therapy with glucocorticoids.\nA CURIOUS CASE OF CYANOSIS. C.T. Ko 1 , N. Spell 1 ; 1 Emory University, Atlanta, GA (Tracking ID #76425) LEARNING OBJECTIVES: 1. Recognize methemoglobinemia as a cause of cyanosis, even without G6PD deficiency. 2. Diagnose and treat methemoglobinemia by understanding its mechanism of action. 3. Recognize the drugs that can cause methemoglobinemia. CASE INFORMATION: A 26-year-old African-American female with HIV presented complaining of worsening shortness of breath, headaches, and generalized weakness. Her CD4 count was 11/mm3. She had been discharged from the hospital one week prior with treatment for presumptive Pneumocystis carinii pneumonia. Her discharge medications included clindamycin and primaquine. On physical exam, she was a thin, cachetic, pleasant female in mild respiratory distress. Vital signs included a temperature of 102.5F (39.2C), blood pressure 90/50 mm Hg, pulse 124 beats/minute, and respiratory rate 24 breaths/minute. Her pulse oximeter reading was 85% saturation on room air and 89% saturation with a 100% nonbreathing oxygen mask. She had conjunctival and mucosal pallor as well as cyanosis of her nail beds, diffuse shoddy lymphadenopathy, and tachycardia with no associated murmurs/rubs/ gallops. Lung fields were clear to auscultation with adequate airflow. Laboratory evaluation revealed white blood cells 3400/mm3 (47N, 46L, 6M), hemoglobin 7.0 g/dl, hematocrit 20.2%, platelets 130,000/mm3, and reticulocytes 1.4%. Her hematocrit one week prior was 35.5%. Chemistry profile showed total bilirubin 1.0 mg/dl, LDH 1374 U/L, and G6PD 10.6. All other chemistry values were within normal limits. Chest X-ray was unchanged from her previous films with minimal bilateral reticular prominence likely secondary to mild interstitial scarring. Arterial blood gas on room air showed pH 7.40, pCO2 28 mm Hg, pO2 73 mm Hg, oxyhemoglobin 73%, carboxyhemoglobin 0.2%, and methemoglobin 25%. Primaquine was discontinued, and methylene blue was administered. Methemoglobin level was 13.6% within one hour. One week later in outpatient follow-up, methemoglobin level was 2.6%. IMPLICATIONS/DISCUSSION: Methemoglobinemia causes cyanosis that is unresponsive to oxygen therapy. In addition, pulse oximeter readings are not accurate when there is methemoglobin in the blood. Etiologies of methemoglobinemia include medications, chemical agents, and hereditary causes. The goal of therapy in methemoglobinemia is to reduce heme iron in order to restore normal oxygen transport. year-old Asian-American woman presented with three weeks of progressive muscle weakness. She had difficulty combing her hair, lifting her arms or getting out of a chair. At presentation she could not walk. She had a dusky, almost dirty appearing rash on her chest and around her eyes. An erythematous rash was noted on the dorsal surface of the MCP and PIP joints of her hands. She could not lift either thigh off the bed. Laboratory values: CK 14,245, RF 273, ANA + >1:640 (speckled pattern), AST 605, ALT 207, ALK PHOS 95, LDH 150. Hepatitis B CAb +, Hep Sab +. Anti-RNP, Anti-Jo, Anti-SSA, Anti-SSB and anti-DS DNA were all negative. A presumptive diagnosis of dermatomyositis was made, a confirmatory muscle biopsy performed and prednisone begun. Malignancy workup was negative. She improved slightly and was discharged to a rehabilitation facility. IMPLICATIONS/DISCUSSION: Dermatomyositis (DM) is a rare idiopathic inflammatory myopathy that has a 2:1 female preponderance and presents most commonly in the fifth decade. The incidence of DM is 1:100,000. Pathogenetically, DM is associated with immune complex deposition in a perivascular and perifasicular distribution. The etiology is unknown although viruses have been implicated. The typical presentation of DM is proximal muscle weakness in association with classic dermatological findings. Gottren's papules, as seen in our patient, are the most commonly seen skin rash. The classic heliotrope rash is the most specific manifestation of DM but is only rarely seen. Other findings include acanthosis nigricans,`m echanics hands,'' diffuse flat erythema in a shawl like distribution, and periungual erythema. The elevated liver enzymes in this patient are problematic since DM alone can raise parenchymal liver enzymes. The hepatitis B profile is not suggestive of an acute infection and unlikely to be causing the elevated liver enzymes. Hepatitis B, however, has been implicated in the pathogenesis of some cases of DM and raises the question of a delayed slowly progressive humorally mediated immune reaction. The relationship between the two conditions is unknown although a few select reported cases suggest it may be more than coincidental. There was no history of smoking, travel, or prolonged immobilization. His ROS was otherwise negative. His medications were aspirin, metaprolol, lisinopril, and sulfasalazine. After initial treatment for pulmonary edema at an outside hospital, he was transferred to our institution. He was intubated due to worsening hypoxia. A bronchoscopy was performed: bronchoalveolar lavage (BAL) was bloody. VS: Temp -378C, BP -124/60 mmHg, HR -110/min, SaO2 -98% on assist control ventilation. Exam was notable for bilateral rales and absence of S3 or JVD. Initial labs revealed Na-127, creatinine -1.0, CK-MB -15, Troponin I -10.2. EKG was normal; CXR showed interstitial pulmonary edema. Echocardiogram showed EF of 35%. A highresolution chest CT showed ground glass appearance consistent with ARDS. He was treated with ceftriaxone and azithromycin. His hospital course was complicated by right middle and anterior cerebral artery ischemic stroke, ARF (BUN/Cr 118/3.3), anemia and ultimately shock. He expired on the ninth hospital day. Autopsy reports confirmed thrombotic microangiopathy consistent with HUS. BAL culture subsequently grew influenza A. IMPLICATIONS/DISCUSSION: Influenza, occurring as outbreaks in winter seasons, is usually self-limited; but causes increased morbidity and mortality in high-risk populations. Such high-risk group includes patients with underlying cardiac, pulmonary, and immunosuppressive diseases, nursing home residents and older than 65. Primary viral pneumonia is the most severe pulmonary complication that tends to occur in individuals with elevated left atrial filling pressures, and pulmonary diseases. Systemic complications include rhabdomyolysis, Reye syndrome, transverse myelitis, and myocarditis. Other rare complications, such as hemolytic uremic syndrome and toxic shock syndrome worsen the clinical outcome. Apart from the tissue culture, the virus can be detected in throat swabs and by serologic methods. This high-risk patient presented with acute severe respiratory failure from primary influenza A pneumonia complicated by HUS leading to renal failure and anemia. Increasing the pretest probability by clinical evaluation will be helpful in initiating treatment earlier in the course rather than be fooled by the flu. LEARNING OBJECTIVES: 1. To recognize the clinical and pathologic presentation of talcosis, also known as foreign body granulomatosis. To recognize the sequelae of talcosis, specifically panlobular emphysema, acute pulmonary hypertension, ARDS, and spontaneous pneumothorax. 2. To recognize signs and symptoms of factitious disorders (Munchausen's). 3. To discuss differences between factitious disorders, somatoform disorders and malingering. CASE INFORMATION: A 50 year-old Oregon man with an extensive past medical history including chronic pain syndrome involving his hip, knee, and groin, total hip replacement surgery with multiple revisions and post-surgical complications presented with a recent history of hypertensive episodes and syncope. He was referred to VMMC for evaluation of a possible pheochromocytoma. In addition to syncope, the history and physical revealed an antecubital abscess and fevers. The patient was admitted and an Infectious Disease consult ordered. He reported that In March, 2002, he developed increasing frequency of attacks characterized by chill, abrupt fever, hypertensive episodes, and syncope. In mid-March, he noted swelling, redness and warmth in his right antecubital fossa and continued to have fever every few days. The patient's hospital course was complicated by multiple episodes of fever >39 oC, witnessed and unwitnessed syncopal episodes, frequently infliltrated intravenous lines, polymicrobial blood cultures, increasing oxygen needs, infected PICC, and atrial fibrillation. His medical work-up was extensive. He ultimately underwent a chest CT which showed multiple 2-3mm, bilateral pulmonary nodules. The patient had thoracoscopic lung biopsy of several nodules. The pathology revealed near complete occlusion of small and medium vessels by extracellular refracticle material. The patient was confronted with these findings and confessed to having crushed and injected oxycontin on several hundred occasions.\nOccurs when tablets are pulverized and injected. The most common fillers used in tablets are talc, cornstarch, cellulose. These fillers cause an initial arteritis which is associated with rapid influx of neutrophils around the intravascular foreign body. The influx of neutrophils is followed shortly thereafter by granuloma formation as talc particles migrate through the vessel wall to the surrounding perivascular and interstitial tissue. The most common radiographic finding is widespread, 2-3mm well defined nodules typically in the mid lung. The clinical presentation is typically non-specific and may mimic URI with: cough, dyspnea, increased sputum. Sequelae of talcosis includes: panlobular emphysema, acute pulmonary hypertension, ARDS, and spontaneous pneumothorax. Treatment options are limited but include: Dexamethasone and D-penicillamine. Steroids appear to have a limited (if any) role. Vasodilatation with hydralazine may help those with pulmonary hypertension. FACTITIOUS DISORDER-A form of feigned illness which differs from malingering (which has a external incentive-avoiding work, financial gain) in that it has no other incentive other than to be a patient and experience the sick role. Both factitious disorder and malingering differ from somatoform disorders in that they are voluntarily produced. Munchausen's Syndrome is the most extreme (and highly publicized) form of factitious disorder. Patients with factitious disorder often appear more comfortable than their condition would warrant. They may be pleasant, cooperative and receptive to ANY intervention-even if painful and risky. Nurses often note that they often have no visitors/phone calls suggesting anti-social or isolating behaviors. They may also give a medical history that is unbelievable or``too good to be true''. Be suspicious when pt's deny you the right to access old records or speak with previous providers. Yesterday she had been seen in the clinic and diagnosed with trichomonas vaginitis. The resident had dutifully told her that this was a sexually transmitted disease and that both she and her husband needed to be treated simultaneously. Unfortunately, because of her husband's health they had not had sex in over ten years. Now her husband was accusing her of infidelity, a fact she denied vehemently. The Case: The patient is a 59 year old white woman with a 1 week history of burning on urination and a mild vaginal discharge, described as thin and clear. She denied fever or flank pain. Her social history was significant for part-time work as a housekeeper which took her out of the house periodically. Her husband had been disabled for 10 years with a back injury and there had been no sexual activity since the injury. The pelvic examination was remarkable only for a thin watery discharge with a mild odor. Cervix and adnexa were benign. A wet prep revealed numerous trichomonads. IMPLICATIONS/DISCUSSION: Trichomonas vaginalis infection classically presents as a vaginitis/cervicitis with a thin, yellow, frothy, foul smelling discharge often times associated with dysuria. Nevertheless, up to 40% of infections are asymptomatic. The association with sexual activity is well documented. As an illustration of this, the incidence of infection in the general population has been reported as high as 5-10%, while in venereal disease clinics the incidence jumps to 25% and up to 70% among prostitutes. However, unlike other sexually transmitted diseases, the age distribution of of trichomonas infection is bimodal. For example, most women with gonorrhea are in their early 20's, with less than 2% over the age of 40. Trichomonas infections also peak during years of maximal sexual activity but there appears to be a second peak around the time of menopause. Indeed, >30% of patients are over the age of 40. It has been hypothesized that this represents women infected at an early age who harbored the trichomonas asymptomatically but present with symptoms at a time when the vaginal milieu changes (eg. menopause) allowing the trichomonas to propagate. There are several reported cases of newborns and apparent virgins with trichomonas. These likely represent either transmission from infected mothers at the time of birth or a nonsexual source. Studies from the British literature (Burgess,1963; Whittington 1957) establish that trichomonas can survive for up to 45 minutes on a toilet seat, although transmission by this route has never been proven. Has she been faithful? Clearly there is valid justification to believe that she is telling the truth. year-old man presented with a five weeks of bowel and bladder incontinence and parethesias of the lower extremities. He had a history of diabetes mellitus and HIV. Two weeks prior he had presented for evaluation of urinary hesitancy and paresthesias of the lower extremities. He was diagnosed with diabetic peripheral neuropathy and released. On this admission, his neurological examination revealed decreased sensation in the lower extremities, absent ankle reflexes, an ataxic gait, saddle anesthesia, and decreased anal sphincter tone. An MRI of the spine revealed an extradural mass extending from L4 to S4. Biopsy confirmed the neoplasm to be Burkitt's lymphoma. IMPLICATIONS/DISCUSSION: Although spinal cord compression is a medical emergency, access to an MRI may be limited. The physical examination is instrumental in establishing the diagnosis and expediting appropriate therapy. Although diabetes and HIV are common causes of peripheral neuropathy, they are not causes of the cauda equine syndrome (CES). A methodical physical examination detected saddle anesthesia and sphincter impairment, establishing the diagnosis of CES. Compression of the roots can be caused by a ruptured disk, infection, fracture, or tumor. HIV patients are at increased risk for tumors associated with co-sexually transmited viral infections such as HHV6 and HHV8. CES is a neurosurgical emergency, requiring immediately operative decompression of the spinal canal. A timely diagnosis of this syndrome is essential, as patients with paraneoplastic impingement may not regain function beyond the deficit at presentation.\nCORONARY DISEASE. E.J. Lee 1 , K. Barnard 1 ; 1 University of Pittsburgh, Pittsburgh, PA (Tracking ID #75596) LEARNING OBJECTIVES: Recognize gender differences in the presentation of myocardial ischemia and consider microvascular spasm as a cause for chest pain in women with angiographically normal coronary arteries. CASE INFORMATION: A 54 year-old Asian-American woman with history of chronic back pain presented with retrosternal chest pain. The pain was sharp, radiated to her left arm, awoke her from sleep, and lasted for one hour. She had had similar pain the previous day, lasting an hour before spontaneously resolving. Neither episode was associated with nausea, vomiting, diaphoresis, or exertion. Another similar episode occurred one year ago. Echocardiogram at that time showed mild hypertrophy and normal overall function, while coronary catheterization revealed normal coronary arteries and an ejection fraction of 65%. The patient had no history of diabetes, hypertension, hyperlipidemia, smoking or family history of coronary artery disease. On initial evaluation in the Emergency Department, the patient was without chest pain. Her electrocardiograph showed normal sinus rhythm with no ischemic changes. Chest radiograph was normal. Total CPK was 71 and troponin was less than 0.2. She was admitted to rule out myocardial infarction. Shortly after being admitted, the medicine house staff were called to evaluate the patient for``seizure-like activity.'' The patient was found in severe emotional distress with taut extension of her upper and lower extremities, and clawing of her hands bilaterally. She was alert, oriented, and able to follow commands throughout this episode which resolved within minutes. Electrolytes showed a calcium of 9.7, magnesium of 1.7, phosphate of 3.3, and potassium of 3.8. An electrocardiograph at this time showed ST depression in leads II, III, and AVF. Repeat electrocardiograph showed a similar pattern of ST depressions. The patient was transferred to the cardiac step-down unit with a presumptive diagnosis of unstable angina and started on anticoagulation therapy. Aspirin and beta-blockers were held due to reported allergies. Subsequent electrocardiographs showed normal ST segments in all leads. Second and third sets of cardiac enzymes were negative. Cardiac catheterization the following day revealed normal coronary arteries and an ejection fraction of 60%. The patient was discharged home the following day in good condition. IMPLICATIONS/DISCUSSION: There is abundant data to suggest there are gender differences in the risk factors, presentation, and management of cardiac disease. It has been shown that negative affect and emotional distress may increase risk of ischemic heart disease and interfere with effective cardiac rehabilitation. Mental stress has been known to cause ischemic changes on electrocardiograph and abnormalities in regional and global left ventricular function. These effects have been associated with microvascular spasm. A mental stress-induced ischemic episode may have occurred in this patient. A coronary catheterization that showed normal coronary flow would not be uncommon in such a case. It is important to realize that despite angiographically normal coronary arteries, this may be an atypical presentation of true underlying coronary disease.\nTHROMBOTIC THROMBOCYTOPENIC PURPURA-HEMOLYTIC UREMIC SYNDROME (TTP-HUS) AFTER A SINGLE DOSE OF QUININE. J. Lee 1 , M. Krasnoff 1 ; 1 Dartmouth Hitchcock Medical Center, Lebanon, NH (Tracking ID #74449) LEARNING OBJECTIVES: 1. Identify a potentially fatal complication of a commonly prescribed therapy for nocturnal leg cramps. 2. Recognize quinine as the most common cause of drug-associated TTP-HUS. CASE INFORMATION: The patient is a 66 year old previously healthy female who presented to the emergency department with the acute onset of nausea, vomiting, abdominal pain, diarrhea, and disorientation 2.5 hours after taking a single dose of 260 mg of quinine for nocturnal leg cramps. Over the next few hours, she developed severe hypotension, requiring pressors. Labs were notable for WBC 3.6 Plt 123,000 Bun 25 Cr 1.9 T bili 1.7 PTT 75 PT > 100. A CT scan of the abdomen and pelvis was unremarkable. The patient was admitted to the ICU with a presumed diagnosis of sepsis of unclear etiology; she was started empirically on IV ampicillin/sulbactam. Over the next few days, the patient developed microangiopathic hemolytic anemia, severe thrombocytopenia (Plt 12,000), and acute renal failure (Cr 8.4). Cultures of sputum, urine and blood showed no growth. Her clinical diagnosis was felt to be TTP-HUS; her antibiotics were stopped and she was begun on daily plasmapheresis. Dialysis was instituted. After two weeks of therapy, the patient's anemia, thrombocytopenia, renal failure, disorientation, and coagulopathy were all improving or resolved. An adverse event report was sent to FDA MedWatch. IMPLICATIONS/DISCUSSION: Nocturnal leg cramping is a common complaint encountered in the primary care setting. Despite quinine being frequently prescribed, its potential for severe adverse reactions is largely underrecognized. Quinine is the most common cause of drug-associated TTP-HUS. Common presenting symptoms include the acute onset of abdominal pain, nausea, vomiting, diarrhea, fevers and chills. The symptoms usually begin abruptly, several hours after the ingestion of quinine. A single tablet is sufficient to cause TTP-HUS. In addition to the expected findings of TTP-HUS, low-grade DIC and leukopenia can also be seen. The mechanism of quinine-associated TTP-HUS is presumed to be caused by drug-dependent antibodies. Treatment involves avoidance of quinine exposure, plasmapheresis and supportive therapy. While available by prescription, quinine is also found in beverages, such as quinine water, tonic water, and gin and tonic. In addition, there is an extensive list of herbal remedies containing quinine including cinchona tree, peruvian bark, and china bark. Physicians should carefully question any patient with TTP-HUS for quinine exposure, especially since patients may not report it among their medications. In addition, physicians need to counsel patients regarding future avoidance of all quinine-containing compounds, since repeat ingestion of quinine may cause relapse of TTP-HUS.\nPATIENT. D. Lehman 1 , J. Mills 1 , C. Sorensen 1 , J. Whitesel 1 , R. Reves 1 ; 1 Denver Health and Hospital Authority, Denver, CO (Tracking ID #75102) LEARNING OBJECTIVES: To recognize that tuberculosis can present with unusual clinical manifestations, often sub-acute or chronic, especially in the immunocompromised context, such as HIV infection. CASE INFORMATION: 48 y/o male with heterosexually acquired HIV infection and a CD4 count of 306, presented with a 1-month history of penile and rectal pain and 1 week of no urethral voiding. He noted urine coming from the rectum with feces. Voiding cystourethrogram showed a prostatic-rectal fistula and a Foley catheter was placed. He was born in Mexico and had a negative PPD 10 years earlier. Four months earlier he had presented with dysuria, an enlarged prostate, pyuria and microscopic hematuria. CT scanning then showed a 2.7 by 3.0 cm hypodensity within an enlarged prostate that, in the absence of fever and toxicity, appeared clinically not consistent with a prostatic abscess. No urine culture was done, but he received 10 days of levofloxacin. Fistula evaluation by GI with colonoscopic biopsy showed chronic inflammation and necrosis. Transrectal ultrasound showed a large prostate with prostatic calculi with no cyst or abscess. Granulomas and acid-fast bacilli (AFB) were found in biopsies obtained during an unsuccessful surgical attempt to repair the fistula. Bilateral infiltrates were found on a post-operative chest X-ray. AFB were found in urine and sputum and cultures grew Mycobacterium tuberculosis (TB). Standard 4-drug therapy for TB was successful and the fistula closed without further surgery after 6 months of directly observed treatment and Foley drainage. IMPLICATIONS/DISCUSSION: Delayed and missed diagnoses of active pulmonary and extrapulmonary TB are common due to the relative rarity of the disease and a lack of familiarity with its protean manifestations. TB of the genitourinary tract accounts for about 1% of reported cases of TB in the US. Extrapulmonary forms (27% of US cases) are more common among children and patients with HIV-infection or other disorders of immunity. The keys to earlier recognition of active TB are awareness of: 1) the manifestations of the disease, 2) risk factors for previous exposure and latent TB infection, and 3) risk factors for progression from latent TB infection to active disease. In retrospect this patient had all three of these features. The clinical features of active TB, both pulmonary and extrapulmonary are variable, but chronic and sub-acute presentations are common. Signs of systemic toxicity are helpful if present but are commonly absent. TB of the urinary tract should be suspected in patients with unexplained``sterile'' pyuria and/or hematuria and in those with unexplained chronic inflammation and fibrosis. This patient had a common risk factor for infection, having been born in Mexico, and had HIV infection as a risk factor for progression to active TB. Although the GU tract is a relatively common extra-pulmonary site for TB, there is little mention in the literature of fistula as a complication of TB prostatitis. The most likely etiology of a prostaticrectal fistula is surgery, rarely by infection. Radiological studies revealed what appeared to be a solid posterior occipital mass consistent with a malignant lesion and surgical intervention was pursued. At craniotomy he was found to have an abscess; smears revealed yeast forms and cultures grew Cryptococcus neoformans. CSF results were WBC 255, protein 112, glucose 27 and cryptococcal antigen 1:64. Serum cryptococcal antigen was 1:128. He disclosed no risk factors for immunodeficiency which was confirmed by negative studies for HIV-1 and 2 antibody, HIV-1 and 2 PCR, HTLV-1 and 2 antibody and HTLV-1 and 2 PCR. Of note, his CD4 count was 79 and his total IgG, IgG1 subclass and IgG2 subclass levels were low. IgA and IgM levels were at the lower limits of the normal range. He received two weeks of amphotericin B and improved appropriately. Lumbar puncture at that time revealed: WBC 440, protein 111, glucose 30 and cryptococcal antigen 1:2. Repeat CD4 count was 191 and serum cryptococcal antigen was 1:64. His therapy was changed to fluconazole and he continued to improve. IMPLICATIONS/DISCUSSION: Although possible in immunocompetent hosts, cryptococcal meningoencephalitis presents most commonly in patients with HIV infection, drugassociated immunosuppression, leukemia and complement deficiency. This infection has rarely been reported in cases of hypogammaglobulinemia. Cryptococcus neoformans infects through aerosolization producing a primary respiratory infection that spreads hematogenously to distant sites. In hypogammaglobulinemia of the type shown in this patient, the proposed host defect stems from dysfunctional T-cell activity resulting in fundamental difficulties with complement fixation, opsonization and reduction of circulating antigens. We propose the possibility that the hypogammaglobulinemia facilitated the development of this patient's CNS disease. These immunosuppressed patients present with fever, headache and malaise similar to, but typically more intense and shorter in duration than infections in the immunocompetent host. Response to treatment with amphotericin B and fluconazole are similar to those in immunocompetent hosts.Close monitoring and medication adjustments per clinical response are essential. Thus, this case provided an unusual initial presentation of hypogammaglobulinemia as cryptococcal meningoencephalitis. LEARNING OBJECTIVES: To recognize filariasis in the differential diagnosis of nephrotic syndrome. CASE INFORMATION: A 46 year old man who had recently emigrated from West Africa presented with a three month history of lower extremity pitting edema. His physical examination was significant for anasarca. Laboratory data revealed a white blood cell count of 9900 with 58% eosinophils, an albumin level of 1.2 g/dl as well as a BUN and creatinine of 16 and 1.0 respectively. An initial urinalysis revealed protein level of greater than 300 mg/dl and microhematuria. A subsequent 24-hour urine collection revealed 7.6 grams of protein confirming a diagnosis of nephrotic syndrome. The differential diagnosis of nephrotic syndrome with eosinophilia includes lymphoma and various parasite infections. While parasitic serologies were pending, chest and abdominal CT scans were normal ruling out a diagnosis of lymphoma. A renal biopsy showed membranous glomerulonephritis with diffuse IgG deposits in the subepithelium. Subsequent peripheral blood smears showed obvious microfilariae of the Loa loa species, indicating active infection. Further laboratory studies revealed the absence of other conditions associated with membranous glomerulopathy. Therefore, it was concluded that the nephrotic syndrome and the membranous glomerulonephritis were most likely caused by the filarial infection. The patient was treated with metolazone and lisinopril to treat the anasarca/ nephrotic syndrome and given ivermectin to treat the filariasis. At follow-up two months later, the edema had resolved and his urinalysis had almost normalized. IMPLICATIONS/DISCUSSION: Loa loa is one of eight filarial species and occurs mainly in the rain forests of Africa. Adult worms develop and mate to form microfilariae, which appear in the blood. Whereas other filarial species cause the classic symptoms of lymphedema, the loaisis species is more commonly associated with pruritus and localizd areas of subcutaneous swelling. Various nephropathies, including nephrotic syndrome, are rare and likely result from immune complex deposition within the glomerulus. In tropical countries, infectious diseases are common causes of the nephrotic syndrome. As demonstrated in this case, one should consider the diagnosis of filariasis in patients that present with anasarca and/or nephrotic syndrome and who have a history of travel to endemic countries for filariasis. year-old white female with a history of von Willebrand disease presented with 3 weeks of severe fatigue. She denied history of trauma or abnormal bleeding although she reported easy skin bruising. On physical examination, her temperature was 37.4 degrees Centigrade and BP 102/72 mmHg. She appeared thin and pale. Neck was supple, without thyromegaly. Lungs were clear. Heart was regular, without murmurs or gallops. Abdomen was benign without masses, tenderness or hepatosplenomegaly. Extremities had no cyanosis, clubbing or edema. Neurological examination was non-focal. CBC revealed a hemoglobin of 10g/dl, platelet of 33,000/mm3, and WBC of 3500/mm3 with 13% blasts. Bone marrow biopsy found 40% blasts with positive CD33 and CD34. She was diagnosed with acute myeloid leukemia. Chemotherapy with cytarabine was started. Five hours later, she developed severe stabbing left upper quadrant abdominal pain radiating to the back and left shoulder, aggravated by body movement, only slightly relieved by intravenous hydromorphone. One hour later she became hypotensive with a BP of 90/50 mmHg. Hemoglobin dropped from 9.0 to 7.1g/dl in 4 hours. Electrocardiogram showed sinus tachycardia and no acute ischemic changes. Abdominal series was unremarkable. An abdominal CT scan was reported unremarkable initially, but further review demonstrated blood surrounding the spleen. Emergent laparotomy found acute rupture of a normal sized spleen (8.0  5.5  2.3 cm). Splenectomy was performed. She was transfused a total of 6 units of packed red blood cells. Hemoglobin remained stable after the surgery. She developed acute respiratory distress syndrome and died of respiratory failure and cardiac arrest within a week. IMPLICATIONS/DISCUSSION: The spleen is an immunologic organ commonly involved in hematologic diseases. Splenic rupture can be classified into 1) traumatic; 2) spontaneous; and 3) pathologic. Pathologic splenic rupture occurs in a spleen affected by a disease without previous trauma. The presentations of splenic rupture are the result of intra-abdominal hemorrhage, hypotension, tachycardia, peritoneal irritation signs, and fever. The abdominal pain could radiate to the left shoulder (Kehr's sign). Pathologic rupture of a spleen affected by a hematologic malignancy is a rare event and it is especially unusual for a normal sized spleen to rupture. The risk of splenic rupture increases with age and size of the spleen. The release of the enzymatic content of cells shortly after induction chemotherapy may lead to splenic damage, followed by rupture of the spleen. The present patient had received cytarabine just 5 hours before the abrupt onset of abdominal pain. Other risk factors of bleeding such as thrombocytopenia and von Willebrand disease might also have contributed to her catastrophic event. The diagnosis of pathologic splenic rupture begins with a high index of suspicion. A patient with risk factors presenting with left upper abdominal pain should be observed closely and investigated. Plain abdominal X-ray, ultrasound and CT scan are valuable tests. Aspiration of blood by abdominal puncture could confirm the diagnosis. For insidious rupture, liverspleen scintigraphy is of value. The only therapy is splenectomy. A 67-year-old male was well until 6 weeks prior to presentation, when he developed diffuse extremity deep aching pain and difficulty with walking, maintaining his balance and doing buttons. He denied diplopia, but had trouble looking to the left. He denied weakness, numbness, sensory loss, trouble with speech or articulation, or bowel or bladder dysfunction. On physical examination, he had normal vital signs. He was alert, cooperative, but somewhat slow. A 2  2 cm non-tender supraclavicular lymph node was palpable. Heart, lungs and abdomen were unremarkable. Extremities had no cyanosis, clubbing or edema. Eyes could not move past the midline on leftward gaze. Motor strength was 5/5 in all muscle groups tested. Vibratory sense was diminished distally in the right lower extremity. A heel-to-shin testing was ataxic. He could not sit nor stand unaided. Romberg's sign was noninterpretable even with the eyes open. Plantar responses were flexor bilaterally. Cerebral spinal fluid reveaked glucose 93, lymphocyte 4, IgG 15.1, and protein 100. Anti-Hu antibody was positive. Brain MRI showed a small old lacunar infarct. Chest CT scan identified a 1 ± 2 cm right suprahilar pulmonary nodule, 3 right upper lobe nodular densities less than 1 cm each, and a prominent 3.5 cm nodule in the right pretracheal area. EMG and nerve conduction studies demonstrated a sensory motor polyneuropathy. Lymph node biopsy showed neoplastic cells staining with synaptophysin and chromogranin, supporting the diagnosis of large cell neuroendocrine carcinoma. The patient was treated with carboplatin and etoposide for three doses and IVIgG for five doses. The symptoms were stabilized. IMPLICATIONS/DISCUSSION: Paraneoplastic encephalomyelitis (PEM) is a frequent remote effect of cancer characterized by neuronal loss and inflammatory infiltrates in the nervous system. The onset is usually subacute and causes severe neurological dysfunction which antedates the diagnosis of cancer. The presenting neurological syndrome is usually sensory neuropathy, cerebellar dysfunction and cortical encephalitis. Diagnosis of the primary cancer remains challenging and requires a high index of suspicion. Usually, it is made by the demonstration of specific paraneoplastic antibodies. Anti-Hu antibody recognizes a family of RNA-binding proteins expressed in the nuclei of neurons and cancer cells. It has been used as a diagnostic marker, but no evidence suggests it causes neuronal damage. Effective treatment of the tumor with chemotherapy is an independent predictor for stabilization of PEM. Large cell neuroendocrine carcinoma is a highly aggressive tumor that usually occurs in smokers, and may be either central or peripheral. It has been suggested that surgically resectable tumors be excised and that advanced stage lesions be treated with combination chemotherapy. Immunotherapy has been proven effective in some patients in tumor regression and PEM stabilization or improvement. The prognosis is poor, with a median survival of 11 months. LEARNING OBJECTIVES: Recognize that the clinical manifestations and radiologic findings of peritoneal tuberculosis (TB) may mimic ovarian carcinoma. Recognize that an elevated CA-125 level may be more suggestive of tuberculous peritonitis than ovarian carcinoma in certain populations such as young women, HIV-infected women and women from areas endemic for tuberculosis. CASE INFORMATION: A 29 year-old Vietnamese woman with no significant past medical history presented to the emergency department with a 10 day history of fever, non-productive cough, abdominal pain, and increasing abdominal girth. A PPD placed as an outpatient was reactive to 16mm. She emigrated from Vietnam ten years prior, had no recent travel history, and had no recent sick or TB contacts. Family history was negative for cancer. On physical examination, the patient was febrile to 102.6F with a pulse of 111 BPM. Pulmonary exam was clear to auscultation. Abdominal exam was significant for mild distention, and tenderness in the epigastric and right upper quadrant regions without rebound or guarding. Gynecologic exam was normal. Chest x-ray was normal. A CT scan of the abdomen and pelvis showed diffuse intraabdominal ascites with omental caking, strongly suggestive of metastatic disease from ovarian carcinoma. Pelvic ultrasound was negative for adnexal masses. Ascitic fluid analysis showed a predominantly lymphocytic exudate. There were no malignant cells, acid-fast bacilli (AFB) or other organisms detected in the fluid. Fluid culture yielded no growth. Multiple sputum smears were negative for AFB. A CA-125 level was elevated to 546 U/ml. A CT guided biopsy of the omentum revealed granulomatous inflammation and well-defined granulomas with central necrosis ± histology consistent with tuberculous peritonitis. The patient was started on a regimen of isoniazid, rifampin, ethambutol and pyrazinamide. On follow-up two weeks after discharge, the patient reported feeling well with complete resolution of the fever and abdominal pain. IMPLICATIONS/DISCUSSION: Peritoneal tuberculosis is an uncommon entity. The common clinical presentation is ascites or an abdominal mass. The elevated CA-125 and radiologic appearance of the omentum, as in the case of our patient, can be highly suggestive of ovarian carcinoma. However, it is well established that many other conditions, including peritonitis can elevate the CA-125. When these features are present in a patient who is at high risk for developing tuberculosis and who is at low risk for developing ovarian cancer then the diagnosis of tuberculosis must be considered. Establishing the diagnosis of peritoneal tuberculosis can be difficult as ascitic fluid is rarely positive for AFB and even culture of biopsy specimens may be negative. Diagnosis is often made on the basis of suggestive histopathology and clinical features. Clinicians must maintain a high index of suspicion for tuberculous peritonitis in patients presenting with ascites and abdominal masses, especially in young women (in whom ovarian carcinoma is uncommon), HIV-infected patients and patients from areas endemic for tuberculosis. In these cases, empiric antituberculosis therapy can be considered while further workup is pending. CA-125, while nonspecific for peritoneal tuberculosis, can be useful in monitoring response to therapy and would be expected to return to normal levels.\nCHRONIC SINUSITIS AND BEYOND. A. Loewen 1 ; 1 University of Calgary, Calgary, Alberta, Canada (Tracking ID #77075) LEARNING OBJECTIVES: To review an approach to the patient with chronic infection. To discuss the manifestations of Wegener's Granulomatosis. CASE INFORMATION: A 33 year old male with a 4 month history of chronic sinusitis which is refractory to antibiotics, and an episode of suppurative otitis media, presents with left sided Bell's palsy and multiple boils. There is microscopic hematuria with normal renal function. Drainage and biopsy of sinuses demonstrates necrotizing granulomatous inflammation. Anti PR3 level is 18.5 kEu/L. A diagnosis of Wegener's granulomatosis is made. The patient later develops hemoptysis while on therapy with prednisone and cyclophosphamide. IMPLICATIONS/DISCUSSION: An approach to the patient with apparently chronic infection must include infectious causes, underlying immunosuppressed states and noninfectious causes. Wegener's granulomatosis is a vasculitis of small arteries and veins and usually presents as a pulmonary-renal syndrome. In this case it presented with upper airway involvement, pyoderma gangrenosum and cranial nerve palsy, butpulmonary and renal symptoms were minimal. A broad differential diagnosis can lead to the correct diagnosis, avoiding unnecessary interventions, and hasten administration of appropriate therapy.\nG. Loukatous 1 , J. Wiese 1 , J. Aliota 1 ; 1 Tulane University, New Orleans, LA (Tracking ID #77080) LEARNING OBJECTIVES: 1. Identification of the clinical signs and symptoms of Vibrio Vulnificus infection 2. Recognition that early and aggressive intervention is essential to optimal patient outcome CASE INFORMATION: A 42-year-old Vietnamese man presented with a two-day history of fever and a painful left leg. He denied trauma or stasis involving the leg. His pulse was 110; blood pressure 110/50; temperature 38.0 C. There was a 10 cm eccyhmotic lesion on his left lower leg. Despite therapy with cefazolin his condition deteriorated. Over 12 hours, the ecchymosis extended as the skin lesion rapidly developed multiple fluid-filled bullae. General surgery was consulted for surgical debridement. Upon further questioning, the patient noted a recent history of raw oyster ingestion. Wound and blood cultures returned positive for Vibrio vulnificus. He was started on tetracycline. Although he subsequently developed multi-organ failure, he recovered after 22 days of aggressive medical treatment and continued surgical debridement. IMPLICATIONS/DISCUSSION: Early diagnosis and treatment is the critical component of a successful outcome from a Vibrio vulnificus infection. The key to diagnosis is combining an astute history with an observant physical examination. In sea-side cities, the consumption of oysters or exposure to the water may be so common as to preclude the patient from thinking to offer this historical clue unless prompted. Recognizing the appearance of Vibrio, then, is the key to diagnosis. Erythematous and ecchymotic skin lesions that rapidly evolve to hemorrhagic vesicles and bullae should alert the physician to the possibility of a Vibrio infection. Like necrotizing faciitis, vibrio invades the deep fascia, destroying the facial structures as it rapidly extends. Ecchymosis, bullae and anesthesia reflect the loss of vessels, skin partitions and nerves. Early intervention with appropriate antibiotic therapy and aggressive wound debridement is essential in such cases. Even with early intervention, bacteremia from V. vulnificus carries a 50% mortality that increases to 90±100% in the presence of hypotension. A 34 year old non-smoking, HIV-negative Hispanic male, who received his last dose of chemotherapy for lymphoma 1 week prior, presented with 3 days of increasing cough, dyspnea on exertion, and fever. He denied any sick contacts, TB exposure, recent travel, or pet exposures. His vital signs were:T 99.6 F, BP 115/78, P 100, R 20, and Pulse Ox 96% on room air. Respiratory exam showed unlabored breathing and clear lungs. His studies showed: WBC 1.9 (N 51%, L 12%, M 35%), Hb 9, Hct 25.9, Plt 44, and LDH 1461. Chest xray showed bilateral hilar fullness not seen on prior films. Differential diagnosis on admission was pulmonary infection (community acquired, atypical, and fungal pneumonia; TB; PCP) versus recurrence of lymphoma. Hospital course:Ceftriaxone and azithromycin were empirically started on Day 1. By Day 2, the patient remained febrile and showed signs of decompensation with decreasing BP and increasing oxygen need to 4 liters. A room air ABG at that time showed: pH 7.49, pCO2 36, PO2 43, saturation 83%. Chest CT showed progression to diffuse bilateral alveolar infiltrates without adenopathy. Trimethoprim/sulfamethoxazole was added empirically for PCP. The patient was then transferred to the ICU for prophylactic intubation and diagnostic bronchoscopy showing positive Pneumocystis carinii direct fluorescent antibody on lavage. All other studies (biopsy, brush, and sputum/blood samples) were negative for bacterial, fungal, and malignant processes. Prednisone was subsequently added. The patient improved dramatically with extubation on Day 3 and discharge within the week. IMPLICATIONS/DISCUSSION: PCP is usually seen in AIDS patients with CD4 counts <200 without chemoprophylaxis. Unfortunately, it may not be readily considered in the differential in immunosuppressed, non-HIVpatients. The above vignette serves to remind us to not exclude PCP in the differential because a delay in evaluation could be fatal. Our patient fared well because we recognized his predispositions (hematologic malignancy and immunosuppression from chemotherapy) early in his hospital course which led to prompt treatment. The calculated absolute lymphocyte count was 228, making the CD4 count likely <200. LEARNING OBJECTIVES: Formulate a differential diagnosis of facial swelling using clinical and laboratory data. Initiate early and aggressive treatment based on the presumed diagnosis of facial swelling. Recognize that individuals with recurrent severe infection should be tested for immunodeficiencies. CASE INFORMATION: A 19 year old male was admitted in August, 2001 with face and lip swelling and pain. He has no significant past medical history or family history. He smokes cigarettes, drinks alcohol, and uses cocaine and marijuana. On physical exam he is afebrile, with a pulse of 132, respirations of 20 and a BP of 110/70. Oxygen saturation initially was 100% on room air. His left face, mouth, and neck are swollen and firm, his left eye was swollen shut and his tongue was swollen. He had no cervical adenopathy and clear lung fields. Initial impression was angioedema and treatment involved epinephrine, solumedrol, and with worsening respiratory status, intubation. Fever climbed to 103.3, treatment was started with ampicillinsulbactim and clindamycin. With positive blood cultures the diagnosis was now thought to be necrotizing fasciitis. Supportive care including TPN continued until he was extubated and discharged after 3 weeks. Patient never followed up and was readmitted in May, 2002 with a similar presentation and was found to have common variable immunodeficiency. Upon discharge, he still has not followed up and is not being treated. IMPLICATIONS/DISCUSSION: Necrotizing infections of the skin and fascia involve tissue destruction, systemic toxicity, and a high mortality rate. There is a tendency to underestimate the severity and extent of infection. We now see more angioedema and might miss this clinical presentation. Normal complement studies make hereditary angioedema unlikely and normal tryptase makes angioedema from other causes unlikely. Treatment of necrotizing fasciitis involves airway control, IV antibiotics, and early, aggressive surgical debridement. Individuals with common variable immunodeficiency frequently present with recurrent infections with encapsulated bacterial pathogens as in our patient. Those with recurrent infections should be checked for the disorder and treated with IV immunoglobulin. LEARNING OBJECTIVES: This case report illustrates a rare diagnosis and an often overlooked complication of a benign therapy. Prompt diagnosis is essential to preventing long term sequelae. CASE INFORMATION: A fifty-four-year-old man presented with nausea and vomiting for one month. He was having more than five non-bloody emeses per day, often within 10 minutes of eating. Emesis was not always associated with eating. He stated that he was unable to keep down any food. He denied fevers, chills, rigors, or night sweats. He described low-grade headaches with associated tinnitus, an 18-pound weight loss and generalized weakness with increased somnolence as well as new onset erectile dysfunction. He complained of intermittent epigastric abdominal pain, and had had a previous laparotomy for abdominal pain approximately five years ago. He is a lifetime non-smoker and denied taking any medications. Evaluation revealed total calcium of 16.6 with ionized calcium of 7.7 and a creatinine of 4.9. Unenhanced CT revealed a band of calcification in the midline of the anterior abdominal wall measuring approximately 6 cm in length and 1.4 cm in diameter. In addition, there were numerous small mesenteric and retroperitoneal lymph nodes along with a 1cm node in the suprapubic anterior abdominal tissue. He had a tiny stone in the left kidney and all the bones were noted to be hyperdense. His hemoglobin was 11.7 with a normal peripheral smear, erythrocyte sedimentation elevated at 38. B12/folate, LDH and T-and B cell surface markers were normal. PTH-RP was non-detectable and PTH was appropriately suppressed. He was treated with IV fluids, furosemide and pamidronate, with normalization of his calcium at time of dismissal. Upon further questioning, discovered that he was ingesting 6 to 7 grams of calcium per day for the last 20 years. IMPLICATIONS/DISCUSSION: These findings were consistent with milk-alkali syndrome. The recent emphasis on calcium therapy for prevention of osteoporosis and the availability of calcium carbonate has made milk-alkali syndrome the third leading cause of hypercalcemia.\nThis presentation is consistent with the subacute form of milk-alkali syndrome referred to as Cope's Syndrome. These patients are exposed intermittently for many years and present with symptoms of both acute and chronic hypercalcemia. This patient responded to therapy and his creatinine remained minimally elevated at 1.8 at a six-month follow-up visit.\nA FIFTY-SEVEN YEAR OLD FEMALE PRESENTING WITH AGRAPHIA FROM CEREBRAL EMBOLISM OF UNUSUAL ETIOLOGY. E. Martorell 1 , P. Abraham 1 ; 1 Mayo Clinic, Jacksonville, FL (Tracking ID #75540)\nLEARNING OBJECTIVES: 1) To describe an unique presentation of a cardiac myxoma.\n2) To recognize cardiac myxomas in the differential diagnosis of acute embolic cerebrovascular accident in adult patients. 3) To assess, diagnose and manage cardiac myxomas early in order to prevent potential mortality and morbidity. CASE INFORMATION: A 57-year-old right-handed woman presented to the emergency department with a three-hour history of new onset writing difficulty without additional symptoms. On physical exam her vitals were normal except for an elevated blood pressure of 156/73. Her head, eye, mouth, neck, skin, cardiovascular and pulmonary examinations were unremarkable. Her neurological examination was completely unremarkable with the exception of a mild impairment of finger to thumb movements (apraxia) on the right hand. She was able to write her own thoughts, but had difficulty due to significant agraphia. MRI demonstrated multiple acute and sub-acute ischemic as well as hemorrhagic lesions without surrounding edema. Electrocardiogram and chest films were normal. Baseline CBC, coagulation studies, chemistry studies and rheumatologic serologies were within normal limits. Carotid duplex showed mild stenosis of the carotid arteries bilaterally. A tranesophageal echocardiogram revealed a large multilobulated, motile left atrial mass affixed via a stalk to the left interatrial septum consistent with atrial myxoma. The patient subsequently underwent urgent thoracotomy. A mucinous dark red polypoidgelatinous 5cm  2cm  1cm mass was resected from the left atrium. Biopsy revealed a benign cardiac atrial myxoma. The patient remained neurologically stable and was discharged home on day #10. IMPLICATIONS/DISCUSSION: Myxomas are twice as common in women as in men and most frequently develop between the third and sixth decades of life. About 1% of ischemic strokes in young people are caused by atrial myxomas. Although embolic complications from myxoma most often affect the brain in more than two third of cases, our patient is unique because of her mild, single neurological deficit and absence of the typical presenting signs and symptoms characteristic of this tumor such as obstructive cardiac signs (54±95%) and constitutional symptoms (90%). Systemic emboli occurs in 10±45% of myxoma patients. Cardiac myxoma should always be considered in the differential diagnosis of embolic stroke in adult patients because rapid diagnosis allows curative surgical resection and prevents further morbidity and mortality. heparin. An IVC filter was placed on day #2 because of large clot burden and concern for further pulmonary emboli. Given his right ventricular hypertrophy and likelihood of future pulmonary emboli, the patient was discharged on life-long therapy with coumadin. IMPLICATIONS/DISCUSSION: The incidence of pulmonary emboli in the US approximates 400,000 cases a year, with another 800,000 cases remaining undiagnosed. In one large series of patients with no pre-existing pulmonary disease, the incidence of dyspnea was 73%, pleuritic chest pain 66%, cough 37%, tachypnea 70%, tachycardia 30% and rales 51%. Laboratory, EKG and CXR findings are routinely nonspecific. The untreated mortality for PE is~30%, while the mortality in treated cases ranges from 2 to 8%. It is imperative that clinicians maintain a high index of suspicion for pulmonary emboli when evaluating the patient with dyspnea. Cerebral aneurysms are an extrarenal complication of APKD, with rupture causing 11% of deaths in theses patients. The prevalence of cerebral aneurysms increases with age, averaging around 10% in this population. Patients with a family history of subarachnoid hemorrhage or cerebral aneurysm are at greatest risk. In one study, asymptomatic cerebral aneurysms were found in 22% of patients with a family history, versus 5% of patients with no family history. Risk of rupture increases with size of the aneurysm and with location in the posterior circulation, with values ranging from 0.5 to 4% per year. As the size of the aneurysm is directly related to age, it is rare to detect an aneurysm in patients younger than age 30. Magnetic Resonance angiography is the recommended method of screening. APKD should be considered in patients with a strong family history of renal disease. Screening for cerebral aneurysms in patients with APKD is controversial. Many investigators recommend screening after the age of 30. Other authors recommend screening only for patients with a family history of cerebral aneurysms. Recommended screening intervals vary from every 3 to every 10 years. LEARNING OBJECTIVES: 1. Transient myocardial dysfunction has been described in many non-cardiac illnesses, including sepsis, subarachnoid hemmorhage (SAH), Acute Respiratory Distress Syndrome (ARDS), and and post operatively in non cardiac patients. 2. Mechanisms include oxygen supply and demand mismatch, circulating myocardial depressants, and calcium overload secondary to catecholamine surges. 3. Resolution is usually complete over ten to fourteen days, but elevated troponins may be a predictor of imcomplete recovery. CASE INFORMATION: The patient is a 20 year old previously healthy college student who presented to the emergency department with a three day history of increasing myalgias, sore throat, and diffuse erythematous rash with desquamation of her finger tips. She rapidly developed multi system organ failure, and required temporary mechanical ventilation, continuous veno-venous hemodialysis, and high doses of three pressors for blood pressure support. We had high clinical suspicion for toxic shock syndrome, which was later confirmed by positive TSST-1 antigen. On hospital day #3, she developed a wide complex tachycardia. Her ejection fraction was estimated at 15±20% by echocardiogram. Serial troponins peaked at 33.3. By the time of discharge, 2 1/2 weeks later, her ejection fraction had increased to 34±40%. Her ejection fraction was 30% at four weeks, and 30% at 4 months after initial presentation. She is maintained on a beta blocker, and an agiotensin converting enzyme inhibitor. IMPLICATIONS/DISCUSSION: Transient myocardial dysfunction has been described in a multitude of non-cardiac illnesses, including subarachnoid hemmorage (SAH), Acute Respiratory Distress Syndrome (ARDS), trauma, sepsis, and post operatively in non-cardiac surgical patients. It is a common complication of sepsis. A variety of non-coronary artery related mechanisms have been proposed, icluding oxygen supply and demand mismatch, circulation of various myocardial depressants such as cytokines, and high catecholamine surges leading to calcium overload. While transient myocardial dysfunction has been described in both ARDS and severe sepsis, there is only one other case report in the literature specifically related to toxic shock syndrome. The usual course of sepsis-related myocardial dysfunction is complete resolution over ten to fourteen days. The fact that this patient had an elevation in troponin, and that she still had a depressed ejection fraction four months after presentation, suggests that she may have experienced some degree of ischemic myocardial injury. The magnitude of the persistence of the myocardial insult attests to thd dangers of toxic shock syndrome, even in patients who recover from the acute sepsis and respiratory failure. A 52 year old homeless male presents with multiple complaints. He last saw a doctor ten years ago. His main complaints include right hand numbness and weakness, particularly while he's working. Additionally, he reports occasional dizziness and headaches. On further review of systems, he describes claudication symptoms, difficulty swallowing, especially solid foods, but denies weight loss. His past medical history includes diabetes and hypertension. However, he last received medications over ten years ago. He does drink alcohol daily and smokes cigarettes. On exam, his blood pressure reads 150/100 in the left arm and 130/70 in the right arm. His heart rate is 76, respiratory rate is 6, and he is afebrile. Significant findings on exam include right carotid, right subclavian, and right and left femoral bruits. He also has diminished pulses in the right radial and right brachial arteries, as well as decreased pulses in his feet. An EKG suggests enlarged left ventricle hypertrophy. He eventually received an angiogram, specifically to see if his subclavian bruits and right hand symptoms were significant. His right subclavian artery was aberrant and appeared to wrap behind his esophagus. He also had a severe stenosis of his right subclavian artery proximal to the right vertebral take-off. Furthermore, an exam of the lower extremities revealed severe stenosis in his right iliac artery that received angioplasty. Given the stenosis on angiogram and evidence of flow-reversal, it appeared that subclavian steal syndrome accounted for several of his symptoms, such as the dizziness, headaches, right hand claudication, and exam findings. Coincidentally, the aberrant subclavian seemed to explain his symptoms of dysphagia. Although the diagnosis of subclavian steal syndrome can be deduced by his history and exam, the aberrant subclavian was unexpected, but along with his stenosis, accounts for many of his symptoms. IMPLICATIONS/DISCUSSION: DISCUSSION: Subclavian stenosis can cause symptoms related to subclavian steal. The vertebral artery on the side of the stenosis creates retrograde blood flow from the brain stem by essentially stealing blood from the contralateral vertebral artery. The prevalence of an aberrant right subclavian has been reported as 0.5%-2.9%. The finding of dysphagia has also been reported. This is termed dysphagia lusoria, and was originally described in the 18th century. It translates as jest of nature. The treatment is surgical resection if it causes significant symptoms. He admitted to intravenous drug use, using his right thigh for access. His temperature was 101 F, pulse 110, blood pressure 90/60. His right thigh was erythmatous, edematous and tender, with 10  8 centimeter posterolateral area of fluctulance. The rest of the exam was unremarkable. Labs: WBC 50,000 (93% neutrophils), hemoglobin 7.1, CPK 104, creatinine 5.9. Blood and wound cultures were drawn. MRI showed necrotizing fascittis of posterior/lateral thigh with overlying cellulits, no osteomylelitis. Surgical exploration revealed necrotic skin and subcutaneous tissue extending down to muscle and fascial level, involving half the circumference of thigh. Wide excisional debridement of thigh was performed. Blood and wound cultures grew group A streptococcus sensitive to cefazolin. After antibiotic treatment and two subsequent debridements, the patient improved. Upon discharge, his WBC and creatinine were normal. IMPLICATIONS/DISCUSSION: There are two types of necrotizing fascittis. Type one, which is caused by mixed aerobic and anaerobic bacteria, is associated with diabetes mellitus, cervical necrotizing fasciitis, and fournier's gangrene of perianal area. Type two necrotizing fasciitis, due to group A streptococcus, is associated with history of blunt trauma, varicella, intravenous drug use, penetrating injury and possably with nonsteroidal antiinflammatory drugs. Necrotizing fasciitis should be suspected in patients with severe unexplained pain increasing over time, skin changes (erythema, red-purple discoloration, blisters, bullae), fever, systemic toxicity, myalgias, diarrhea and anorexia. Lab tests show leukocytosis with marked left shift and elevated CPK and creatinine. Imaging studies are most helpful in type one, where gas formation occurs. MRI may overestimate deep tissue involvement. Thus, the only way to make a definitive diagnosis is via surgical exploration. Surgery is indicated for severe pain, toxicity, fever and elevated CPK. Early surgery may improve the outcome, but mortality rates are high even in cases of optimal therapy. Repeat explorations and debridement should be performed until all necrotic tissue has been removed. Additional therapies include antibiotics and intravenous immune globulin.\nNON-HODGKIN'S LYMPHOMA PRESENTING AS FLANK PAIN. K.Y. Miskel 1 ; 1 Hospital of the University of Pennsylvania, Philadelphia, PA (Tracking ID #73961)\nLEARNING OBJECTIVES: 1) Recognize an atypical presentation of lymphoma 2) Recognize that systemic non-Hodgkin's lymphoma is a common HIV-related malignancy CASE INFORMATION: A 33 year old healthy male with no significant past medical history presented with three weeks of bilateral flank pain. His flank pain was intermittent but had progressively worsened, with radiation to his groin. The pain was associated with movement. He denied fevers/chills, nausea/vomiting, dysuria, or hematuria. Social history was negative for tobacco, alcohol, or illicit drugs. He was homosexual and involved in a long-term monogamous relationship. He had a family history of nephrolithiasis. On initial exam, he was afebrile with a benign abdominal exam, no costovertebral angle tenderness, a normal rectal exam, and guiac negative stool. A urinalysis was significant for +++blood and no leukocyte esterase or protein.\nAn abdominal xray showed a normal bowel gas pattern with no renal calculi. He was presumed to have radiolucent nephrolithiasis and was advised to follow-up closely. Five days later, he presented to the emergency room with unremitting flank pain, new suprapubic pain, dysuria and fevers to 103±104 F. His only medications were ibuprofen and percocet (prescribed recently for his flank pain). On exam, he appeared uncomfortable, had a T 99.2 F, oral thrush, and bilateral cervical/axillary lymphadenopathy. His abdomen was soft, mildly distended, and diffusely tender to palpation, particularly in the suprapubic area. He had scattered papules with central umbilication on his face, buttocks, and thighs. Laboratory studies were significant for a WBC 5.6, hemoglobin 11.9, creatinine 2.6, AST 124, ALT 158, alkaline phosphatase 627, and total bilirubin 1.5. Abdominal CT scan showed bulky retroperitoneal and pelvic lymphadenopathy, bilateral hydronephrosis, and a circumferentially thickened bladder wall. Bone marrow biopsy and bladder wall biopsy confirmed the diagnosis of stage IV immunoblastic non-Hodgkin's lymphoma involving the bone marrow, GI tract, liver, spleen, and bladder. HIV was positive with a CD4 count of 138 and a viral load of >500,000. IMPLICATIONS/DISCUSSION: Non-Hodgkin's lymphoma occurs in 10-30% of AIDS patients, the most common type being high-grade diffuse large B cell (immunoblastic variant) or Burkitt's-like. Patients usually present with stage IV disease and frequently have involvement of the bone marrow, CNS, liver, and gastrointestinal tract. Although HIV associated systemic lymphoma frequently presents with rapid nodal enlargement and B symptoms, it has no pathognomonic features. This previously healthy male had HIV risk factors as well as molluscum contagiosum and oral thrush that suggested the diagnosis of HIV infection. In addition to chemotherapy, immune reconsitution with highly active anti-retroviral would be necessary for effective treatment.\nALPHA-INTERFERON THERAPY AND DELIRIUM. D. Misra 1 , S. Young 1 , C. Schleupner 1 ; 1 New Hanover Regional Medical Center, Wilmington, NC (Tracking ID #75477) LEARNING OBJECTIVES: Recognize delirium as one of the neuro-psychiatric adverse effects of alpha interferon therapy. We intend to share our experience because it may pose as a diagnostic challenge for clinicians. CASE INFORMATION: We present the case of a 57 yr old caucasian male who had previously been diagnosed with malignant melanoma. He had undergone local excision and was found to have positive lymph nodes. He was started on adjuvant alpha interferon therapy, initially at a dose of 40 million units a day which he could not tolerate primarily because of constitutional symptoms. His symptoms included severe arthralgias, myalgias and headaches. He received this dosage for only 4 days, which was then discontinued and subsequently started back on a reduced dose of 20 million units a day after a drug free interval of one week. He tolerated the lower dose without any side effects. The following week, he was started back on 40 million units a day of interferon which he received for five days. Within a couple of days, pt was experiencing severe headaches along with myalgias and arthralgias. This became worse and patient presented with delirium, agitation and combativeness. He required intravenous sedation and eventually endotracheal intubation for airway protection. His workup including blood counts, basic metabolic panel; urinalysis, drug screen, CT scan of the head and CSF examination were all within normal limits. Serum transaminases were mildly elevated but this was unchanged since the beginning of interferon treatment. His stay in the intensive care unit lasted 24 hours. The following day, he was back to his baseline cognitive level and was discharged home. IMPLICATIONS/DISCUSSION: Alpha interferon is a glycoprotein produced by leukocytes which has broad spectrum antiviral, immunologic and antiproliferative properties. Therefore, it is commonly used in treatment of hairy cell leukemia, hepatitis, renal cell carcinoma and malignant melanoma. The spectrum of side effects is wide and depends on various factors including the dose of the drug, age of the patient and the type of interferon. Our literature search revealed very few case reports of interferon induced delirium. Neuro-psychiatric adverse effects occur with doses higher than 30 million units per day. The temporal relationship of the onset of delirium with high dose interferon treatment in our patient stands out prominently. We believe that this occurrence should be brought to the notice of physicians prescribing interferon so that patients presenting with neuro-psychiatric manifestations are diagnosed and managed appropriately. A 51 year-old man with an ischemic cardiomyopathy presented to the emergency department after experiencing multiple shocks from his automatic implantable cardiac defibrillator (AICD). In the ED, the patient developed ventricular tachycardia at a rate of 162 bpm with a BP of 132/102. His AICD began antitachycardia pacing and DC countershocks without success. The patient was loaded with amiodarone and placed on a 2mg/ min infusion of amiodarone. Sinus rhythm was restored. Lab values included normal liver function tests and a creatinine of 2.1. The patient stabilized. After 24 hours, the amiodarone infusion was reduced to 1 mg/min. Twelve hours later, the patient developed confusion and severe dyspnea. Lasix was given without improvement. The patient developed fever, hypotension, and became lethargic. The exam showed a quiet, tender abdomen. The HCO3 had fallen to 6 meq/L, and the creatinine had risen to 3.2 mg/dl. The ALT was 1947 U/L, the AST was 2629 U/L, the bilirubin was 4.2, and the PT was 16.9s (INR 2.8) . The patient was diagnosed with fulminant hepatic failure. Dopamine and a bicarbonate infusion were begun. Antibiotics were started. The patient was intubated and vitamin K was given. Amiodarone was considered to be a possible cause of the fulminant liver failure. Thus, 72 hours after admission, the amiodarone infusion was stopped despite persistent ventricular ectopy. The patient's status declined and he died on the fifth day from complications from a liver biopsy. IMPLICATIONS/DISCUSSION: Fulminant hepatic failure develops in less than 2 weeks after an insult to the liver. The diagnosis of liver failure should be entertained in the presence of encephalopathy associated with jaundice, coagulopathy, acidosis, fever and hypotension. In this patient, clinical suspicion was confirmed by the rapid change in the liver function tests. The viral hepatidities and drug/toxin exposure are the most common etiologies of fulminant liver failure. In this patient, the leading considerations were ischemic and drug/toxin exposure. A review of the literature revealed at least twenty fatalities from acute hepatic failure following the administration of high dose parenteral amiodarone over the last twenty years. The chronology of exposure and onset of hepatic failure were consistent with amiodarone induced hepatotoxicity in this patient. Regardless of the cause, the treatment of acute liver failure requires removal of the exposure and supportive care for the expected complications. Despite aggressive support, mortality remains at 60-90% without transplantation. LEARNING OBJECTIVES: To recognize that brown recluse spider bites may cause pyoderma gangrenosum and pathergy due to persistent neutrophil mediated injury. CASE INFORMATION: A 47 year-old woman presented with a six month history of recurrent, painful, nonpruritic ulcers on the dorsa of both wrists and the right calf. Past history revealed that six months prior, while cleaning debris from an old tavern, she was bitten on the hand by a spider. The spider was captured and identified as a brown recluse spider. Within hours she was ill with nausea, vomiting, malaise and fever. She improved with supportive treatment. The lesion cleared but weeks later, began to reoccur following minor trauma such as that caused by mosquito bites. On physical examination we discovered pyoderma gangrenosum like lesions that were confirmed by biopsy. Long-term therapy with dapsone was started. IMPLICATIONS/DISCUSSION: Pyoderma Gangrenosum was first described by Brunsting et al in 1930. The etiology is unknown in fifty percent of the cases but associated with Ulcerative Colitis and Behcet's Disease. Spider bites rarely lead to pyoderma gangrenosum and even more rarely lead to recurrent lesions and pathergy. Brown recluse venom has been purified and studied and is known to persist in some wounds for a relatively long time. The precise mechanism of the lesions is not known although neutrophil inhibitors such as nitrogen mustard and dapsone can mitigate or prevent the reaction. The development of new lesions at distant sites from the inciting lesion is unexplainable and suggests an immune mediated response and not a local reaction. The observed clinical response to dapsone suggests an ongoing neutrophil mediated injury. Currently dapsone is being carefully tapered. It is unknown if the lesions will persist and argue for a permanent ongoing cell mediated injury or cease and suggest a self limited condition as remaining venom is cleared.\nPLASMAPHERESIS FOR THE TREATMENT OF HYPERTRIGLYCERIDEMIA. C. Carosella 1 , C. Karmen 1 , S. Warshafsky 1 , Y. Murray 1 ; 1 New York Medical College, Valhalla, NY (Tracking ID #76460) LEARNING OBJECTIVES: To recognize the value of plasmapheresis in the treatment of triglyceride-induced acute pancreatitis. CASE INFORMATION: A 50-year-old Hispanic woman with a history of insulin-dependent diabetes mellitus, coronary artery disease and hypertriglyceridemia presented to the emergency room with severe abdominal pain, nausea, vomiting and poor oral intake all gradually worsening over the prior two weeks. She had been treated with a combination of atorvastatin, gemfibrozol, and niacin with only marginal success in controlling her lipid levels. Physical exam revealed an ill-appearing woman complaining of abdominal pain. Bowel sounds were hypoactive, and the abdomen was diffusely tender. The serum was grossly lipemic. Laboratory studies revealed a glucose of 1321 mg/dl, amylase of 1008 mg/dl, lipase 2001 mg/dl, and triglycerides 9198 mg/dl. CT scan of the abdomen was consistent with acute pancreatitis. Her condition rapidly deteriorated with respiratory failure, requiring ventilator support, and renal failure, requiring dialysis. She was transferred to the University Hospital for plasmapheresis. One plasmpheresis treatment with continuous infusion of heparin lead to a substantial and sustained reduction of triglyceride level to less than 150 mg/dl. The patient steadily improved with decreased dependence on the ventilator, increased urine output, and decreased swelling of the pancreas. Unfortunately, the patient suffered a cardiac arrest on the sixteenth hospital day. IMPLICATIONS/DISCUSSION: Plasmapheresis has been used to treat hypertriglyceridemia when adequate diet and drug therapy fail. In this case plasmapheresis was highly effective in removingthetriglyceridesand mayhave ledto thepatient'ssteady,albeit temporary,improvement. (1) to recognize hyperammonemia due to acute valproic acid intoxication (2) to manage hyperammonemia due to acute valproic acid intoxication by understanding its mechanism.\nCASE INFORMATION: The patient is a 42 year-old male who ingested 90 500 mg tablets of valproic acid following an argument with his wife. The exact time of ingestion was unclear, but was felt to be 6 to 8 hours prior to presentation. The patient's wife assumed that he was sleeping, but when found to be unresponsive again several hours later, he was brought to the emergency department. On physical examination, the patient was unresponsive, hypotensive, and without hyperreflexia or clonus on neurological examination. Laboratory values revealed normal liver function tests, hypocalcemia, valproate level of 1157 (ref 50±150 ug/ml), and an ammonia level of 270 (ref 9±33 umol/L). The patient's electrocardiogram was notable for QT prolongation. CT scan of the head and lumbar puncture were unremarkable. Intravenous fluids were administered and dopamine was initiated for blood pressure support. The patient's respiratory status declined, he was unable to protect his airway and therefore intubated. Carnitine was administered for the hyperammonemia on a daily basis. The valproic acid and ammonia levels declined over the next 48 hours and his mental status improved. He was extubated after approximately 72 hours. The patient did well and was transferred to psychiatry. IMPLICATIONS/DISCUSSION: Hyperammonemia results when valproic acid is metabolized to propionic acid, which inhibits carbamyl phosphate synthetase, the first enzyme involved in the urea cycle. Valproic acid also binds to carnitine, a molecule important in the metabolism of long-chain fatty acids and ketoacid analogues of various amino acids, thereby depleting serum concentrations of carnitine. Valproic acid also causes increased renal production of ammonia by reducing the synthesis of glutamine. Administration of exogenous carnitine is thought to decrease ammonia levels by binding to valproic acid, and also by relieving the inhibition of carbamyl phosphate synthetase and thus urea synthesis. Management of valproic acid intoxication is largely supportive. Patients who present early may benefit from gastric lavage and a single dose of activated charcoal; however, multiple-dose activated charcoal does not increase the elimination of valproic acid. Other interventions may involve blood pressure support with intravenous fluids and vasopressors, correction of electrolyte abnormalities, and correction of acid/base disorders (commonly an anion gap metabolic acidosis). Mechanical ventilation may be necessary in patients who require airway protection. In the majority of cases, this management strategy results in favorable outcomes. Hemodialysis and charcoal hemoperfusion are additional treatment options. Typically, extracorporeal removal is employed in patients with renal abnormalities, hypotension refractory to all supportive measures, severe metabolic abnormalities, active seizure, or those who are persistently comatose. However there are no controlled trials that demonstrate an improvement in outcome with these measures. year old female initially presented to her primary physician 5 years ago with a blood pressure of 159/98. Repeat readings were similar and she was started on hydralazine 25 mg. There was a modest effect from hydralazine, but control was never acheived. Four months later hydralazine was changed to losartan 50 mg. No significant lowering was obtained. Losartan was increased one month later to 100mg and two months later hydrochlorothiazide 25 mg was added. Her blood pressure stabalized to 140/90 for 6 months. Then a gradual increase in her blood pressure was noticed, so diltiazem was added. Her physical exam and lab values were unremarkable. Due to failure of multiple antihypertensive medications,a magnetic resonance arteriogram of the renal artery was conducted which revealed renal artery stenosis. An angiogram was conducted showing segments of stenosis and dilatation of the right renal artery, consistent with the diagnosis of fibromuscular dysplasia. Renal artery angioplasty was performed, and follow up blood pressure normalized on two medications. IMPLICATIONS/DISCUSSION: This is an elderly female with hypertension refractory to medications. A secondary cause was sought out with an imaging study, which showed a unilateral fibromuscular dysplasia of the right renal artery. It is unusual for a women in this age group to present with this type of secondary hypertension. Patients with fibromuscular dysplasia respond well to angioplasty. LEARNING OBJECTIVES: 1. The reader should be able to list three common causes for hypophosphatemia. 2. The reader should be able to explain why phosphate repletion is typically not be necessary for hypophosphatemia from respiratory alkalosis. CASE INFORMATION: Hyperventilation can precipitate life-threatening alkalosis and severe hypophosphatemia that does not require phosphate repletion. A 36 yo female who was status post cadaveric tracheal transplant and multiple stent placements presented with suspected stent re-stenosis, complaining of dyspnea, increased work of breathing, and inability to clear secretions. Her respiratory rate was 30±34 with normal oxygenation. Emergent bronchoscopy revealed only minimally occlusive concretions, however peri-procedure labs revealed an arterial blood pH of 7.71 and a phosphate of 0.6 mg/dL. The patient reported weakness and paresthesias in all four extremities plus arthralgias in her hands. The patient was started on a NaPO4 infusion @ 0.6 mmol/kg over 6 hrs. She was given supportive care and reassurance for her hyperventilation. After only three hours, the PO4 was 3.3 mg/dL and the infusion was stopped. Repeat ABG showed a pH of 7.49, with improvement in pCO2 from 14 to 27 mmHg. The patient's symptoms resolved. IMPLICATIONS/DISCUSSION: Severe hypophosphatemia, defined as a serum PO4 <1.5 mg/dL, has been reported in 0.22±2.15% of adult hospital admissions. It occurs more commonly in alcoholics and malnourished patients and has a variety of causes, including respiratory alkalosis. In respiratory alkalosis, carbon dioxide readily diffuses from the intracellular space resulting in higher intracellular pH. This activates glycolysis and the formation of phosphatecontaining compounds. Phosphate is used from the circulating inorganic phosphate pool, depleting extracellular stores. This process induces relative hypophosphatemia. This contrasts with metabolic alkalosis where excess extracellular bicarbonate is only poorly diffusible and does not significantly raise intracellular pH. Severe hypophosphatemia can be a lifethreatening disturbance but may not represent total body phosphate depletion or require supplementation. An understanding of the basic physiology guides therapy. 67 year old male initially seen in the emergency room five days before admission for right lower extremity redness, pain and swelling. He was given pain medication and sent home. He returned the following day and two days later with persistent symptoms and was given appropriate oral antibiotics for presumed cellulitis. An ultrasound showed no thrombosis. After his fourth ER visit he was admitted for intravenous antibiotics. Additionally, over the past three months he had noted bilateral lower extremity pain with walking over one block. Work up to date included normal resting and exercise ankle/brachial indices a CT scan of the lumbar spine, which showed disc bulges at L3-4 and L4-5 with mild spinal stenosis. When we met him, he complained of severe pain despite oral narcotics and also endorsed difficulty with his gait over the past week. On physical exam, temperature was 98.60 and the right lower extremity had diffuse anterior erythema without distinct borders. There were no breaks in the skin but onychomycosis was present on bilateral toes. There was 2+ edema on the right and none on the left. Dorsalis pedis pulse was noted only by doppler on the right, but was intact on the left. Neurologic exam revealed significant difficulty with dorsiflexion of the right foot which was presumed to be secondary to pain or possibly related to his known spinal stenosis. Labs revealed a normal white blood cell count, elevated C-reactive protein, small blood in the urine with 0±5 red cells and a creatinine slightly above his baseline. Plain radiographs were normal. Broad spectrum intravenous antibiotics, elevation and pain control were initiated at admission. After two days, the redness and swelling improved but a foot drop and pain persisted. Neurology felt his foot drop could be consistent with his known disc disease but also suggested surgical evaluation. Anterior compartment syndrome was subsequently diagnosed based on a direct compartment pressure reading of 45 mmHg. Because of the delay in diagnosis, the fact that pulses were present and that there was no tissue necrosis or abscess seen on subsequent scanning, the decision was made not to operate. The patient's pain and swelling gradually resolved over weeks but he was left with a permanent foot drop. IMPLICATIONS/DISCUSSION: Compartment syndrome is a surgical emergency that must be detected early to avoid potential serious sequelae. Causes are many and include closed trauma (most commonly), vascular injury and reperfusion, burns, prolonged surgery or traction, overexertion/exercise, and infection. It occurs most commonly in the arm or leg but can be seen in the foot, abdomen, retroperitoneum and mediastinum. Recognition depends on clinical suspicion and can be remembered as the five P's: pain (severe and out of proportion to injury); pulselessness, paralysis (excluding concomitant nerve injury); paresthesia and pallor. Laboratory may show myoglobin in the urine or elevated creatine kinase. Imaging studies may help rule out other causes but direct measurement of compartment pressures are needed for diagnosis. Also well described is the syndrome of chronic compartment syndrome, which is typically thought of as an overuse injury in runners but remains as an interesting possible explanation for this patient's claudication history. LEARNING OBJECTIVES: Early recognition and treatment of Alcohol Withdrawal Syndrome (AWS) may reduce perioperative morbidity and mortality. Intravenous Ethanol (IVE) is still used for the peri-operative management AWS. This case illustrates the importance of 1) identifying patients who require AWS treatment and using evidence-based treatments early to prevent AWS peri-operative complications, 2) exploring the use of alcohol to treat perioperative AWS, and 3) discussing optimum treatments of AWS with patients and family prior to surgery. CASE INFORMATION: T.O. is a 65 year-old man with history of hypertension, congestive heart failure and peripheral vascular disease with bilateral thigh claudication admitted for aortobifemoral bypass. Initial documentation of alcohol use consisted of an admission nursing note stating that the patient drank alcohol. After an uncomplicated surgery, he was successfully extubated and on post-operative day (POD) 2 was transferred to a general surgical bed. On POD 3, he became increasingly agitated and combative. AWS was suspected and Lorazepam 1±2 mg PRN was ordered. Over the next three days he did not improve and was returned to the SICU for re-intubation for airway protection and IV benzodiazapine treatment. After being informed of his clinical condition, the patient's son requested his father receive alcohol to prevent delirium tremens since his father had not consented to``detoxification''. A 10% ethanol drip was begun to 100 cc/h on POD 8 and on POD 9 he was successfully extubated. Although he became more alert and interactive, attempts to wean his IVE caused increased agitation. On POD 11 he was transferred to a general surgical bed, his IVE was discontinued, and he was ordered 12 ounces beer PRN. On POD 12, he continued to be confrontational and was discharged to home. IMPLICATIONS/DISCUSSION: AWS is a complex constellation of signs and symptoms related to autonomic hyperactivity in alcohol dependent patients whose alcohol intake is decreased or discontinued. Severe AWS includes life-threatening delirium tremens that may be exacerbated by the stress of surgery. Pre-or peri-operative detoxification from alcohol may also increase surgical morbidity. Symptom triggered, appropriate dosing of benzodiazapines are effective first line treatment for AWS. However, intravenous and oral alcohol use continues to be used in the perioperative period. Although multiple case reports exist detailing the use of IVE or oral alcohol to prevent withdrawal symptoms, no clinical trials exist to demonstrate its efficacy in management of peri-operative AWS. The use of IVE in the perioperative period is complicated by electrolyte disturbances, pancreatic complications, and difficulty in objectively measuring treatment efficacy. Use of oral alcohol may also reinforce unhealthy outpatient alcohol drinking behavior. Insufficient evidence-based studies exists to guide clinicians regarding the optimum management of peri-operative AWS and until such time, alcohol treatments will continue to be used.\nCenter, San Jose, CA (Tracking ID #75786) LEARNING OBJECTIVES: To diagnose nephrotic syndrome in a diabetic. To assess the clinical complications of nephrotic syndrome. To recognize the clinical challenges of the obese patient. CASE INFORMATION: A 52 year old morbidly obese male presented to his primary care physician with four days of facial and upper extremity edema. He had developed orthopnea, paroxysmal nocturnal dyspnea, and dyspnea with exertion. His past medical history included poorly controlled diabetes, chronic lower extremity edema, and untreated obstructive sleep apnea. An initial chest CT demonstrated pleural effusions and a soft tissue mediastinal mass thought to be compressing the superior vena cava. Mediastinoscopy of the mass demonstrated no tissue findings; a repeat chest CT, with dye injected in both arms simultaneously, revealed the mass was likely lipomatosis. Further workup of the pleural effusion included an unremarkable echocardiogram and an ultrasound-guided thoracentesis. The pleural effusion was transudative and negative for cytology. The patient's urine revealed 3+ proteinuria and oval fat bodies, and a spot urine protein/creatinine ratio demonstrated over 4 grams of protein.\nThese findings, along with hypoalbuminemia, were consistent with nephrotic syndrome. The patient's symptoms improved with furosemide diuresis. However, the patient was rehospitalized a week later, complaining of acute shortness of breath. He was already known to be hypoxic, probably from obstructive sleep apnea. A ventilation perfusion scan was performed, and demonstrated a new lingular mismatch of intermediate probability for a pulmonary embolism. The patient stayed in the hospital until five days of oral coumadin overlapped with a heparin drip, as his obesity prevented accurate dosing of low-molecular-weight heparin. IMPLICATIONS/DISCUSSION: Diabetes is a common cause of nephrotic syndrome. The diagnosis can be made by quantification of urine protein excretion; oval fat bodies suggest hyperlipidemia from a fall in oncotic pressure and decreased clearance of very low density lipoproteins. Clinical symptoms include peripheral edema, pleural effusions, and hypercoagulable conditions like pulmonary embolism. The pathophysiology of these clinical symptoms may be due to sodium retention from renal disease and hemostatic abnormalities such as urinary loss of antithrombin III. The diagnostic workup was made more difficult for this patient due to complicating conditions related to his morbid obesity, such as the lipomatosis and obstructive sleep apnea. As obesity increasingly becomes a public health epidemic, generalists need to recognize the extensive clinical challenges for the obese patient. LEARNING OBJECTIVES: 1) To discuss the differential diagnosis of hypoglycemia.\n2) To recognize that accidental sulfonylurea ingestion is a cause of hypoglycemia in adult nondiabetics.\n3) To recognize that polypharmacy issues are common and are not limited to a patient's own prescriptions. CASE INFORMATION: An 80 year old male on multiple medications presented with severe hypoglycemia. He had no prior history of diabetes and never used alcohol. During his initial hospitalization, the patient was found to be hypogonadal and diagnosed with cholangitis. Diagnosis was based on hypothermia, choledocholithiasis and ductal dilatation although blood cultures were negative and initially there was no leukocytosis. Cortisol levels were normal and no abdominal tumors were found by CT. Insulin levels and c-peptide levels were elevated. The patient's medications did not include any associated with hyperinsulinism. The patient's glucose normalized after two days of treatment with a glucose drip, antibiotics, and stone extraction with sphincterotomy. Upon discharge, the leading diagnosis was sepsis-induced hypoglycemia. The patient was rehospitalized five months later with another episode of hypoglycemia. A sulfonylurea level was sent and returned positive for glipizide. It was then discovered that the patient was erroneously taking his wife's glipizide. The patient has had no further episodes of hypoglycemia. IMPLICATIONS/DISCUSSION: The differential diagnosis of hypoglycemia includes hormone deficiencies, enzyme defects, acquired liver disease, malnutrition, sepsis, malignancy, medications, and hyperinsulinism. Hyperinsulinism can be caused by sepsis, insulinoma, autoantibodies and medications. These medications include exogenous insulin, sulfonylureas and other sulfa drugs, and also quinine and pentamidine. Hypoglycemia due to accidental oral sulfonylurea ingestion in non-diabetics is more common in the pediatric rather than the internal medicine literature. Drug-induced hypoglycemia should be considered whenever the patient has potential access to hypoglycemia-inducing agents. Polypharmacy is a wellrecognized cause of adverse drug events among the elderly. However, estimates of polypharmacy are limited to an individual's medications obtained by prescription or overthe-counter. It should be recognized that individuals with multiple medications may inadvertently consume medications other than their own.\nA CASE OF ASCITES AND UNILATERAL LEG SWELLING. B. Taqui 1 , D. Oxman 1 , L. Kaplan 1 ; 1 Temple University, Philadelphia, PA (Tracking ID #76436)\nLEARNING OBJECTIVES: 1. Recognize iliac vein compression (May-Thurner) syndrome as rare cause of left leg swelling in patient with negative venous dopplers 2. Recognize potential for endovascular damage and thrombosis resulting from condition 3. Recognize endovascular stenting as potential cure CASE INFORMATION: 38 year old Caucasian male with history of alcohol abuse presented with three days of abdominal pain and increasing abdominal girth. Serum lipase was markedly elevated and paracentesis showed ascitic fluid consistent with pancreatic ascites. He was started on total parenteral nutrition. He subsequently developed left leg and scrotal swelling. Lower extremity venous duplex imaging was negative for deep venous thrombosis, but contrast venography revealed compression of the iliac vein by the right iliac artery. The patient underwent endovascular stenting of the femoral vein with complete resolution of leg and scrotal swelling. His swelling has not recurred since then. His pancreatitis and ascites also gradually resolved. IMPLICATIONS/DISCUSSION: The potential for obstruction of the left common iliac vein by the overlying right common iliac artery against the pelvic brim was first noted by Virchow. Later, May and Thurner described the``iliac compression syndrome.'' In this patient, it was believed that the ascites coupled with intravascular volume burden of parenteral nutrition led to the patients symptoms. The syndrome, which can be acute or chronic, predisposes patients to thrombosis. Thus, contrast venography should be considered in patients with unilateral left lower extremity swelling and negative duplex imaging. Therapy with endovascular stenting of femoral vein offers patients the chance of cure. angiogram showed a beaded segmental narrowing involving the right internal carotid artery from the bifurcation to the base of the skull. Fibromuscular dysplasia was considered, but a repeat angiogram 3 days later showed that the vessels had returned to normal. The patient was discharged on a calcium channel blocker and coumadin. IMPLICATIONS/DISCUSSION: Stroke in young adults is usually due to cardiac embolism, dissection of arteries, vasculitis, and conditions associated with hypercoagulable states. Reversible cerebral segmental vasoconstriction' also known as Call-Fleming syndrome is a rare cause of stroke or TIA and is characterized angiographically by multiple areas of reversible segmental arterial narrowing. Symptom onset can be spontaneous, but has also been associated with pregnancy, migraine, and use of sympathomimetic drugs. Most patients present with headache. Symptoms can last from few minutes to 6 months. The mechanism of vascular narrowing is not understood. Repeat angiogram may be needed to demonstrate the reversible nature of the angiographic changes. LEARNING OBJECTIVES: 1. Consider scurvy in the differential diagnosis when patients appear to have vasculitis, especially in populations at high risk. 2. Appreciate that early diagnosis, facilitated by taking a good dietary history, is important, as scurvy is potentially fatal. CASE INFORMATION: A 54-year-old schizophrenic man presented to rheumatology clinic with a vasculitis-like skin rash, joint swelling and tenderness, edema of a lower extremity and poor dentition. He was a chronic smoker and had a history of alcoholism. He was not on any medication other than haloperidol on a prn basis. An extensive work up for vasculitis was negative. The patient was referred for biopsy of his skin lesions and scurvy was clinically suspected and confirmed with a low vitamin-C level. Biopsy did not show evidence of vasculitis. The patient's symptoms and signs resolved rapidly with oral vitamin-C. IMPLICATIONS/DISCUSSION: Scurvy is a disorder caused by vitamin C deficiency. Its protean manifestations include petechiae, ecchymoses, hyperkeratoses, arthralgias, malaise, fatigue, impaired wound healing, hair with corkscrew deformity and swollen, red purple gums. Our case is unusual because our patient presented predominantly with joint symptoms and a vasculitic-appearing rash. The diagnosis was missed by the rheumatologist, but was later made based on a dermatologist's suspicion. Scurvy is rare in industrialized nations but can occur in certain high-risk groups because of poor intake of vegetables and fruits or an increased need for vitamin C. High-risk groups include the elderly, pregnant and lactating women, infants, the urban poor, the malnourished, food faddists, alcoholics and those with cancer. Scurvy has also been reported in psychiatric patients with depression, schizophrenia and anorexia nervosa. Schizophrenic patients are predisposed to abnormal dietary patterns, food fads and self neglect which subject them to a risk of scurvy. Our patient was at risk because of underlying schizophrenia, a history of alcoholism, and malnutrition, which predisposed him to poor intake. His smoking history probably contributed to his deficiency by increasing vitamin C needs. LEARNING OBJECTIVES: Recognize that pulmonary edema is one of the many causes of localized upper lobe infiltrates, especially when patients at risk for heart failure have been in dependent positions for prolonged periods of time. CASE INFORMATION: A 52-year old man with type-2 diabetes and severe atherosclerotic cardiovascular disease, who had had a toe amputated for an infected ulcer two days before, was admitted with a 3-day history of orthopnea and dyspnea on exertion. He had been on prolonged bed rest to facilitate healing of his foot. On admission he was hypoxic, with coarse crackles at the apices of the lungs and decreased breath sounds at the lung bases. There was no elevation of JVP and the rest of the cardiovascular exam was unremarkable. CXR showed upper lobe alveolar shadows bilaterally with bilateral pleural effusions and Kerley B lines. Spiral CT of the chest done to rule out pulmonary embolism showed bilateral pleural effusions and bilateral nodular upper lobe infiltrates without evidence of embolism. Atypical pulmonary edema was suspected and the patient was propped up in bed, and given furosemide. His symptoms and CXR abnormalities resolved in 2 days. Natriuretic peptide was found to be markedly elevated at 857 pg/ml and an echocardiogram confirmed LV dysfunction with an EF of 20%. The atypical presentation of this patients pulmonary edema was explained by a gravitational effect on the pulmonary circulation, caused by his prolonged assumption of the Trendelenberg position as part of the management of his diabetic foot problems. IMPLICATIONS/DISCUSSION: Pulmonary edema presents classically with bilateral parahilar alveolar shadows producing a bat wing appearance on CXR. In situations characterized by gravitational effects or alterations in lung perfusion, as in COPD or pulmonary embolism, pulmonary edema may present in less typical ways. Acute mitral regurgitation has been reported as causing right upper lobe pulmonary edema and unilateral pulmonary edema has been attributed to the gravitational effects of posture. These atypical presentations can be confused with pneumonia and aspiration. To make the correct diagnosis in patients with atypical CXR's, clinicians must rely on other clinical and radiologic signs of heart failure and take a careful history. Workup revealed pulmonary infiltrates, anemia with left-shift, and a sputum KOH preparation demonstrating round, non-staining elements felt to be consistent with blastomycosis. After four days of intravenous amphotericin, the patient developed progressive pulmonary infiltrates requiring ventilatory support. Subsequent evaluation with broncho-alveolar lavage revealed alveolar hemorrhage and red cell ghosts within macrophages; these findings were consistent with a vasculitic rather than an infectious disease process. Eleven years later, the patient presented with hemoptysis, fatigue and arthralgias leading to initiation of immunosuppresants for presumed autoimmune disease. After discharge, however, sputum cultures grew Blastomyces dermatitidis, and followup chest CT revealed bibasilar consolidation with cavitary changes prompting a reduction in steroid dose and initiation of anti-fungal therapy. IMPLICATIONS/DISCUSSION: The literature contains multiple cases of blastomycosis mimicking a variety of illnesses, from neoplastic to autoimmune disease. Certain confounding factors can limit the differential diagnosis: (i) the indolent course of blastomycosis with nonspecific presenting symptoms may catch the unwary off-guard, (ii) blastomycosis may present in a manner similar to a variety of disease processes, (iii) and finally, blastomycosis may be superimposed upon another illness, clouding both diagnosis and management.\nACUTE TUBULOINTERSTITIAL NEPHRITIS ASSOCIATED WITH CLOPIDOGREL. N. Palanichamy 1 , C. Kumar 1 ; 1 Oakwood Healthcare System, Dearborn, MI (Tracking ID #76611) LEARNING OBJECTIVES: To describe clopidogrel induced acute renal failure, a previously unreported drug reaction. CASE INFORMATION: An 89 year-old white male presented with hematuria and acute renal failure. His Medications were aspirin, plavix, metaprolol, prinivil, lasix and flomax. He was placed on clopidogrel two weeks prior. He stopped the drug the day before admission. His creatinine level was 3.9 mg/dl (baseline creatinine 1.3 mg/dl). Urinalysis showed proteinuria, leukocytes and erythrocytes. 24-hour protein excretion was 756 mg. A renal ultrasound showed no obstruction. Over the course of the next week his renal function deteriorated (creat 8.9 mg/ dl). Renal biopsy showed a widened, edematous interstitium with moderate lymphocytosis, plasma cells and regenerative tubules and was compatible with tubulointerstitial nephritis (AIN). Dialysis was started and the patient was placed on steroids. With treatment the creatinine decreased to 2.6 mg/dl, the dialysis stopped and the steroids tapered. Based on the history and presentation clopidogrel was thought to be the etiology of the nephritis in this case. IMPLICATIONS/DISCUSSION: To our knowledge, this is the first case of clopidogrel induced acute tubulointerstitial nephritis. The rapid deterioration in the renal function two weeks after starting clopidogrel and the absence of confounding factors suggests the diagnosis in the presence of the biopsy findings. Clopidogrel affects platelet aggregation by inhibiting the ADP receptor and the subsequent ADP-mediated activation of the glycoprotein GPIIb/IIIa complex. The precise mechanism of the renal dysfunction is unknown but, like ticlopidine, a chemically similar drug with known adverse renal effects, may be caused by cell-mediated injury. Guerciolini et al. (1985) demonstrated a positive lymphocyte stimulation test in explaining agranulocytosis associated with ticlopidine. The other known mechanism of drug induced AIN, antibody mediated injury and circulating immune complexes are less likely causes insofar as the immunoflorescent studies in our case were nonspecific. In addition to the more commonly known side effects of clopidogrel physicians now need to realize that in rare instances it may cause renal dysfunction. LEARNING OBJECTIVES: Think about malignancy in the differential diagnosis of leukemoid reaction. CASE INFORMATION: A 68 year old Caucasian male with no significant past medical history presented with complaints of swelling of both legs, vague abdominal pain, watery diarrhea and increased urinary frequency of 6 weeks duration. He also reported a weight gain of 50 pounds over the past 6 months. On physical examination, he was afebrile, pulse was 81/min and blood pressure was 145/82 mm Hg. He had pallor and massive lower extremity edema extending upto the scrotum. There was no lymphadenopathy. Abdominal examination revealed a large hard mass extending anteriorly from the right lumbar region to the opposite flank and pelvis. Laboratory data-Hb 8.7gm/dl, Total WBC 74,800/cu mm, Differential±40% neutrophils, 56% bands and 4% monocytes, platelets 195 K/cu mm, LAP score 217. Urinalysis was normal. Alkaline phosphatase 144 IU/L, total serum protein 6.3 g/dl, C-reactive protein 13.3 mg/dl. Both blood and urine cultures were negative. Peripheral smear±leukocytosis, mature granulocytes and band forms, no blasts. CT scan of the abdomen showed a large, lobulated retroperitoneal mass extending to the posterior aspect of the right kidney, measuring 9Â8Â15 cm and another left sided pelvic mass, 14Â10Â17 cm, contiguous with the urinary bladder and pressing on the IVC. Multiple low-density lesions were also noted in the liver and spleen. Bone marrow biopsy revealed a myeloid predominant hypercellular marrow, no blast cells. A CT guided biopsy from the hypodense lesion in the liver revealed a metastatic, poorly differentiated malignant fibrous histiocytoma (MFH), pleomorphic type, positive for vimentin and negative for cytokeratin and alpha-fetoprotein on immunostaining. The IL-6 and IL-2 levels were normal. The patient was started on chemotherapy with cyclophosphamide, dexamethasone, adriamycin and dacarbazine. He developed disseminated intravascular coagulation on the 5th day of chemotherapy and expired. IMPLICATIONS/DISCUSSION: This case is unique because of the association of malignant fibrous histiocytoma with leukemoid reaction (LR). Several cases of soft tissue sarcomas with leukocytosis have been reported, but less than 15 cases of MFH with this phenomenon were found in the review of literature. The relationship between cytokine levels and LR has not been consistently demonstrated in the cases reported. Although, in our patient, serum cytokine levels were noninformative and histologically, the metastatic component of the tumor was not significantly different from the``non-inflammatory'' fibroxanthosarcomas, the absence of any other cause of leukocytosis highlights its association with MFH. The prognostic significance of leukocytosis with these tumors is yet to be defined, an added survival benefit of the inflammatory component is debatable. year-old male with a history of 30 pack-year tobacco use and positive tuberculin test, presented with nonproductive cough and night sweats for several months. Physical exam was normal. Serum immunofixation tests and acid fast stains of sputum were normal. Pulmonary function test (PFTs) showed moderate obstructive airway disease, and transthoracic echocardiogram was unremarkable. Chest x-ray (CXR) exam revealed a rounded opacity in the right lung apex. Computed tomography (CT) showed a 2.5 cm right apical mass and multiple small nodules seen in both lung fields. Transbronchial biopsy of the apical mass was consistent with pulmonary nodular amyloidosis. Follow-up CT imaging showed no significant change after one year. Case 2: A 73 year-old Caucasian male with a history of 40 pack-year tobacco use, had a CXR for preoperative evaluation for blepharoplasty. He denied cough, weight loss, and night sweats. Physical exam and laboratories were all within normal limits. PFTs were consistent with mild obstructive airway disease. CXR exam showed an incidental finding of a round density in the right middle lung field. CT revealed a 1.5 cm round opacity in the right middle lobe. Transbronchial lung biopsy of the mass was consistent with pulmonary nodular amyloidosis. Follow-up CT imaging showed no significant change after one year. IMPLICATIONS/DISCUSSION: Pulmonay amyloidosis may present in one of three ways:\n(1) diffuse, interstitial process associated with primary, systemic amyloidosis and a median survival of 16 months after diagnosis; (2) Endobronchial amyloid causing obstruction that is usually amenable to treatment with laser therapy; and (3) isolated, pulmonary nodular amyloidosis that is not associated with systemic disease and follows a benign course. Our two cases are examples of pulmonary nodular amyloidosis. Pulmonary disease is uncovered as an incidental finding with one or more nodular opacities seen on chest imaging suggesting a malignant process. Transbronchial lung biopsy may establish the diagnosis with light microscopy showing amorphous, extracellular deposits that stain pink with hematoxylin and eosin. Under polarized light, amyloid produces apple-green birefringence. In our two cases of primary nodular amyloidosis, elevated monoclonal proteins were not found, consistent with the absence of systemic involvement in this presentation of disease. Further, follow-up chest imaging has revealed no change in the pulmonary nodular opacities, reflecting the benign course of this disease. year-old homeless male with a history of untreated Schizophrenia was brought in for evaluation of fatigue and altered mental status. On presentation, the patient was noted to be disheveled and confused with initial BP 128/61, P88, RR 17, and T99.9. Physical exam was remarkable for numerous nits on hair and clothing, and skin with innumerate number of excoriated, hemorrhagic papules. Rectal exam revealed no masses but guaiac negative brownish stool. Initial laboratories included: WBC 13.7, HGB 3.7, HCT 11.5, PLT 375, MCV 72, RDW 16, and 71% PMN. Gastric lavage was negative for occult blood. Computed tomography of head and a diagnostic lumbar puncture were normal. Serum anemia work-up was remarkable for low ferritin (11) and iron (76). Folate and vitamin B12 levels were normal. The remaining laboratories, imaging studies, and cultures were all within normal limits. He was treated for pediculosis with permethrin and received blood transfusion with an appropriate increase in blood counts. His mental status and leukocytosis significantly improved as pediculosis and anemia were treated. Blood counts has remained stable after treating pediculosis. IMPLICATIONS/DISCUSSION: The 3 most common arthropod exoparasites in humans are Pediculus capitis (head louse), Pediculus corporis (body louse), and Phthirus pubis (pubic louse). They belong to the order Anoplura, the sucking lice that feed on blood approximately 5 times per day for approximately 40 minutes each time. Hemorrhagic macules or papules develop at the sites of feeding lice, and vertical excoriations due to scratching and postinflammatory hyperpigmentation are common (aka, vagabond's disease). These lice can be transmitted directly from person to person. They are also a potential vector for transmission of diseases such as typhus, louse-borne relapsing fever, and endocarditis. Bacterial superinfection at the sites of infestation is also common. Zoonotic pediculosis (louse infestation) has been reported as a major cause of anemia in domestic animals. This possibility, however, has not been reported in humans. Our case presents a homeless male wtih severe iron-deficient anemia associated with chronic pediculosis. This case supports the hypothesis that louse infestation is an uncommon cause of anemia in humans. To our knowledge this is the first case report of its kind (Internet and Medline search from 1960 to present). year-old woman presented with chest pain and syncope after exertion. The chest tightness was associated with shortness of breath, nausea, and diaphoresis. She first experienced exertional angina at age 18 during military training. The episodes had increased in frequency since. Her vital signs were normal. The cardiac examination was normal with the exception of a a left-sided heave and a loud, persistently split S2 that widened with inspiration. An electrocardiogram demonstrated normal sinus rhythm, right axis deviation, and T-wave inversion in the inferior and anterior leads. Three troponin assays were negative. Echocardiography revealed right ventricular enlargement, dilated pulmonary arteries with pressures of 50 mm Hg, normal chamber sizes, normal valves, and no septal defects. A ventilation/perfusion scan, ANA and RF were normal. A right heart catheterization confirmed pulmonary artery hypertension with a systolic pressure of 70 mmHg and a normal wedge pressure. A bubble study looking for small septal defects was normal. Angiography confirmed the presence of dilated pulmonary arteries without evidence of thromboembolic disease. IMPLICATIONS/DISCUSSION: Primary pulmonary hypertension is a diagnosis of exclusion, with a prevalence of one to two people per million. Because of its low incidence and protean nature, the most cost effective strategy is to exclude common etiologies of pulmonary hypertension such as pulmonary embolism, valve disease, lupus, CREST and left heart failure. Right heart catheterization is the best diagnostic test. The median survival after diagnosis is 2.5 years. Intravenous epoprostenol is the drug of choice, but oral vasodilators are also used. Anticoagulation has been shown to prolong life; some patients may also respond to oral vasodilators. Anticoagulation improves mortality by decreasing thombosis and thromboembolism. This patient was started on a trial of oral bosentan and warfarin. Since primary pulmonary hypertension has no cure, an exhaustive search for secondary causes is warranted in any patient with this diagnosis. Other than these findings, her neurological examination was unremarkable. MRI of the head revealed multiple foci of hyperintensities in the white matter; bilateral enhancement of the tentorium and cranial nerves III, V, VII; and unremarkable orbits (Fig 1,2,3) . Cerebrospinal fluid (CSF) revealed a protein level of 600 mg/dl; WBC 210/uL (lymphocytes 95%) and glucose 91 mg/dl. Serum and CSF were positive for Lyme antibodies on ELISA. The result was confirmed by western blot assay on the CSF. The patient was started on intravenous ceftriaxone 2 gm every 24 hrs. On the 4th day of antibiotic therapy, she developed left sided LMN facial palsy. After 4 weeks, a repeat CSF study showed significant improvement: the protein level fell to 82 mg/dl and WBC count to 33/ul. Six weeks after ceftriaxone therapy, the diplopia, trigeminal neuralgia and facial palsy resolved completely. IMPLICATIONS/DISCUSSION: Though facial palsy is a relatively common presentation of Lyme disease, multiple cranial neuropathies may be the initial manifestation and must be borne in mind. The diagnosis can be easily missed in such cases without any antecedent history of tick bite or rash. A history of travel or residence in an endemic area is a useful clue. In addition, MRI may play an important role in the diagnosis. Our patient's MRI at admission revealed bilateral enhancement of meninges and cranial nerves III, V and VII indicating evidence of inflammation that preceded the full clinical expression. To our knowledge, multiple cranial neuropathies as the initial manifestation and evidence of gadolinium enhanced MRI lesions of the involved cranial nerves have been rarely reported. Recognizing these subtle imaging findings, combined with high clinical suspicion is the key to diagnosis and allows early treatment which can help prevent further complications.\nWALDENSTROM'S MACROGLOBULINEMIA AND HEART FAILURE. R. Pearl 1 , J. Wiese 1 ; 1 Tulane University, New Orleans, LA (Tracking ID #77006)\nLEARNING OBJECTIVES: 1. Use the physical examination to determine cost-effective use of echocardiography 2. Use an understanding of physiology to guide diagnostic procedures 3. Diagnose infiltrative cardiomyopathy. CASE INFORMATION: A 73 year-old man presented with right-sided chest pain and shortness of breath. The shortness of breath had worsened over two weeks, with new onset three-pillow orthopnea and lower extremity edema. His vital signs were normal. He had a soft S1, a normal S2, and an S4. Although he had bilateral crackles and an elevated JVP, there was no S3 or inferiorly displaced PMI. His edema was initially ascribed to hypo-albuminemia (Albumin = 2.) although his pit recovery time was greater than 90 seconds. Based upon a suspicion of a restrictive cardiomyopathy, an echocardiogram was obtained. This showed left ventricular hypertrophy and an E-to-A mitral-flow reversal consistent with diastolic dysfunction. His ejection fraction was 20% despite ventricular hypertrophy. A bone marrow biopsy was performed that confirmed the diagnosis of Waldenstrom's macroglobulinemia. IMPLICATIONS/DISCUSSION: The physical examination in concert with an understanding of the physiology underlying disease is the foundation for cost-effective identification of occult diagnoses. The silent S1 suggested a low ejection fraction, and while the S4 without an S3 or inferiorly displaced PMI suggested a non-compliant hypertrophic ventricle. Both of these conclusions were confirmed by echo. A low ejection fraction despite ventricular hypertrophy suggested an infiltration of the ventricular wall. LaPlace's law states that wall tension is a function of the pressure and radius of a chamber, divided by the thickness of the chamber's wall. Because wall tension is proportional to afterload (like a spring, the ventricle must overcome the weight that has stretched it) a thick ventricle wall should lower wall tension, thereby decreasing the ventricular afterload. The ejection fraction should increase proportional with the wall thickness. Failure to do so suggests that the thickness is due to infiltration with a non-contractile substance. The history of chronic renal insufficiency and anemia suggested the diagnosis of amyloidosis due to Waldenstrom's macroglobulinemia. This clue was instrumental in prompting the bone marrow biopsy that confirming the diagnosis. The finding of a speckled myocardium on echocardiogram is a classic, though late state finding of the disease. A 54-year-old male with a history of leukocytoclastic vasculitis was noted to have a diffuse erythematous rash while being admitted for an unrelated endoscopic procedure. The rash first developed two weeks prior in the patient's right groin and gradually spread to include both legs and his right arm. It had not responded to a course of levofloxacin or over the counter topical ointments. In the month prior to admission the patient had been tapered from 15 milligrams to 12.5 milligrams of prednisone. On admission the patient had warmth, erythema, and mild tenderness to the affected areas. Numerous 1 to 5 millimeter pustules also were present on the erythematous base. The patient was afebrile and had a mildly elevated white count of 11 thousand. Gram stain and culture of the pustules revealed no bacteria. Fungal elements were not seen. A punch biopsy was taken and returned consistent with pustular psoriasis. Acitretin therapy was initiated. IMPLICATIONS/DISCUSSION: Psoriasis is a relatively common dermatologic disorder that occurs in approximately 1% of the North American population. While generally benign, psoriasis can present in an atypical and potentially life threatening manner. Pustular psoriasis can occur in varying degrees of severity with distinct subtypes. These include the relatively limited form of palmoplantar pustulosis to the more severe generalized von Zumbush type. If untreated, disruption of the skin's barrier can lead to large amounts of volume loss and secondary bacterial infections. Systemic involvement including arthritis, heart, liver, and kidney failure has been associated with advanced cases. Triggering events are often unknown, but there has been an association with pregnancy, concurrent infections, and medications. Given the nonspecific somatic complaints and laboratory abnormalities, successful diagnosis requires recognition of the pustular areas on the erythematous base. Our patient had two associated medication risk factors of hydroxychloroquine therapy and corticosteroid withdrawal that likely acted as the inciting event. IMPLICATIONS/DISCUSSION: t is a challenge to manage pregnant patients who require anticoagulation for prosthetic valves. Coumadin is the drug of choice for anticoagulation, however because it freely crosses the placenta it is not considered safe to use in pregnant patients. The teratogenic effects of coumadin usually occur between the sixth and ninth weeks of gestation. The most common abnormalities occur in bone and cartilage development. In the second and third trimesters one can develop abnormalities of the fetal central nervous system, optic atrophy, microcephaly, mental retardation, spastisity and hypotonia. Unfractionated heparin does not cross the placenta because of its high molecular weight. Since it does not cross the transplacental barrier it lacks teratogenic effects and will not anticoagulate the fetus. Low molecular weight heparin is another anticoagulant that should be considered in these patients. However, its use in pregnancy is not well studied. The ideal treatment for a pregnant woman requiring anticoagulation would be to switch from coumadin to heparin prior to conception. The patient shold be hospitalized until she is therapeutic on heparin. The three approaches that are recommended include: 1. Dose adjusted unfractionated heparin throughout pregnancy. Heparin is given subcutaneously every 12 hours and then dose adjusted to maintain two times the normal PTT, or anti-Xa levels of 0.35 to 0.70 U/ml. 2. Dose adjusted low molecular weight heparin throughout the pregnancy so as to keep a four hour post injection anti-Xa level at 1.0 U/ml. 3. Unfractionated or low molecular weight heparin therapy until the thirteenth week, with a change to coumadin until the middle of the third trimester and then restarted on unfractionated or low molecular weight heparin until delivery. LEARNING OBJECTIVES: 1) Recognize the cutaneous manifestations of sarcoidosis 2) Review the differential diagnosis of a nodular cutaneous lesion in a immigrant from subsaharan africa 3)Review the pathophysiology of cutaneous sarcoidosis CASE INFORMATION: A healthy 21 year old South Sudanese woman, 4 weeks post-partum, who had spent 2 years in an Ethiopian refugee camp before immigrating to the U.S one year ago, presented with a rash on her left cheek of over 2 years duration. She had noticed the rash first back in Sudan and had persisted during her refugee camp stay. She reported that the rash had been non-pruritic, non-exudative, indolent and asymptomatic. She had no history of photosensitivity, exposure to chemicals or insect bite. A week's trial of anti-fungal topical application was unsuccessful. Her recent pregnancy was uneventful including negative HIV, Hep.BSAg and VDRL serology. Her PMH was negative for major medical illnesses and prior eruptive lesions. She did have a history of positive PPD 1 year ago with negative CXR following immigration to the U.S. She had not been treated with anti-tuberculous agents. Her ROS was otherwise unremarkable. Exam revealed a hyperpigmented nodular lesion approximately 1cm wide and 4 cms long in a semi-circular distribution over the left malar area. The rash was further noted to be non-tender, normothermic with mild induration. Physical exam revealed a healthy 21 year old woman with normal cardiac, pulmonary, gastrointestinal and neurological exam, with no evidence of lymphadenopathy and a similar rash elsewhere. Her CBC was within normal limits. Given the patient's ethnicity and travel history, leishmaniasis and cutaneous mycobacterial infections were included in the differential. Consideration was also given to Sarcoid and variant discoid lupus. (Syphilis was considered unlikely, given recent negative serology). A diagnostic excisional biopsy revealed sarcoid-like granulomas surrounded by mild chronic lymphocytic infiltration. Special stains (including AFB, giemsa, PAS, Fite, Giemza and warthin-starry) did not reveal pathogenic microorganisms. Patient was initiated on a local topical steroid application (clobetasol propionate) for two weeks with dramatic resolution of the rash. A diagnosis of Sarcoidosis was therefore made by exclusion and dramatic response to treatment. IMPLICATIONS/DISCUSSION: The protean manifestations of Sarcoidosis include a) Erythema nodosum (most common, although non-specific lesion). b)Lupus pernio (classically involving the rim of the nose and associated with infiltration of the nasal mucosa causing ulcerations or even fatal airway obstructions). c) Papules, nodules or plaques (as seen in the above patient). d) Psoriasis like rash. e) lesions in scars and tattoos. The learning objectives of this presentation are to review the cutaneous manifestations of Sarcoidosis, the differential diagnosis of nodular cutaneous lesions in an imigrant population and the pathophysiology of Cutaneous Sarcoidosis. Considering the growing immigrant population in the U.S, the differential of an indolent chronic rash should include parasitic infestations. However, in patients of African decent, the extremely variable masquerading rash of sarcoidosis should be considered as an important part of the differential. Hemochromatosis is the most common adult genetic disorder. Early diagnosis enables rapid treatment and an improved prognosis. The first clinical manifestation is represented by arthropathy in 45% of the cases. The articular features of are often nonspecific and associated with a delay in diagnosis that may compromise outcome. Treatment of the arthritis with analgesics is often disappointing, but identification of the underlying disease permits institution of life-saving phlebotomy therapy. The aim of this report is to highlight the fact that patients with premature osteoarthritis should alert the physician to screen for hemochromatosis in order to formulate the correct diagnosis before the development of severe internal organ involvement. MRI of the head was done to rule out pituitary masses and was normal. After extensive review it was found that she received an injection of Kenalog 40 mg to the right shoulder from another MD six weeks prior to presentation. Without any other exogenous sources of steroid, it was concluded the pt had experienced cushingoid symptoms from the Kenalog injection followed by secondary adrenocortical insufficiency at presentation. IMPLICATIONS/DISCUSSION: Therapy with pharmacological doses of glucocorticoids is the most frequent cause of secondary adrenocortical insufficiency. The usual presentation is similar to that of primary adrenocortical insufficiency, with two important exceptions. Since pituitary secretion of ACTH is deficient, the characteristic hyperpigmentation of Addisons disease is absent. The clinical features of mineralocorticoid deficiency are usually absent in the unstressed state, and therefore volume depletion, dehydration, and electrolyte abnormalities are usually absent. Hypotension is also less severe, except in acute presentations. Hyponatremia can be present, and is usually due to water retention and inability to excrete a water load rather than to sodium loss. The clinical features of ACTH and glucocorticoid deficiency are nonspecific and consist predominantly of weakness, lethargy, easy fatigue, anorexia, nausea, and occasionally, vomiting. Patients may also describe arthralgias, myalgias, and exacerbation of allergic responses. With acute decompensation, severe hypotension or shock may occur and be unresponsive to vasopressors unless glucocorticoids are administered. At that time he also described shortness of breath, insomnia, anorexia, and a sense of anxiety. The exam revealed an agitated but otherwise well-appearing man with tachycardia. Mr J had a positive urine cocaine screen. He was admitted and ruled-out for myocaridal infarction (MI) with serial troponins and electrocardiogram. Two months later, Mr J returned with similar complaints. Again his urine was positive for cocaine and his serum was negative for troponins. Over the next three months he returned to the ED four more times with a new resting tremor and a weight loss of over sixty pounds. Cocaine screens remained positive. The patient refused treatment for substance abuse and expressed dissatisfaction with the quality of care he received. Finally in September of 2002 a Thyroid work-up was performed, demonstrating a TSH <0.1 and a free thyroxine >6.5. A radioiodide uptake scan confirmed the diagnosis of Graves' disease. The patient had symptomatic relief with beta blockade and propylthiouracil. He is currently scheduled for thyroid ablation. IMPLICATIONS/DISCUSSION: An estimated 1.5 million Americans (0.7%) abuse cocaine chronically. In 1998, cocaine contributed to Emergency Department visits on 152,000 occasions (1). Cocaine abuse is a potentially life-long chronic disease. Acute cocaine intoxication may mimic other diseases, preventing or delaying their diagnosis. An easy explanation for Mr. J's tremor, weight loss and agitation were already available, thus a thyroid work-up was delayed. Cocaine abuse may draw the physician's attention to conditions caused by chronic abuse rather than unrelated medical problems. MI should be ruled out, but not without considering hyperthyroidism. In one study of 165 patients with cocaine intoxication and psychotic symptoms, only 18% were given the proper diagnosis of schizophrenia on the initial evaluation (2) . Finally, some physicians may perceive substance abuse as a primary and overriding health care problem. Mr. J did not wish to address his cocaine abuse and still rightly expected treatment of his other medical problems. In conclusion, many misperceptions may cloud the diagnostic process with patients who abuse cocaine: confusion of symptoms with drug effects, distraction from unrelated medical conditions and prioritizing abstinence above other medical care. LEARNING OBJECTIVES: Recognize the potential for endocarditis after an endometrial sampling procedure. CASE INFORMATION: Antibiotic prophylaxis is not recommended prior to endometrial sampling procedures or even dilation and curettage because of the very low risk of infectious complications. We report a case of acute bacterial endocarditis caused by Streptococcus agalactiae following endometrial sampling in a woman with no known valvular abnormalities. A 59-year-old woman with a history of abnormal uterine bleeding who had undergone office based endometrial biopsy about two weeks earlier was found unresponsive at home and was brought to the hospital. She was noted to have a temperature of 102.5 degrees Fahrenheit; she was awake, but did not speak and withdrew her left side more than her right side to painful stimuli. She also had petechiae in her conjunctiva, fingers and toes. Laboratory evaluation showed white blood cell count of 21,400 cells/mm3, normal electrolytes and glucose, and normal liver enzymes. CT scan of the head was normal at presentation. On hospital Day 3, blood cultures from admission grew beta-hemolytic Group B Streptococcus from all samples. Transthoracic and transesophageal echocardiograms demonstrated a 4Â3 cm lesion on the posterior leaflet of the mitral valve with moderate mitral regurgitation. Repeat CT of the head demonstrated a large left middle cerebral artery infarction, but no evidence of abscess. The patient responded to antibiotics, but died of complications before valve replacement surgery could be performed. IMPLICATIONS/DISCUSSION: Endometrial assessment by means of biopsy or sampling of endometrial cells is a minimally invasive alternative for dilation and curettage or hysteroscopy. Techniques using the Pipelle endometrial sampling device are the most popular due to high sensitivity and specificity compared to other office based procedures, and low rates of complications. In dilation and curettage, studies show transient bacteremia in 5% of patients and up to 0.34 in 100 patients have fever complicating their procedure. We hypothesize that our patient's endocarditis was caused by bacteremia resulting from her endometrial sampling, making this a very unusual complication of a common procedure. There is no data supporting the use of antibiotic prophylaxis for office based (i.e. Pipelle) or operating room (i.e. dilation and curettage) endometrial sampling. Unfortunately for our patient, mortality rates for Group B Streptococcus endocarditis approach 40%, similar to those associated with staphylococcal species.\nWEIGHT fatigue, and night sweats. He denied using tobacco or intravenous drugs. On physical examination, he had orthostatic hypotension and a new 2/6 systolic ejection murmur. He did not demonstrate peripheral stigmata associated with endocarditis (Osler's nodes, Janeway lesions, or splinter hemorrhages). Pertinent labs for the hospitalization included a hemoglobin of 10.2 g/dL, hematocrit of 30.1%, and mean corpuscular volume of 81.9 fL (further labs confimed iron deficiency). An erythrocyte sedimentation rate (ESR) was 35; C-reactive protein was 12.5 and albumin was 2.0. WBC, BUN, creatinine, liver function tests, chest radiograph, and EKG were normal. PSA, TSH, and morning cortisol levels were normal. PPD and HIV screening were negative. Esophagogastroduodenoscopy and colonoscopy did not show any abnormal pathology. CT scans of the head, neck, chest, abdomen, and pelvis were unremarkable except for a small pericardial effusion and an old ischemic left cerebellar infarct. On day six of his hospital stay, the patient had a fever with a Tmax of 39.0 and a bandemia of 14% with a normal WBC count. Two sets of blood cultures were drawn and diphtheroids, identified as species other than Corynebacterium jeikeium, grew in all specimens. A transthoracic echocardiogram revealed an aortic valve vegetation, moderate aortic regurgitation, and an ejection fraction of 60 to 65%. A diagnosis of subacute bacterial endocarditis was made and the patient was treated with a four week course of intravenous ampicillin. Surveillance cultures drawn five days after starting antibiotics were negative and the patient remained afebrile for the rest of his hospital stay. IMPLICATIONS/DISCUSSION: Bacterial endocarditis is a common entity encountered in the hospital setting. The diagnosis may be missed as it can mimick a variety of conditions due its protean clinical manifestations and the diversity of pathogens associated with it. Recognition of these signs and symptoms can enable the clinician to make an early diagnosis and pave the way for intervention and further reduction in morbidity and mortality. These include constitutional symptoms such as fever, fatigue, and weight loss; new and/or changing murmurs; embolic phenomenon such as Janeway lesions, mycotic aneurysms, and peripheral emboli to multiple organs; and immune complex mediated phenomena such as glomerulonephritis, Osler's nodes, and arthritis. Elevated ESR and C-reactive protein, and a positive rheumatoid factor often accompany these presentations. Anemia is present in 70 to 90% of cases. Weight loss is found in up to 25% of cases and often leads to an exhaustive work up for malignancy, tuberculosis, and other chronic diseases. If there is a high index of suspicion, diagnosis can be made by positive serial blood cultures or the presence of valvular vegetations on echocardiogram. Streptococci, enterococci, and staphylococci are isolated in the vast majority of cases. Other pathogens are rather uncommon and include the HACEK group (Hemophilus, Actinobacilus, Cardiobacterium, Eikenella, and Kingella), Coxiella, Neisseria, Pseudomonas, Salmonella, Streptobacillus, Bacteroides, Corynebacterium and other anaerobic gram-negative bacilli. There are case reports of native and prosthetic valve endocarditis caused by diptheroids including C. jeikeium, C. urealyticum and C. striatum. Of the nondiphtheriae corynebacteria, C. jeikeium is the most worrisome as it is resistant to multiple antibiotics and warrants treatment with vancomycin. Diagnosis of the causative organism is imperative for initiation of therapy with appropriate antibiotics and possible surgical intervention. LEARNING OBJECTIVES: After listening to this vignette presentation the leaner will: 1. Recognize the importance of NSAID-induced adverse effects in the lower GI tract. 2. Add NSAIDs (including the selective COX-2 inhibitors) to the differential diagnosis of rectal bleeding and intestinal strictures. CASE INFORMATION: A 58 year old female with a history of GERD treated with a proton pump inhibitor, hypertension, hyperlipidemia, and hypothyroidism was prescribed celecoxib 200mg/day for mechanical low back pain. After approximately a month of therapy she presented with bloody diarrhea. She denied fever or chills. There was only minor abdominal cramping. The patient has a strong family history of colon cancer and had undergone a surveillance colonoscopy 2 years ago. This was normal except for a few small diverticula.\nAbdominal examination was unremarkable. Rectal exam revealed a small external hemorrhoid and no masses, red blood was found on the exam glove. Stool culture, ova and parasites, and WBCs was negative. The patient discontinued the celecoxib and the bleeding diminished. A flexible sigmoidoscopy demonstrated a few 5 mm ulcers in the descending and transverse colon. Biopsies revealed nonspecific acute and chronic inflammation. No granulomas or crypt abscesses were seen. The bloody diarrhea completely resolved. The patient remains off of the celecoxib. Her back pain remains problematic. IMPLICATIONS/DISCUSSION: Upper GI tract ulceration with hemorrhage is a wellrecognized complication of NSAIDs. This serious side effect prompted the development of the selective COX-2 inhibitors which are touted as being safer in this regard. (However, this claim has recently been questioned.) Less known important GI adverse events attributed to NSAIDs include strictures and ulcerations of the small and large intestine. This case demonstrates symptomatic ulcerations of the colon attributed to celecoxib, a COX-2 specific NSAID. The patient's clinical presentation, and histology are consistent with that described with nonselective NSAIDS. Clinicians must consider NSAIDs in the differential diagnosis of lower GI tract bleeding, ulceration, or strictures. Studies are needed to determine if COX-2 specific inhibitors are safer than non-specific NSAIDs regarding the lower GI tract. This may become especially relevant in assessing the role of COX-2 inhibitors in the prevention of colon cancer. Although she had a family history of diabetes, a previous fasting glucose before beginning olanzapine was 85 mg/dL. Her exam was significant only for a non-obese, dehydrated appearing female who was orthostatic and tachycardic. Total glucose was 1122 mg/dL and measured serum osmolality was 320 mOsm/kg. A venous blood gas showed pH 7.38 and pCO2 41. Other labs included positive serum ACE test, trace urine ketones, and (in mEq/L) serum HCO3 23, sodium 122 and potassium 5.4 (anion gap of 19). IMPLICATIONS/DISCUSSION: As early as 1926, patients with schizophrenia have been known to have an increased risk of developing hyperglycemia, a process which was exacerbated with the introduction of phenothyiazines in 1956 and the atypical anti-psychotics in 1994. Increasingly, non-schizophrenic patients taking atypical anti-psychotics are demonstrating increased episodes of hyperglycemia and the complications thereof. One proposed mechanism for this hyperglycemia is through drug interference with cellular glucose transport proteins leading to increased insulin resistance and impaired glucose utilization in the periphery. This effect is not dose dependent and has its onset from 10 day to 18 months after the initiation of the drug. The literature is replete with examples of DKA in schizophrenic patients taking atypical anti-psychotics but does not specifically report the development of non-ketotic hyperosmolar syndrome. Additionally, patients taking atypical anti-psychotic medicines for diagnoses other than schizophrenia±including bipolar disorder and major depression with psychosis±risk developing hyperglycemia.\nA CASE OF STAPHYLOCOCCAL COLITIS PRESENTING AS TOXIC SHOCK SYNDROME. T. Rutkoski 1 , C. Mueller 1 ; 1 University of Cincinnati, Cincinnati, OH (Tracking ID #77132)\nLEARNING OBJECTIVES: 1) Recognize the presentation of toxic shock syndrome.\n2) Realize its occurrence in males and nonmenstrual females. CASE INFORMATION: MZ was an 18 y.o. caucasian female with a PMH of depression and PTSD who presented to the ER with complaints of fever, rash and abdominal pain. Outpatient medications included only quetiapine. The patient was currently living in a women's shelter b/c of reported sexual abuse and she denied tobacco, alcohol, and drugs. Initial PE was significant for fever, hypotension, conjunctival injection, rash, diffuse abdominal tenderness, and a negative pelvic exam. Labs revealed an elevated WBC count, a renal panel with elevated AG, mild LFT abnormalities, negative CSF tap, negative tox. screen, and a contaminated U/A with ketones. The patient was initially treated with ceftriaxone and aggressive fluid management. She did develop anemia and mild pulmonary edema. Her illness gradually improved. She was discharged home and follow up in the office 5 days later revealed desquamation of her hands. Her infectious w/u was negative except for a stool cx growing S. aureus. Toxic Shock Staphylococcal Toxin-1 was negative, but Staph aureus B toxin was positive. IMPLICATIONS/DISCUSSION: The initial cases of staphylococcal TSS were described in young menstruating females in the late 1970's. Much was learned about this disease in a short time. Soon an association was found between highly absorbent tampons and TSS. These tampons were removed from the market in the early 1980's. Since then the number of cases of TSS has declined greatly. Today, however the number of reported cases is relatively stable. These cases can be found in the menstruating and nonmenstruating female, the young and old, and the male and female patient. There are very few cases of Staph TSS secondary to colitis in the literature. Overall disease mortality has also improved throughout the years but remains as high as 15% in some studies (although more likely 2±5% by most accounts). It is therefore important to think of this disease in the differential diagnosis and treat it appropriately. The patient was started on a diltiazem drip for rate control and advanced to metoprolol for long term management. She was also started on aspirin for thrombus prophylaxis. No underlying etiology for her arrhythmia was identified. However, an extensive review of the patient's family history revealed that her father had been diagnosed with heart failure secondary to Chagas' disease back in El Salvador. A sample of the patient's blood was sent for serology for Chagas' disease and returned positive with a titer of 1:512. The patient was placed on nifurtimox. Although it was believed that she may have been in the subacute phase of her illness, treatment was initiated as a means of preventing advancement of her disease to chronic illness. IMPLICATIONS/DISCUSSION: Chagas' disease is a protozoan infection due to Trypanosoma cruzi and is primarily related to three problems: megaesophagus; megacolon; and cardiac disease. Chagas' myocarditis is the most common form of cardiomyopathy in Latin-American countries and therefore has become a considerable public health problem for many areas. It is estimated that over 750,000 years of productive life are lost annually because of premature deaths due to this disorder. The presentation of Chagas' disease consists of an acute and chronic phase, separated by an indeterminate phase. The latter describes a patient who has positive se-rology, but no symptoms, physical signs, or laboratory evidence of organ involvement. Biopsy was consistent with candida without evidence of aspergilus. IMPLICATIONS/DISCUSSION: Aspergillosis encompasses a variety of manifestations of infection. The major entities include invasive disease, acute bronchopulmonary aspergillosis (ABPA) and pulmonary aspergilloma. Disease due to aspergillus organisms is uncommon and usually occurs in the setting of immunosuppression for which neutropenia (ANC < 1500 TH/ MM3) and corticosteroids are the most common predisposing factors. The most common manifestation of invasive aspergillosis involve the lung and contiguous structures. The central nervous system and gastrointestinal system (especially esophagus and bowel) are the first and second most common target of dissemination respectively. Symptoms of aspergillus esophagitis are usually consistent with severe odynophagia similar to those of candidiasis. Isolated esophageal aspergillosis is very rare. There are two case reports published describing this unusual presentation, one involving a patient with acute leukemia and another occurred in a patient 25 days post bone marrow transplantation. This case is of interest because though our patient was steroid dependent she was not neutropenic prior to and at the time of presentation (ANC > 1500 TH/MM3). Further she was asymptomatic in regards to her fungal infection and did not have evidence of aspergillus involvement at other sites. In addition esophageal involvement usually occurs as a secondary complication of severe invasive lung disease. To our knowledge, this is the first case to describe isolated esophageal aspergillosis in a patient without the overt risk factors for invasive aspergillosis. Health is now moving forward in fostering high quality language programs that will help eliminate racial and ethnic disparities in healthcare and will promote access to optimal healthcare for all. Legislation has been introduced into the State legislature regarding cultural competency training and additional recommendations to the State are being drafted to expand this model program. Our hospital is now implementing an on-going training program for all interpreters and staff prior to enlisting their services, have hired bilingual greeters for the hospital, and a bilingual patient advocate. We have also initiated a cumulative and ongoing series of research projects in the field of cross-cultural -old Oriental man presented with complaints of cramping abdominal pain, tenesmus (immediately after eating) and constipation with pellet like stools of 3 weeks duration. His past history included Coronary Artery Disease and Radical Prostatectomy for Prostate Carcinoma 8 years ago with regular follow up. Physical examination was normal except for an irregular rectal mass partially occluding the lumen of the rectum. A preliminary clinical diagnosis of rectal carcinoma was made. Flexible sigmoidoscopy showed a cauliflower like ulcero-nodular mass occupying the lumen of the rectum. Biopsies of which were consistent with Adenocarcinoma. CT scan of the pelvis showed a mass adherent to the posterior wall of urinary bladder in the region of trigone, partially obstructing lumen of the rectum and also causing bilateral hydroureters and bilateral hydronephrosis. A subsequent serum PSA was 138 ng /ml, the previous value being 22 ng/ml. Based on this data a diagnosis of prostate carcinoma was made. Because of the extent of disease and underlying heart disease palliative measures were chosen for management including the stents in rectum and bladder to relieve obstruction. IMPLICATIONS/DISCUSSION: Finding a rectal mass on clinical examination is not uncommon. Our case is unusual in that the rectal mass was of prostatic origin, presenting 8 years after initial diagnosis. Though rectal involvement by prostatic carcinoma is present in 0.56% to 11.5% of cases on postmortem studies, the presence of Denonvillier's Fascia usually inhibits it. Commonest modes of involvement of rectum are a bulging prostate mass with intact rectal mucosa or an annular stricture, ulcerating masses being the rarest and accounting for less than 1% of these cases. Knowledge of this is important as it can avoid a misdiagnosis of primary rectal cancer, especially in patients who do not have a history of Prostate cancer or those with a history of prostate cancer who have received radiotherapy. LEARNING OBJECTIVES: 1. To recognise that superwarfarin causes prolonged coagulopathy needing high dose Vitamin K treatment for weeks to months 2. To suspect superwarfarin overdose as a cause of coagulopathy resistant to vitamin K and fresh frozen plasma treatment CASE INFORMATION: A 50 years old gentleman with a past medical history of depression and recurrent suicidal attempts presented to emergency room (ER) with history of hematuria, epistaxis and bruising of skin after suicidal attempt with anticoagulant ingestion. Physical examination revealed widespread ecchymosis of skin and heme positive stool but was otherwise unremarkable. Laboratory studies revealed hemoglobin (Hbg) 4.8 g/dl (13.7±16.5 g/dl), prothrombin time (PT) >50 sec (11.5±14.3 sec), INR too high to be reported, and activated partial thromboplastin time (APTT) >150 sec (21±37 sec). Patient was thought to have ingested warfarin, the most commonly available anticoagulant. His anemia was corrected with blood transfusion and coagulopathy treated by fresh frozen plasma (FFP) and Vitamin K. After 6 days of treatment his Hbg was12.2 g/dl, PT = 14.7sec, INR = 1.26, and APTT = 50 sec. Vitamin K was discontinued and he was discharged home after psychiatry consultation. His epistaxis and bruising of skin returned after 1 week resulting him to revisit ER. Patient denied any further intake of anticoagulant since discharge, however confirmed that the anticoagulant he had ingested 4 weeks ago with suicidal intent was infact superwarfarin (rodenticide) subsequently identified as brodifacoum. His blood studies revealed Hbg 7.9 g/dl, PT >200 sec, INR = 397.5 and APTT = 95.3 sec. He was again treated with FFP, blood transfusion and Vitamin K. This time he was discharged on long term high dose (100 mg/day) oral Vitamin K and out patient follow-up. IMPLICATIONS/DISCUSSION: Superwarfarins were developed in 1970's to overcome warfarin resistance in rats. They are long acting, fat-soluble anticoagulants, and 100 times more potent than warfarin. Their half-life varies from 16 to 69 days compared to 37 hours for warfarin. Brodifacoum is the most commonly used superwarfarin thus the commonest cause of superwarfarin poisoning. Its poisoning is mostly accidental however it can result from suicidal attempt, industrial exposure or deliberate self-poisoning with denial (Munchausen syndrome). Clinical features of brodifacoum poisoning are varied and include epistaxis, hematurea, gastrointestinal bleeding, ecchymosis, hemoptysis, spontaneous abortion and intracranial bleeding. PT, INR and PTT are usually severely prolonged but correct on mixing study. The diagnosis may be confirmed by detecting brodifacoum in blood however it is usually missed on the initial screening test for superwarfarin and may need a special assay. Physicians must have a high index of suspicion of superwarfarin exposure when patients have prolonged unexplained coagulopathy resistant to vitamin K replacement and FFP, along with undetectable warfarin levels and depletion of vitamin K dependent coagulation factors. After correction of initial life threatening coagulopathy by transfusion of blood products these patients should be placed on long-term high dose Vitamin K therapy and closely followed in outpatient clinic.\nTRANSPLANT PATIENT. M. Schaeffer 1 , E. Warm 1 ; 1 University of Cincinnati, Cincinnati, OH (Tracking ID #74228) LEARNING OBJECTIVES: 1) To understand that patients undergoing solid organ transplantation require immunosupressive medications and should undergo thorough evaluation for infectious diseases with consideration of subclinical parasitic infections. 2) To understand that patients taking glucocorticoids have compromised cell-mediated immunity and are susceptible to life-threatening infections by organisms that typically cause mild or no disease in healthy individuals. CASE INFORMATION: Solid organ transplant patients require immunosuppression and are susceptible to many life-threatening infections. We report a case of fatal fulminate infection with Strongyloides stercoralis 3 months following orthotopic heart transplantation. A 54-yearold male underwent orthotopic heart transplantation for ischemic cardiomyopathy. Over the next 6 weeks he required increasing doses of prednisone, tacrolimus and mycophenolate mofetil for graft rejection. 8 weeks after transplant he suffered a perforated colonic diverticulum, and underwent emergent surgery. On postoperative day 14 he developed hypotension, respiratory failure and decreased mental status. S. stercoralis was identified in stool and bronchoalveolar aspirate, and Gram negative bacteremia was found. LP revealed motile S. stercoralis larvae in CSF and Gram negative meningitis. Brain MRI revealed multiple areas of signal change consistent with extensive new infarcts. After 10 days of supportive care and antibiotics, treatment was withdrawn at the family's request and the patient died. IMPLICATIONS/DISCUSSION: Strongyloides Hyperinfection Syndrome (SHS) occurs when massive infestation by S. stercoralis occurs in the lungs, GI tract and rarely in the central nervous system (CNS). Strongyloides stercoralis is a helminthic parasite endemic to the southeastern region of the United States. S. stercoralis has a complex life cycle in the human involving the skin, circulatory system, lungs, and gastrointestinal tract. Compromised host defenses leads to susceptibility to SHS. Along with this massive invasion of S. stercoralis, secondary Gram-negative bacteremia and sepsis are common. CNS invasion is not common but has devastating consequences, as seen in our patient. Perhaps the most damaging aspect of CNS invasion by S. stercoralis occurs when bacteria cross the blood-brain barrier attached to the parasite or within the gut of the worm leading to secondary bacterial meningitis. Although pre-transplant patients infection screening occurs, thorough assessment for parasites is often lacking. In patients in endemic areas and with risk factors for subclinical parasitic infestation, consideration should be given to further evaluation prior to surgery and initiation of immunosupressive medications. Overall mortality of SHS is greater than 80%.\nNEUROSYPHILIS PRESENTING AS FECAL INCONTINENCE. E. Schmidt 1 , B. Mathis 1 , E. Warm 1 ; 1 University of Cincinnati, Cincinnati, OH (Tracking ID #74057) LEARNING OBJECTIVES: 1) Recognize various manifestations of tertiary syphilis. 2) Recognize treatment benefits of neurosyphilis. 3) Recognize that cases of tertiary syphilis may increase in prevalence in the clinic setting following the rise in cases of early syphilis seen in the past two decades. CASE INFORMATION: A 66 year-old African American male with history of treated syphilis at age 25 presented to an outpatient clinic following a hospital stay for recent subcortical stroke and non-Q wave myocardial infarction. He reported fecal and urinary incontinence and subjective memory deterioration for an undetermined amount of time. On exam he was found to have no anal sphincter tone and was unkempt. The patient was admitted to the hospital, and had a spine MRI that was negative for any lesions. He was found to have a SED rate of 117 mm/h, a positive RPR with a 1:1024 titer, and a positive serum FTA-Abs. Lumbar puncture revealed a pleocytosis with a predominance of lymphocytes, an elevated protein at 152 mg/dL, and a positive VDRL at 32 dilutions. The patient was treated for neurosyphilis with 14 days of intravenous penicillin G, 24 million units per day. He subsequently was found to be HIV positive. On clinic follow up, the patient had improved anal sphincter tone, improved physical appearance, and improved subjective cognition. His weight improved by 13 pounds and his RPR had decreased to a titer of 1:512. IMPLICATIONS/DISCUSSION: The patient described above had clear benefit of treatment with apparent reversal of some of the neurologic sequelae of his tertiary syphilis. There is lack of data in the literature supporting reversibility of the neurologic complications of this disease, especially the dementia, that is thought to be related to fibrosis, and therefore the thought of the RPR as a screening test for reversible causes of dementia has been questioned. It has been proposed that there may be an increase in the number of cases of tertiary syphilis in light of the increased number of primary syphilis cases seen in the past two decades, as well as an increase in the HIV population. There must be a heightened level of suspicion for this process in anyone with neurologic findings and history consistent with this diagnosis.\nENDOCARDITIS AS A RED-HERRING. N. Schmidt 1 , R. Witzig 1 ; 1 Tulane University, New Orleans, LA (Tracking ID #77065) LEARNING OBJECTIVES: 1. Develop a thorough diagnostic strategy for fever in patients who use intravenous drugs. 2. Diagnose splenic abcess. CASE INFORMATION: A 25 year-old woman presented with lethargy and fever. She reported a history of intravenous drug use and a recent forearm cellulitis. She had a temperature of 38.28C, a systolic murmur, diffuse abdominal pain, and a purulent pustule on the left leg. The WBC was 19,000. An Abdominal CT scan demonstrated several hypodense lesions in the spleen. Subsequent laparatomy confirmed splenic abscesses, and a splenectomy was performed. Blood and splenic tissue cultures grew methicillin-resistant Staphylococcus aureus. Transesophageal echocardiogram did not show evidence of endocarditis. The patient was diagnosed with impetigo and splenic abscesses with subsequent sepsis. IMPLICATIONS/DISCUSSION: Staphlococcal bacteremia is common in intravenous drug users due to unsterile injection techniques. Not all patients with bacteremia, however, will develop endocarditis. The evaluation of a fever in a patient who uses intravenous drugs should extend past the heart if the patient does not meet the Duke criteria for endocarditis. The local skin infection in this patient led to bacteremia and subsequent splenic abscess formation and sepsis. Spleenic abscesses with or without endocarditis are a frequent complication of patients who use intravenous drugs. Almost all patients have fever and abdominal discomfort, but signs and symptoms of a splenic abscess may be vague and overshadowed by underlying endocarditis. The careful attention to the historical complaint and a methodical and thorough physical examination led to the appropriate diagostic test. Treatment requires splenectomy and antibiotics directed against the involved pathogens. Patients with IVDU and fever require careful evaluation not only for endocarditis, but also for bacterial seeding of other organs. Abdominal signs should prompt the search for intraabdominal abscess formation. We present an unusual case of a patient with progressive and evolving postpartum neurologic changes thought secondary to a pregnancy related vasculitic process. A healthy 30 year-old female (G1 P0) underwent a Cesarean section after unsuccessful induction of labor. The pregnancy was uneventful without signs or symptoms of pre-eclampsia. Six hours postpartum she presented with right-sided hemiparesis and language difficulties. A head CT scan showed a hypodensity in the left caudate and anterior internal capsule. The patient was anticoagulated and evaluated for thromboembolic source. By hospital day 4, the patient's symptoms had nearly resolved. On day 5 she developed recurrence of symptoms and was found to have new areas of infarction including the left cerebellum and posterior frontal lobe. She was started on high dose steroids and cerebral angiogram was done which showed findings consistent with vasculitis. Subsequent imaging showed continuous evolving infarcts and intravenous cyclophosphamide was then added. A complete rheumatologic and hypercoagulable work up was performed and unremarkable except for an erythrocyte sedimentation rate (ESR) of 98 mm/hr on admission that decreased to 52 mm/hr prior to initiation of steroids. The patient was continued on steroids and cyclophosphamide, as repeat imaging 6 months postpartum showed new evolving infarcts. 9 months postpartum, no new areas of infarction were noted and repeat angiogram showed some resolution of changes. 18 months postpartum, the patient continues to improve and is slowly being tapered off of immunosuppressive therapy. The patient has never had any other manifestations of vasculitis other than the cerebral findings as above. IMPLICATIONS/DISCUSSION: The pathogenesis of postpartum cerebral vasculitis remains unclear. It is postulated that vasospasm of the vessels, as seen with migraine headaches, may be triggered by hormonal changes unique to the postpartum period possibly producing an autoimmune reaction resulting in a vasculitis. Contributing factors may also include altered hemodynamics, and the relative coagulopathy associated with pregnancy. Diagnosis of this rare but potentially neurologically devastating disease should be confirmed with angiogram as current therapy with high dose steroids and cyclophosphamide is not without risk. Recurrence of disease with subsequent births has been documented and women diagnosed with postpartum cerebral vasculitis should be counseled on potential risks with subsequent pregnancies. LEARNING OBJECTIVES: 1. Recognize the importance of eliciting a careful review of systems when evaluating patients with fever. 2. Consider prostatitis in the differential diagnosis of methicillin-resistant Staphylococcus aureus (MRSA) bacteremia. CASE INFORMATION: A 53-year-old truck driver with diabetes presented with a three-day history of subjective fever and rigors. On review of systems the patient disclosed urinary urgency and dysuria. Physical examination was remarkable for a slightly enlarged, exquisitely tender prostate. Pertinent laboratory testing showed a mild microcytic anemia with a normal leukocyte count, glucose 275 mg/dl, hemoglobin A1C 11.7%, and a normal PSA. Urinalysis revealed no pyuria and urine cultures were negative. However, cultures of prostate secretions, and four blood cultures obtained before the initiation of antibiotics, were positive for MRSA. A transrectal ultrasound showed large, solid, nodules in the right and left prostate likely representing an infectious process. A biopsy of the prostate revealed severe acute and chronic prostatitis. Cultures of the prostate tissue grew MRSA. Notably, sensitivities from all MRSA cultures were identical. The patient was treated with intravenous vancomycin and his clinical status improved. IMPLICATIONS/DISCUSSION: Coagulase negative Staphylococcus is known to cause chronic prostatitis. Additionally, methicillin-sensitive Staphylococcus aures can cause chronic prostatitis, especially prostatic abscesses. However, we are unaware of prior cases of MRSA bacteremia resulting from MRSA prostatitis. The patient's prostate biopsy showing prostatitis, along with the identical antimicrobial sensitivities of MRSA from both the prostatic tissue and peripheral blood, implicate MRSA prostatitis with hematogenous spread. This is a unique case of MRSA bacteremia, which appears to have resulted from MRSA prostatitis. Furthermore, this case highlights the importance of eliciting a careful review of systems and performing a thorough physical examination when evaluating the source of a fever. LEARNING OBJECTIVES: To recognize Hypokalemic Thyrotoxic Periodic Paralysis and its precipitating factors, especially since it is a rare disorder that is treatable if diagnosed. We describe a case of HTPP likely precipitated by a test. CASE INFORMATION: The patient is a 59-year-old African-American female admitted for hypokalemia after a colonoscopy. The colonoscopy was for evaluation of her microcytic, hypochromic anemia and a recent history of a 30 lb. weight loss. The colonoscopy was negative, and a CT scan of the chest, abdomen, and pelvis was done for further evaluation of possible malignancy which was also negative. Review of systems was notable for bilateral lower extremity weakness in addition to the weight loss. Two months prior, she had a persantine thallium stress test for evaluation of chest pain which was positive and was followed by an angiogram which showed only moderate coronary artery disease requiring no intervention. Since the stress test, she had progressive lower extremity weakness associated with muscle cramps and had difficulty walking, standing at length, and had difficulty climbing stairs. Physical exam revealed tachycardia, thyromegaly, 3/5 motor strength of bilateral lower extremities, and areflexia. The remainder of the examination was unremarkable. Labs revealed a potassium level of 2.9 mg/dL, and oral and intravenous potassium replacement was given. ECG revealed a sinus tachycardia with left atrial enlargement. A thyroid workup revealed a TSH of <0.01 and a free T4 of 8.33. We diagnosed hypokalemic thyrotoxic periodic paralysis likely precipitated by iodine in the dye. The patient was started on propylthiouracil 150 mg three times daily and metoprolol. The patient was referred for radioactive thyroid scan and possible radioablation therapy. At discharge, the patient's hypokalemia and symptoms had resolved. IMPLICATIONS/DISCUSSION: HTPP is a rare, sporadic, acquired disorder usually seen in Asians. The incidence is <1% in Americans. The disease is associated with an underlying thyrotoxicosis (commonly Grave's disease or thyroiditis) and hypokalemia. The serum potassium level is low, but the total body potassium remains normal. Thyroid hormone mediates an increase in sodium-potassium ATPase activity and a transcellular potassium influx. This interferes with potassium's involvement in promoting muscle contraction Treatment of HTPP includes antithyroid drugs, beta-blockers, and cautious use of potassium. Definitive treatment involves radioablation therapy. The current vignette helps one to recognize the signs and symptoms of HTPP, and describes a case that was precipitated by dye used in an angiogram. LEARNING OBJECTIVES: 1) Review the differential diagnosis of an enlarged uterus in a non-pregnant woman.\n2) Recognize intravenous leiomyomatosis as a rare cause of intracardiac tumors. CASE INFORMATION: A 56 year-old postmenopausal woman with a history of uterine fibroids presented with two days of exertional shortness of breath and constant retrosternal chest pressure, worse with exertion and lying flat. She denied orthopnea, paroxysmal nocturnal dyspnea or cough. Physical exam revealed normal vital signs, elevated JVD, clear lungs, regular rhythm with II/VI holosystolic murmur at the left lower sternal border, no extremity edema, and an enlarged uterus palpable 4 cm below the umbilicus. Basic labs, cardiac enzymes, and EKG were normal. Chest x-ray showed cardiomegaly and vascular prominence. An echocardiogram revealed normal left ventricular ejection fraction, dilated right atrium (RA) and right ventricle (RV), and tricuspid regurgitation. There were two linear mobile echodensities in the inferior vena cava (IVC) protruding into the right atrium and ventricle. Cardiac and abdominopelvic MRI showed multiple uterine leiomyomas, with a lobulated mass arising from the uterine fundus and extending up the right gonadal vein into the IVC, RA, and RV. The patient underwent total hysterectomy and total excision of the mass through the infrarenal IVC. Pathology of the mass showed intravenous leiomyomatosis. IMPLICATIONS/DISCUSSION: The differential diagnosis of an enlarged non-pregnant uterus includes endometrial and myometrial processes. Advanced endometrial carcinoma is the main endometrial cause. Myometrial causes are divided into benign and malignant. Benign conditions include leiomyomas (i.e. fibroids), adenomyosis, and stromal adenomyosis. Malignant causes include leiomyosarcoma and endometrial stromal sarcoma. Uterine conditions capable of extending into the IVC and heart are endometrial stromal sarcoma and intravenous leiomyomatosis. Intravenous leiomyomatosis is a rare benign condition in which nodules of benign smooth muscle tissue grow within the veins of the myometrium and may extend into the uterine and hypogastric veins. Rarely, these tumors extend up the IVC into the heart. Only 43 cases of intravenous leiomyomatosis with cardiac extension have been reported in the English literature. Symptoms of cardiac extension include venous obstruction, right-sided heart failure, and rarely, sudden death. Successful therapy entails total excision of the tumor, as residual tumor could result in recurrence. 3) Identify Salmonella as a rare infectious cause of spondylodiscitis. CASE INFORMATION: A 65-year-old woman presented with four days of low back pain and fever. The pain radiated down her left leg and was aggravated by movement. She denied trauma, weakness or numbness. She had a temperature of 104, blood pressure of 154/76, and heart rate of 115. Physical exam showed lumbar paraspinal spasm with no focal tenderness. Neurological exam was normal, including straight leg test. White cell count was 14000 with 90% neutrophils. ESR was 64. Blood and urine cultures were negative. Lumbar x-rays were normal. An indium scan done as part of the workup for fever and leucocytosis showed increased uptake at L4-L5. MRI of the spine revealed disc herniation at L4-L5, but no spondylodiscitis. Even though the source of fever was uncertain, the patient was treated empirically with vancomycin with resolution of fever. However, the pain persisted and she underwent discectomy. Pathological exam showed acute discitis, but cultures were not done. Despite the surgery, her back pain worsened, and repeat imaging showed destruction of L4 and L5 vertebral end plates, with prevertebral, epidural and paraspinal abscess. A CT-guided biopsy was done, and Salmonella javiana grew from the drained fluid. The patient had hemoglobin C trait but no immunocompromising conditions. She was treated with fluoroquinolones and slowly improved. IMPLICATIONS/DISCUSSION: Most cases of back pain are due to simple benign etiologies. However, certain red flags prompt the search for more serious causes. These include advanced age, malignancy, signs of infection, and progressive neurological symptoms. Spondylodiscitis is the simultaneous infection of the vertebral body and disc space. MRI is the most sensitive and specific imaging modality, with a sensitivity of 91% during the first two weeks of symptoms. Radionuclide scanning is done when MRI cannot be performed. It is very sensitive but not as specific. CT-guided biopsy should be pursued in cases of suspected spondylodiscitis, even with a normal MRI. Most cases of spondylodiscitis are due to Staphylococcus aureus. Other organisms include Streptococcus, E. coli and Salmonella, with the most frequent serotypes being Salmonella typhimurium and S. choleraesuis. Predisposing factors for salmonellosis include sickle cell disease, HIV, immunosuppressive therapies and previous trauma. Spondylodiscitis due to S. javiana has not been previously reported. Hemoglobin C disorders have not been associated with Salmonella osteomyelitis.\nTHE YOUNG WOMAN WITH NUMB FEET. J.J. Silverman 1 ; 1 University of Pennsylvania, Philadelphia, PA (Tracking ID #75939) LEARNING OBJECTIVES: 1) Recount the most common causes of a symmetric polyneuropathy; 2) Perform the initial evaluation of a polyneuropathy 3) Recognize an uncommon presentation of hypothyroidism. CASE INFORMATION: A 28 year-old female with no significant past medical history presents complaining of bilateral foot and hand numbness for the past six weeks. The sensation begins mid-foot, occurs intermittently throughout the day, and she specifically notes worsening symptoms walking on the treadmill at the gym. She reports similar but milder symptoms in her hands, which are not related to activity. Review of systems is otherwise notable for chronic constipation and longstanding irregular menses (since menarche at age 14). Family history is negative for polyneuropathies, cancer and thyroid disease. She smokes two packs of cigarettes per week, does not drink alcohol or use drugs, and denies toxic exposures. She has no history of blood transfusions, reports regular condom use with her two prior sexual partners, and is not presently sexually active. She takes fexofenadine as needed for seasonal allergies. Physical exam revealed a well-appearing young woman, who was afebrile, with a blood pressure of 130/80 and a heart rate of 74. Neurologic exam was notable for decreased sensation to light touch and pinprick in the plantar aspect of her right foot, as well as decreased vibratory sense in her right foot. The remainder of the physical exam was unremarkable. Labs were notable for a TSH of 109, FTI of 2.9, Thyroxine of 2.7 and T3U of 1.06. IMPLICATIONS/DISCUSSION: Polyneuropathy is not an uncommon problem in the primary care setting. The differential diagnosis is quite broad, but chronic systemic disorders, hereditary causes and inflammatory diseases account for the majority of cases. Chronic medical conditions commonly associated with polyneuropathies include diabetes mellitus, malignancy, collagen vascular disease, organ failure, B12 deficiency, alcoholism, AIDS, toxic exposures and plasma cell dyscrasias. Though the history can direct the initial evaluation, for most cases it will include a CBC, basic metabolic panel, liver panel, ANA, SED rate, B12, folate, lead level, RPR, HIV, hepatitis serologies and TSH. If this initial evaluation is inconclusive, an EMG and neurology consult are warranted. Finally, it is important to realize that hypothyroidism is protean in its manifestations, and can present without its most commonly recognized signs and symptoms.\nSHORTNESS OF BREATH: A``NEGATIVE'' LOWER EXTREMITY DOPPLER STUDY. C.W. Simpkins 1 , C. Passaretti 1 , A. Zaas 1 , J. Cofrancesco 1 ; 1 Johns Hopkins University, Baltimore, MD (Tracking ID #76910) LEARNING OBJECTIVES: 1. Assess subacute progressive dyspnea in a young woman. 2. Recognize the presentation of pulmonary sarcoidosis. 3. Recognize the pathology and pathophysiology of early pulmonary sarcoid. CASE INFORMATION: A 48 year old African American female with hypertension, gastroesophageal reflux disease, bipolar disorder, a remote history of lithium therapy and history of total abdominal hysterectomy and bilateral salpingoophorectomy on hormone replacement therapy (HRT), presents with slowly progressive dyspnea on exertion, orthopnea, and paroxysmal nocturnal dyspnea for one year. Lower extremity duplex ultrasounds were negative for blood clot but did show inguinal adenopathy. Review of systems included loud snoring, possible apneic episodes during sleep, nonrestful sleep, intermittent lower extremity swelling, nonproductive cough for 1 week, but no daytime somnolence, fevers, chills, or sweats. She had an 18 pound intentional weight loss over the past year. No history of alcohol, tobacco, or illicit drug use. Physical exam: temperature 100.5 degrees F, blood pressure 146/57 mmHg, pulse 67 bpm, respiratory rate 20, pulse oximetry 95% on 2 liters supplemental oxygen. Patient was a mildly obese woman in no acute distress, with no palpable adenopathy, slightly elevated jugular venous pulse, normal air movement except for rare bibasilar crackles, a prominent P2 heart sound, and no peripheral edema, cyanosis, or clubbing. Arterial blood gas on room air: pH 7.44/PaCO2 35/PaO2 55/bicarb 23. Ventilation/perfusion (V/Q) scan was low probability for pulmonary embolism. Pulmonary function tests showed moderate combined obstructive and restrictive physiology with severe gas transfer defect. Computed tomography (CT) of the chest showed enlarged right atrium, bulky mediastinal adenopathy, and diffuse patchy ground glass infiltrates without effusion or consolidation. Echocardiogram showed normal left ventricular (LV) size and function, moderate dilatation of the right ventricle (RV) and right atrium (RA) with flattened septum. A diagnostic procedure was performed. IMPLICATIONS/DISCUSSION: Initial differential diagnosis included pulmonary embolus (young woman on hormones), sleep apnea (given the pulmonary hypertension), and hypertensive cardiomyopathy with heart failure. However, the negative V/Q and CT, and the bulky lymphadenopathy and patchy ground-glass infiltrates were suspicious for pulmonary sarcoid. Bronchoscopy with lavage and biopsy confirmed this diagnosis with negative cultures, peri-mucosal granuloma and reactive histiocytes. The patient was diuresed, started on high dose steroids with slow taper, and is doing well. The diagnosis, pathophysiology and treatment of pulmonary sarcoid will be discussed. LEARNING OBJECTIVES: 1) Review the differential diagnosis of normocytic anemia. 2) Recognize the cause of normocytic anemia by mixed processes of microcytic and macrocytic anemia.\n3) Appreciate the wide and unusual manifestations of Vitamin B12 deficiency. CASE INFORMATION: A 56 year-old black male with history of hypertension presented to the outpatient clinic with six month history of intermittent low grade fever. On review of systems, he denied cough, chest pain or shortness of breath, palpitations, gastrointestinal bleed, urinary or bowel symptoms, arthritis, weight loss, numbness or weakness. Vital signs revealed temp 100.5, pulse 116, blood pressure 158/96, respirations 18. Physical exam was significant for pale conjunctiva, guaiac negative stool, and slight decreased sensation to light touch, pinprick, and vibration in the lower extremities. Labs showed Hgb 6.7, MCV 87 with normal WBC and platelet with blood smear displaying nucleated rbcs, anisocytosis, poikilocytosis, and isolated macrocytes. A work up including chemistries, cultures of blood and urine, ANA, ESR, and TSH was unremarkable. A vitamin B12 level was 108 pg/mL (normal 180±914 pg/mL) and hemoglobin electrophoresis showed beta thallasemia. With Vitamin B12 supplementation, the patient's fever resolved. IMPLICATIONS/DISCUSSION: The differential diagnosis of normocytic anemia includes chronic diseases, inflammation, endocrinopathies and renal diseases. In some cases, the simultaneous processes of microcytic and macrocytic anemia should also be in the differential diagnosis of normocytic anemia. The abnormal blood smear with size variation of erythrocytes and neuropathy led to the work up and concomitant diagnosis of beta thallasemia and Vitamin B12 deficiency. The clinical manifestations of Vitamin B12 deficiency include fatigue, glossitis, vomiting, diarrhea, dementia, and neuropathies, but fever is not considered a typical feature. From the literature, fever was found in approximately 40% of patients with megaloblastic anemia caused by folate and Vitamin B12 deficiencies in two case studies. The mechanism of the fever is uncertain, but one proposed theory is the increased activity and ineffective erythropoiesis in the bone marrow. Fever resolves rapidly with treatment and failure to do so should prompt a search for other causes. In summary, normocytic anemia can be caused by dual processes of microcytic and macrocytic anemia and fever should be recognized as an atypical feature of Vitamin B12 deficiency.`D LEARNING OBJECTIVES: 1) Differentiate between gynecomastia and breast cancer 2) Recognize the causes and treatment of gynecomastia. CASE INFORMATION: The patient is a 67 year-old male with alcoholic cirrhosis who presented with a painful lump in his right breast. Medications included spironolactone. Social history was significant for one year of sobriety and current tri-weekly use of marijuana. On physical exam, a 3 by 3 centimeter subareolar, tender, mobile, rubbery right breast mass was palpated. A smaller, similiar mass was palpated on the left. There was no nipple discharge or retraction, overlying skin changes, or lymphadenopathy. His testosterone was normal. A diagnosis of bilateral gynecomastia was made, felt to be due to his cirrhosis, compounded by his use of spironolactone and marijuana. The patient stopped both drugs. Two months later, the patient had significant improvement in his pain and decrease in breast tissue. IMPLICATIONS/DISCUSSION: Gynecomastia is common in adult males. The highest prevalence is between 50 to 80 years, with an incidence of 24 to 65 percent. Patients present with a unilateral or bilateral breast mass that may be painful. The most important differentiation is between gynecomastia and breast carcinoma. Carcinoma is less common, usually unilateral, and eccentric rather than symmetric to the nipple/areola. It is firm, fixed and may be associated with skin dimpling, nipple retraction or discharge, and lymphadenopathy. The conditions associated with gynecomastia are felt to represent an imbalance between estrogens and androgens. The most common causes in the adult male are as follows: idiopathic, drugs, cirrhosis, malnutrition, primary hypogonadism, testicular tumors, secondary hypogonadism, hyperthyroidism, and chronic renal insufficiency. Causal drugs include antiandrogens, antibiotics, antiulcers, chemotherapeutics, cardiovascular drugs, and drugs of abuse. The history, physical, and certain diagnostic tests can identify the cause in the majority of patients. Patients with a pain, acute onset, or without an obvious etiology should be evaluated with serum hCG, luteinizing hormone, testosterone, and estradiol levels. An asymptomatic patient found to have gynecomastia incidentally, deemed to be idiopathic, does not require further tests and should be reevaluated in six months. Otherwise, treatment should be directed at removing the underlying cause. Active treatment with androgens, antiestrogens, and aromatase inhibitors is indicated in persistent gynecomastia associated with severe pain, tenderness, or embarrassment. Surgical therapy, including liposuction or excision, is considered in patients who do not respond to medical therapy.\nCAN'T WE JUST LET HER LEAVE? T. Spector 1 , P.P. Balingit 1 , A.G. Gomez 2 ; 1 UCLA San Fernando Valley Program, Sylmar, CA; 2 University of California, Los Angeles, Sepulveda, CA (Tracking ID #76525) LEARNING OBJECTIVES: 1. To recognize and avoid counter-transference with difficult patients. 2. To maintain a professional attitude in difficult circumstances. 3. To discuss when a patient has capacity to refuse treatment. CASE INFORMATION: A 36 year old female with a history of polysubstance abuse presented with 2 weeks of fevers and painful forearm lesions after skin-popping heroin. The patient was an ill-appearing, disheveled woman. Temperature was 38.4C, BP 114/68, heart rate 112. Multiple abscesses were noted on her forearms. A II/VI holosystolic cardiac murmur over the tricuspid area, not heard on previous exams, was also present. EKG and CXR were normal. The patient was admitted with the diagnosis of probable infective endocarditis. Within minutes of arriving to the monitored unit, she demanded to be moved to a room with a TV and swore at the nurses when they refused to give meperidine for her pain. She also demanded that the intern be paged until methadone was ordered for her heroin withdrawal. The intern arrived to the unit after the patient had ripped off her monitor leads, pulled out her IV line and was dressing to leave the hospital. A panicked intern told the resident that the patient was leaving because she felt her needs were being ignored. A psychiatrist was urgently called to determine her capacity to refuse treatment, but she left before the psychiatrist arrived. The resident found the patient outside the hospital. The patient was allowed to vent her frustrations with much vehemence and profanity. Satisfied that her grievances were heard, she allowed the resident to fully explain the benefits of treatment and the risks of refusing care, including death. The patient was alert, oriented, and able to repeat the risks clearly prior to leaving. IMPLICATIONS/DISCUSSION: Most internists are enthusiastic about finding solutions to difficult medical problems. However, a patient's psychological or social problem may not be greeted with the same enthusiasm. It is not uncommon for a patient's personality trait or attitude to invoke anger, disdain or frustration in health workers. Caring for such patients with compassion becomes a challenge. It is important to understand one's own response to difficult patients in order to recognize and prevent counter-transference from interfering with sound and appropriate medical decisions. One must also recognize that the patient's decision to leave without treatment, although a poor one by medical standards, was certainly within her rights. It was important to appease the patient's anger to assure that she made an informed decision to leave against medical advice rather than out of rash frustration and anger. By clearly repeating the risks and consequences, she demonstrated legal capacity to leave and was empowered with the knowledge to return or seek care elsewhere. Definitive guidelines exist that dictate when a physician can treat a patient against his will.\nAN UNCOMMON CAUSE OF HYPOGLYCEMIA. S. Agresta 1 , B. Springgate 1 ; 1 Tulane, New Orleans, LA (Tracking ID #75148) LEARNING OBJECTIVES: 1. Recognize ethanol ingestion as an important cause of sustained hypoglycemia. 2. Provide appropriate treatment for ethanol-induce hypoglycemia. CASE INFORMATION: A 37 year-old man presented after a syncopal episode. Seven months earlier he was diagnosed with alcoholic liver disease. On presentation, he was tremulous and diaphoretic. The remainder of the physical exam was normal. An initial blood glucose was 30 mg/dL. He received one ampule of intravenously D50. Twenty minutes later the symptoms returned. His glucose was 28. Despite additional intravenous dextrose, his glucose continued to drop to between 30 and 40 mg/dl. His AST was 175 U/L, ALT 73 U/L, Hgb A1C 4.6%, and C peptide 15.5 ng/mL (nl). Drug and ethanol screens were negative. After two days of continuous dextrose-containing intravenous fluids, his blood glucose level returned to normal. IMPLICATIONS/DISCUSSION: Alcohol-induced hypoglycemia is an important but infrequently diagnosed sequella of ethanol ingestion. Symptoms are precipitated by a period of fasting followed by ingestion of alcohol. Ethanol inhibits the normal hepatic gluconeogenesis response to starvation-induced hepatic glycogen depletion. As this inhibition may be gradual, hypoglycemic symptoms may develop slowly. Patients may not demonstrate acute alcohol toxicity at the time of presentation. 1 Continuous intravenous glucose is required to prevent recurrent hypoglycemia and to replete hepatic glycogen stores. Chronic alcoholics may also have inhibition of pituitary ACTH, creating a hypo-cortisolemic state, exacerbating the hypoglycemia. surpassed those of the squamous cell type. The general locations of adenocarcinoma metastasis are lymph nodes and liver, and occasionally lung and bone. We report a case of adenocarcinoma of the esophagus with leptomeningeal carcinomatosis. A 56 year old thin white male presented with dysphagia, and a 40 pound weight loss over six months. He also had nausea, vomiting, abdominal pain, rectal bleeding, and diplopia during this period. On exam, he was alert and oriented. He had sluggish reaction of his left pupil, diplopia on downward gaze, guaiac positive stool, and a microcytic anemia (MCV 61). Esophagogastroduodenoscopy showed a mass that extended from the 28th cm of the esophagus to the 37th cm including the GE junction. Whole body CT scan showed an isodense mass in the foramen of Monro, midesophageal thickening, multiple hepatic lesions, and celiac lymphadenopathy. Brain MRI showed leptomeningeal carcinomatosis surrounding the pons and multiple cranial nerves. The patient received urgent whole brain radiation. Upon completion of radiation an Ommaya reservoir was suggested for intrathecal methotrexate. However, his overall quality of life deteriorated rapidly and he chose to receive hospice care. IMPLICATIONS/DISCUSSION: Metastatic sites for esophageal cancer typically involve the liver and lymph nodes and rarely lung and bone. Direct CNS involvement is an extremely rare occurrence. On identification of the leptomeningeal carcinomatosis our patient was able to undergo whole brain radiation to prevent any immediate risk of herniation, but given the extent of his disease his prognosis was extremely poor. The reasons why this adenocarcinoma spread to involve the CNS are unclear, and further study needs to be undertaken. As esophageal adenocarcinoma becomes more common, this type of presentation may be seen more frequently, and clinicians should be aware of this. week. The test took place at 6 AM with an ambient temperature of 75F. At the end of the test the patient collapsed complaining of``complete muscle failure''. He was found to be hypotensive and tachycardic. The patient's history was significant for the use of an herbal supplement called Energel. He had been taking this consistently for the past month to try improve his performance time. Laboratory results initially revealed severe rhabdomyolysis with a creatinine kinase (ck) of 11,000. Acute renal failure secondary to pigment-induced ATN was demonstrated by a serum creatinine (cr) of 2.4 mg/dl and urine positivity for myoglobin. The patient's ck peaked at 240,000 during his hospital course. After 8 days his ck was 45,000 and his cr returned to normal. IMPLICATIONS/DISCUSSION: Energel is a herbal supplement that contains Ma Huang. Ma Huang is the Chinese version of Ephedra which grows in many parts of the world and is used predominantly as a stimulant. Ephedra alkaloids are structurally similar to ephedrine and pseudoephedrine. They stimulate both alpha and beta-adrenergic receptors as well as release norepinephrine from storage sites. Each tablet of Energel contains 330 mg of Ma Huang extract, which is 6% (19.8 mg) of Ephedra. The dose is 2 gelcaps a day. Our patient took this dose. The FDA has banned the sale of Ephedra in large amounts (over 24 mg a day and does not recommend taking this product in small amounts for more than 7 consecutive days. Our patient was taking 40 mg a day for 30 days. Ephedra has caused 17 deaths and hundreds of serious side effects including stroke, myocardial infarction and seizures in previously healthy young adults. To this date there have been no reported cases of rhabdomyolysis associated with Ma Huang. This is an extremely important health concern since Ephedra, in its many forms, is readily available in unscrutinized and easily obtainable health supplements.\nIATROGENIC STROKE AND UNDIAGNOSED DELIRIUM: OPPORTUNITIES TO IMPROVE CARE FOR HOSPITALIZED ELDERS. R. Sudore 1 , C. Landefeld 1 ; 1 University of California, San Francisco, San Francisco, CA (Tracking ID #73989) LEARNING OBJECTIVES: To describe the risk of stroke after cardiac catheterization and to recognize iatrogenic impediments to stroke rehabilitation in elderly patients. CASE INFORMATION: An independent 73 yr-old man with a history of NIDDM and HTN was transferred for cardiac catheterization (cath) after a recent non-Q-wave MI. An uneventful cath revealed 5 vessel disease and an ejection fraction of 30%. 90 minutes post cath he developed right hemiparesis and receptive and expressive aphasia. Head CT was negative and TPA was administered within 2 hours of symptom onset, but without clinical improvement. Two weeks later, neurologic function had not improved; agitation developed and hand restraints and diazepam were started. On consultation, we confirmed the sequelae of stroke, identified ischemic lesions in three toes, and diagnosed delirium. Serum sodium was 128, but other lab studies were normal. After discontinuing diazepam and restricting free water, his agitation resolved, and his dysphagia and receptive aphasia improved. Physical therapy was started. Once his restraints were discontinued his strength improved, though other than feeding, he remained dependent in all activities of daily living. IMPLICATIONS/DISCUSSION: This case highlights the importance of iatrogenic stroke and of undiagnosed delirium. Stroke occurs unpredictably in 0.1% of persons undergoing cath. Stroke is most often due to emboli, as evidenced by this man's cerebral and toe ischemia. His risk factors for stroke from cath included advanced age, extent of coronary artery disease, and depressed ejection fraction. In-hospital delirium incidence on medical wards varies from 11± 33%; the diagnosis is often missed in elderly patients. Delirium increases the risk of death 8fold and the frequency of nursing home placement 5-fold. Diazepam likely led to this man's delirium and slowed his rehabilitation. Also, restraints have been shown to exacerbate delirium, adding to deconditioning and slow recovery. In addition, the incidence of delirium is decreased from 15% to 10% by a multicomponent inter-vention including avoidance of benzodiazepines and restraints, but this intervention is not implemented in most hospitals, including ours. As more elders undergo cath, iatrogenic stroke may occur more often. Simple clinical strategies are indicated to prevent delirium, which is often missed and may slow or prevent rehabilitation from stroke.\nA. Sura 1 , R. Granieri 1 ; 1 University of Pittsburgh, Pittsburgh, PA (Tracking ID #73858) LEARNING OBJECTIVES: 1. To recognize the impact of severely elevated triglycerides 2. To recognize the common causes of acute pancreatitis 3. To evaluate and manage the patient presenting with acute pancreatitis CASE INFORMATION: KD is a 17 year old female with a history of hypertriglyceridemia who presented to the emergency department with severe abdominal pain, nausea, and vomiting. She also complained of dizziness and lightheadedness. She denied alcohol use, medication use, family history of pancreatitis, history of gallstones or trauma. Although her symptoms were similar in character to her previous episodes of pancreatitis, they were much more severe. At the time of presentation, she was febrile to 103 degrees Fahrenheit and orthostatic. Her examination revealed significant abdominal distention, absent bowel sounds, and exquisite diffuse tenderness to palpation. There was no rebound tenderness. Her lab work revealed a hemoglobin of 6.8 (normal for her was 15), WBC 15.8, albumin 2.3, calcium 6.5 and triglyceride level of 11,000. Lab work also showed Na 133, K 3.3, Cl 100, CO2 22, BUN <2, Cr 0.4, glucose 217, lipase 1221, amylase 117, triglycerides >11,000, total cholesterol 245, HDL 14, Ca 7.4, albumin 2.3, Tbili 1.6, AST 25, ALT 22, GGT 83 and alkaline phosphatase 96. CT scan of the abdomen revealed severe necrotizing pancreatitis with necrosis of the pancreatic duct, large amount of ascites, and a large retroperitoneal phlegmon. She was started on imipenem, given blood transfusions, and maintained NPO. She was admitted to the ICU for severe necrotizing pancreatitis. IMPLICATIONS/DISCUSSION: Acute pancreatitis is an acute inflammatory process of the pancreas. It is associated with severe acute upper abdominal pain and elevated serum levels of pancreatic enzymes. Most cases are associated with gallstones or alcohol, but the precise pathogenetic mechanisms are not understood completely. Other causes of acute pancreatitis include biliary sludge, post-procedure, hypertriglyceridemia, hypercalcemia, drugs, trauma, HIV, and hereditary causes (pancreas divisum or a genetic mutation). The diagnosis is made via clinical exam, laboratory findings, and, if needed, imaging studies. CT scan is the most helpful imaging test for the diagnosis and its intrabdominal complications. Serum triglyceride concentrations above 1000 mg/dl can precipitate attacks of acute pancreatitis and may account for 1.3±3.8% of cases. The clinical manifestations of hypertriglyceridemia associated pancreatitis are similar to those seen with other causes with the exception that, for poorly understood reasons, the serum amylase may not be elevated substantially. Clinical assessment of severe pancreatitis is as accurate as most scoring systems. Treatment of acute pancreatitis is aimed at correcting predisposing factors and at the pancreatic inflammation itself. General management consists of supportive care with intravenous hydration, pain management, antibiotics, parenteral or enteral (jejunal) feeding, and in severe cases, necrosectomy.\nACUTE-ONSET ADULT TRACHEOBRONCHOMALACIA. A.C. Sura 1 , M. Ghajarnia 1 , R. Granieri 1 ; 1 University of Pittsburgh, Pittsburgh, PA (Tracking ID #73859) LEARNING OBJECTIVES: 1) To recognize the clinical presentation and diagnostic workup of tracheobronchomalacia in adults. 2) To manage the patient with severe hypoxemia secondary to tracheobronchomalacia. CASE INFORMATION: A 74 year-old male with hypertension and degenerative joint disease presented for preoperative evaluation prior to hip replacement. He endorsed progressively worsening dyspnea with exertion in the past three weeks, orthopnea and lower extremity edema. His oxygen saturation was found to be 69% on room air. He denied fever, chills, chest pain, or chronic cough but endorsed occasional wheezing and cough productive of white sputum in the past two months. He had a distant history of 20 pack-years tobacco use as well as a 50 year history of work in a coal mine. On physical, blood pressure was 147/84, pulse of 69, and respiratory rate of 25; oxygen saturation improved to 91% on non-rebreather facemask. ABG revealed respiratory acidosis with metabolic compensation. The patient developed ventilatory failure with a drop in O2 saturation to 45% and PCO2 of 140. He was intubated, and chest CT revealed left lower lobe collapse. Bronchoscopy showed complete collapse of the left mainstem bronchus. The patient's subsequent hospital course consisted of intermittent respiratory failures requiring repeat intubations. Once extubated, he was maintained on supplemental oxygen and BiPAP. Repeat bronchoscopy found similar findings of flaccid airways. The patient was deemed ineligible for stent placement and high risk for distal collapse given extensive airway involvement. The patient was discharged on oxygen and BiPAP but subsequently weaned off of supplemental oxygen completely. IMPLICATIONS/DISCUSSION: Hypoxemia and hypercarbia refractory to standard treatments for obstructive pulmonary disease may result from airway collapse without any immediately identifiable causes. Well recognized in infants, tracheobronchomalacia (TBM) may also be present in 4.5±15% of all adult patients with respiratory complaints and can mimic chronic bronchitis. It is a disease of middle to late age, more common in males, and in those with a history of chronic respiratory irritation or inflammation but without a clear association to obstructive lung disease. Identified pathology has been limited to a decrease in the longitudinal elastic fibers of the trachea. Bronchoscopy under local anesthesia is the diagnostic gold standard, demonstrating airway narrowing greater than 50% during expiration. Also supportive are pulmonary function tests showing a low FEV1/EIV1 ratio and characteristic notching in the forced expiratory spirogram. Bronchodilators, antibiotics, and mucolytics may be beneficial in patients with concomitant obstructive airway disease or asthma; however, definitive treatment in TBM involves reversing the airway collapse. Avoidance of airway irritants and cough suppression are first line measures. Surgical stenting has been used with success in many cases, but in patients too frail to undergo such procedures or in those with extensive airway involvement, positive airway pressure appears to be the best means of medically managing patients with TBM. Moreover, airway inflammation may play a role in acute airway collapse, and the element of reversibility noted in our patient may point to a role for adjuvant anti-inflammatory medications.\nYOUNG WOMAN. G. Szabo 1 , E. Warm 1 ; 1 University of Cincinnati, Cincinnati, OH (Tracking ID #74064) LEARNING OBJECTIVES: 1) Recognize the potential for lindane toxicity in adults.\n2) Diagnose the signs and symptoms of lindane toxicity in adults. CASE INFORMATION: The toxicity of lindane is well known for the pediatric population, but is generally under appreciated among adults. Inappropriate application of lindane by adults can lead to significant neurologic changes and even death. A 22-year-old thin female without significant medical history was brought to the emergency room by her mother after one day of agitation, visual and tactile hallucinations, paranoia and a five-day history of rash. The rash consisted of pruritic erythematous macules on her abdomen and body. The patient was seen at an outlying emergency room, diagnosed with scabies and given lindane cream. Her rash persisted and she went to another emergency room for evaluation; again she was diagnosed with the scabies and prescribed lindane. 2 days later she presented with the above symptoms. Her exam showed numerous crusted, mildly erythematous erosions and excoriations throughout her body. No vesicles or burrows were noted. She was agitated with paranoia and visual hallucinations. An extensive workup was essentially negative and included a normal lumbar puncture, negative blood cultures and herpes simplex PCR, an unremarkable head CT, no vasculitic change on skin biopsy, and negative serologic studies for RMSF, varicella zoster, syphilis, cryptococcus and HIV. After her sensorium cleared she acknowledged that she left the lindane cream on for 12 to 16 hours, applied it all over her body and did not wash her sheets. She was discharged in stable condition without any neurologic deficits. IMPLICATIONS/DISCUSSION: Although lindane deposits in all lipid tissues, it has a high propensity for the white matter of the brain. Generally, children are at higher risk for toxicity because of their smaller size and lipid deposits. However, thin adults like the one described above, are also susceptible. Lindane absorption is enhanced by its contact with irritated skin, another risk factor also present in our case. Lindane toxicity is a clinical diagnosis and as such must be kept in mind especially in patients presenting with a history of ascabies-like rash and mental status changes.\nS. Tadic 1 , J. Hefner 1 ; 1 University of Pittsburgh, Pittsburgh, PA (Tracking ID #76487) LEARNING OBJECTIVES: 1.) Recognize an unusual presentation of a hypertensive emergency in a young person; 2.) Diagnose an atypical presentation of a pheochromocytoma; 3.) Evaluate and treat a catecholamine-producing tumor. CASE INFORMATION: A previously healthy 27-year-old Vietnamese male was brought to the emergency room with an acute onset of an expressive aphasia and left hemiparesis. He was unresponsive upon arrival and required intubation. A head CT revealed a large subcortical intracranial hemorrhage (ICH) involving the right basal ganglia. The patient underwent an emergent right craniotomy with evacuation of the intracerebral hemorrhage and placement of an extraventricular drain. Postoperatively, he was admitted to the Neuro Intensive Care Unit (NICU) for monitoring. Over the next 72 hours the patient was observed by nursing staff to have periods of intermittent hypertension, treated with prn IV medications and hypotension, treated with fluid boluses (SBP range 88±210). After 4 days in the NICU, the patient was observed to have facial and chest flushing. A pheochromocytoma was suspected. Abdominal MRI revealed bilateral large adrenal masses with increased T2-weighted signal. Results of a 24hour urine collection for metanephrines and catecholamines included: metanephrines 6,320 (26±230 mcg/24 hr); normetanephrines 24,760 (44±540 mcg/24 hr); VMA 16.5 (6 or less mg/ 24 hr); and total metanephrines 31,080 (90±690 mcg/24 hr). Urine dopamine 2,136 (80±440 mcg/24 hr); epinephrine 423 (0±25) and norepinephrine 3,071 (0±100). Calcitonin level was 5 (0-8) and genetic testing for von Hippel-Lindau (vHL) syndrome was negative. The patient developed a right femoral DVT and tested positive for the Lupus Anticoagulant. He underwent a bilateral adrenalectomy and was discharged to home 2 months later on Coumadin and Hydrocortisone. Pathology revealed malignant pheochromocytoma and a metastatic workup was negative. His post-surgical evaluation revealed normal levels of urine metanephrines and catecholamines. IMPLICATIONS/DISCUSSION: Although pheochromocytoma is a rare disease, this case represents the value of bedside evaluation by a physician. Paroxysmal symptoms (i.e. intermittent) can often be treated with prn interventions leading to misdiagnosis or delay in diagnosis. A rare presentation of pheochromocytoma is one of cyclic fluctuations of HTN and hypotension that occurs via an uncertain mechanism. While most pheochromocytomas are sporadic, bilateral tumors are often associated with 2 primary familial disorders: MEN-2 and VHL. Measurement of plasma or urine catecholamines and metabolites are diagnostic in 95% of patients with symptoms. MRI scans are sensitive (98±100%) but are only 70% specific. On T2-weighted images, however, pheochromocytomas appear hyperintense while other adrenal tumors appear isointense. While there is no universally accepted method of preparation for surgery, alpha-adrenergic blockade followed by beta-blockers with surgery 2 weeks later, is common practice. Recurrence rates are 14% at 10 years. LEARNING OBJECTIVES: This clinical vignette discusses the ethical implications of using medication and housing contracts in voluntary case management programs. The Medical High User Case Management Program provides intensive case management, assisting patients with medical adherence, entitlements and housing. When appropriate, case managers will draft housing and medication contracts for opiate pain medications Contracts are done in consultation with primary care providers and the program internist psychiatrist and nurse.\nAs part of these contracts, the program will assist with the refilling of opiates and provide temporary housing. Patients must agree to make scheduled medical and substance abuse appointments. CASE INFORMATION: The patient is a 47-year-old woman with a history of admissions for CHF secondary to cocaine dependence. Her care has been complicated by a history of borderline personality disorder, chronic back pain, a history of upper GI bleeds, major depression and homelessness. The patient's stated goals on enrollment include housing and sobriety. The patient signed a contract that included opiate pain medications, temporary housing, substance abuse treatment and money management. Over the course of the next few months the patient would repeatedly binge on cocaine after receiving her general assistance check at the beginning of each month. The case management program terminated the housing and medication contract and asked the patient to see her primary care physician for all opiate refills. While the patient continued to binge on cocaine, the case manager continued to assist the patient with non-opiate medication refills, appointments with her primary care physician and applications for both housing and a money management. The patient eventually enrolled herself in a residential drug treatment program. She was restarted on her pain medication contract after seeing her primary care provider and is awaiting housing. IMPLICATIONS/DISCUSSION: While we believe that contracts for housing and pain medication refills may be useful in providing external control for seriously medically ill patients, these contracts poses important ethical questions. These contracts may challenge the principle of patient autonomy in patients who retain the legal ability to make medical decisions. These contracts also use housing and pain medications as incentives, items that many feel should be viewed as rights. Therefore these contracts should never deny patients access to the usual standard of care. Instead they should be voluntary and provide additional services that are contingent on the patient's adherence to a care plan. It is also important that contracts be done as part of an ongoing patient provider relationship to insure patient safety. Finally these contracts have the potential to reduce morbidity and mortality, which remains an important ethical imperative. A 48 year old male presented with crushing chest pain of several hours duration and a worsening of his chronic headaches (which had started 8 months ago after a closed head injury). In the previous 30 hours, he had taken 14 tablets of clonidine to try to relieve the headache which he thought was due to his hypertension. On review of systems, he noted a 35 pound weight loss over the past 8 months and admitted to sexual activity with several prostitutes. The only other medication he takes is ibuprofen. His vitals revealed he was afebrile with a pulse of 42 and a blood pressure of 205/133. His exam showed some temporal wasting and grade III hypertensive retinopathy without papilledema. He had an S4 present, but the remainder of his initial cardiac, respiratory, abdominal, neurologic and musculoskeletal exam was unremarkable. Serial ECGs showed sinus bradycardia, left ventricular hypertrophy, and T-wave inversions in the lateral and inferior leads. Initial electrolytes, CBC and serial cardiac biomarkers were also unremarkable. A head CT without contrast showed grey-white matter changes consistent with hypertension. The patient was admitted to the cardiac care unit. A CT scan with IV contrast of the abdomen and thorax showed no evidence of an arotic dissection. His blood pressure was controlled with IV nitroglycerine initially, but then required fenoldopam and nicardipine drips, parenteral hydralazine, procardia, clonidine, and labetolol. He developed a third cranial nerve palsy on the right and his serum sodium dropped from 137 mmol/liter on admission to 121 mmol/liter three days later. CT scans of the head were repeated twice with and without contrast with no change from the initial CT scan. A lumbar puncture was performed and cryptococcal meningitis was diagnosed. A subsequent HIV test was positive. His CD4 count was 12. Amphotericin B was started. Elevated intracranial pressure was controlled by serial lumbar punctures. The patient's cranial nerve palsy showed minimal improvement, but his hypertension improved. IMPLICATIONS/DISCUSSION: The presence of intractable hypertension, bradycardia, and headache suggests increased intracranial pressure as a cause for his hypertension. It is a wellknown cause of hypertension but rarely considered. In this case, his bradycardia was initially thought to be secondary to clonidine. He also lacked any focal neurologic deficits or papilledema on initial presentation to suggest an intracranial pathology. A retrospective casecontrol study published in 1992 showed an increased incidence of diastolic hypertension in AIDS patients with cryptocccal meningitis.\nSIADH. CAUSING OR CAUSED BY PSYCHOSIS? A. Tendler 1 , J. Wiese 1 ; 1 Tulane University, New Orleans, LA (Tracking ID #77076) LEARNING OBJECTIVES: 1. Recognize organic psychosis as a correctable cause of SIADH 2. Distinguish SIADH from from psychogenic polydypsia. 3. Know other causes of SIADH in the psychiatric patient. CASE INFORMATION: A 56 year-old woman with a history of schizophrenia was admitted to the inpatient psychiatry service with paranoid delusions of her brother trying to kill herself and all religious ministers. Her physical examination was normal, including normal neurologic and pulmonary examinations. Her serum sodium was 124 mmol/L and her measured osmolality was 261 mosm/kg. The urine osmolality was 522 mosm/kg. She was treated with risperidone and free water was restricted. She was allowed a liberal salt-containing diet. Her psychosis improved over the next week, with normalization of her serum sodium. IMPLICATIONS/DISCUSSION: Hyponatremia in the schizophrenic patient should not be reflexively attributed to psychogenic polydyspia. Because ADH is liberated from the hypothalamus through the posterior pituitary, anti-psychotic medications acting at surrounding structures may cause SIADH, as may primary psychosis. The urine osmolarity is the best test to distinguish SIADH from psychogenic water drinking, as the concentrating effects of ADH should increase the urine osmolarity. Hyponatremia may cause neurologic impairment, but this impairment is usually in the form of seizures as opposed to psychosis. Treatment of psychosis-induced SIADH is with neuroleptics designed to control the psychosis. Medical consultation for hyponatremia in the psychiatric ward is a common occurrence . Physicians should be aware of this underdiagnosed cause of hyponatremia, and of its successful resolution with anti-psychotic therapy. received no relief, however, and he re-presented with persistent back pain and fevers. Further questioning revealed he had a past history of mitral regurgitation and had had a tooth extraction six months prior. He had a 3/6 systolic murmur at the apex and point tenderness over the lumbar spine. His blood cultures grew Streptococcus viridans. An MRI of the lumbar spine showed edematous changes at T12-L1 with loss of disc space, consistent with discitis. An echocardiogram confirmed endocarditis. IMPLICATIONS/DISCUSSION: Although the diagnosis of urinary tract infection and lower back strain adequately explained his symptoms, each of the two diagnoses was insufficient in explaining all of the symptoms. William of Occam suggested that,``one should not increase, beyond what is necessary, the number of entities required to explain anything.'' Known as Occam's Razor, this principle guides physicians to search for one diagnosis to explain all symptoms, instead of invoking multiple diagnoses. The fever, bacteremia, discitis and immunemediated glomerulonephritis in a patient with valvular abnormality and poor dentition can all be attributed to infective endocarditis.\nAN OIL PIPE CLEANER OUT OF BREATH. D. Tran 1 , J. Wallace 1 ; 1 UCLA±San Fernando Valley Program, Sylmar, CA (Tracking ID #75804) LEARNING OBJECTIVES: 1. Recognize silicosis as a cause of pulmonary fibrosis. 2. Discuss the differential diagnosis of silicosis CASE INFORMATION: 25 y/o Hispanic male presented to ER for increasing dyspnea on exertion. His symptoms began insidiously about a year prior to admission, and have gradually worsened to the point where he can walk less than 1 block. His clinical course, thus far, has been punctuated by periods of exacerbation for which he was treated with antibiotics and prednisone with moderate success. He denies fever, chills, weight loss. There is no history of wheezing, asthma, chest pain, orthopnea, PND, or edema. The patient emigrated from Mexico three years ago. He denies any current or past of tobacco, drug abuse, or alcohol. On further questioning, the patient states that he spent three years in Mexico working as an oil pipè`c leaner''±which involved sandblasting. Physical exam and labs are only significant for a respiratory rate of 32 and paO2 of 70 mmHg at rest. CXR showed diffused bilateral infiltrates. High Resolution CT showed extensive, bilateral, coalescing pulmonary nodules with an upper lobe predominance and non-calcified hilar adenopathy. Patient is PPD and HIV negative. ACE level was 73 (9±67). He underwent video assisted thoroscopic lung biopsy. The pathology showed non-casseating granulomas with bifringent spicules consistent with sicilosis. Fungal and AFB cultures were negative. IMPLICATIONS/DISCUSSION: Silicosis is a type of pneumoconiosis caused by the inhalation of silica dust leading to pulmonary fibrosis. Individuals who work in metal mining or cutting, polishing, or carving stone are at risk. Symptoms usually develop after 3 to 20 years, depending on exposure level. Silicosis can either be simple: asymptomatic and non-progressive, identified by small, round opacities on chest x-ray; or complicated: symptomatic with progression despite discontinuance of the insults. Differential diagnosis includes sarcoidosis, coal worker's pneumonconiosis, neoplasia, TB, and fungal pneumonia. The diagnosis of silicosis is based on careful occupational history, radiographical evidences and pathology. Radiographically, silicosis is characterized by small, round opacities with an upper lobe predominance that can form extensive coalescing masses in advanced cases. The presence of eggshell calcifications on hilar lymph nodes may help distinguish silicosis from other pneumoconiosis. ACE level is elevated in sarcoidosis but can also be elevated in miliary TB, berylliosis, asbestosis, and silicosis. When the diagnosis is in doubt, biopsy can help establish the diagnosis and exclude other causes. On pathology, hyalinized silicotic nodules can be seen. They are formed by macrophages engulfing the particles, releasing cytotoxic enzymes causing fibrosis. There is a high incidence of TB co-infection in individuals with silicosis. Co-infection can be confused with a progression of the silicosis and should be excluded first. There is no effective treatment aside from preventing further exposure.\nA 92 YEAR-OLD WOMAN WHO TOOK A TURN FOR THE WORSE. V. Tsai 1 , R. Ziegelstein 1 ; 1 Johns Hopkins University, Baltimore, MD (Tracking ID #75756) LEARNING OBJECTIVES: To call attention to the possibility that moving a patient soon after a cardiac procedure can result in malposition and malfunction of a permanent pacemaker. CASE INFORMATION: A 92-year-old woman was found unresponsive in her nursing home bed. She was in complete heart block with a ventricular rate in the 20s±30s. Soon after arrival to the hospital, she sustained an asystolic arrest and required emergency temporary pacemaker therapy. The patient did not regain consciousness after electrical pacing at 60 beats per minute. The patient's health care agent consented to the implantation of a permanent pacemaker. After the procedure, a chest radiograph showed an appropriately placed dual chamber permanent pacemaker. The patient did not regain consciousness nor spontaneous movement. Nursing staff turned the patient every two hours to avoid pressure sores. Several days later, a chest radiograph showed that the pacemaker generator was rotated and in the opposite configuration from that noted on the original film. There was no evidence of electrode dislodgment or pacemaker malfunction. IMPLICATIONS/DISCUSSION: Pacemaker-twiddler's syndrome is an unusual complication of permanent pacemaker implantation, first described by Bayliss, et al. in 1968 . It is usually characterized by partial or total loss of pacemaker function secondary to rotation of the pulse generator in its subcutaneous pocket and subsequent electrode dislodgment. The rotation is related to manipulation of the generator by the patient, either consciously or unconsciously. The present report differs from previous cases, since the pacemaker generator rotation could not have been induced by the patient, but was likely the result of routine turning of the patient by the nursing staff in an effort to prevent pressure ulcers. The possibility that turning immobile patients soon after device implantation will result in malposition and malfunction of pacemaker or cardioverter-defibrillator generators is important to recognize, since these devices are being used in an increasing number of elderly patients. Caution should be exercised while moving elderly bedbound patients in the first few days after their cardiac procedure, in order to prevent a potentially life-threatening condition.\nTHE USUAL SUSPECT IN AN UNUSUAL SPOT. J.T. Tseng 1 , P.P. Balingit 1 ; 1 UCLA San Fernando Valley Program, Sylmar, CA (Tracking ID #76488) LEARNING OBJECTIVES: 1. Recognize the clinical presentation of liver abscess. 2. Consider mycobacterial and other atypical organisms as etiologies of liver abscess in the immunocompromised host. CASE INFORMATION: A 41 year old Mexican male with no significant past medical history presented to an urgent care clinic with 1 month of gradually worsening right upper quadrant abdominal pain associated with fevers and night sweats. He described the pain as a dull, constant discomfort without radiation, exacerbated with movement and without change with food intake. Several episodes of nausea and vomiting occurred in the last two days. Intermittent watery diarrhea was present over the past two weeks. He sought medical attention for his abdominal pain one week ago and was prescribed levofloxacin and metronidazole without relief of symptoms. The patient reported frequently visiting Mexico, his last trip being 2 months prior. He denied HIV risk factors including homosexual or promiscuous sexual behaviors, IV drug use, or transfusions. Exam was significant for temperature of 38.8 C and heart rate of 106. Tenderness was elicited upon palpation of the right upper abdominal quadrant. No hepatomegaly was apparent, and a Murphy's sign was not present. CBC was remarkable for lymphopenia. Liver transaminases were within normal limits. No organisms were identified from blood cultures, stool cultures, and studies for ova and parasites. HIV antibody test was positive. Abdominal CT scan revealed a 2  2 cm low-density irregular lesion consistent an with abscess within the right hepatic lobe. Tissue obtained from percutaneous drainage of the liver lesion revealed necrosis and granulomatoid inflammation. AFB stain was positive, and TB was identified in culture. TB was also isolated from the patient's sputum, despite the absence of cough, dyspnea, or other pulmonary symptoms. Four-drug antitubercular therapy was initiated with resolution of the patient's presenting symptoms. IMPLICATIONS/DISCUSSION: Liver abscesses of varying etiologies tend to have similar clinical presentations. Patients typically present with fever, night sweats, and right upper quadrant abdominal pain. Identification of the causative agent becomes critical when patients fail to respond to empiric therapy against pyogenic or amebic abscesses. Serologies are often not helpful in identifying an organism as assays often lack sufficient sensitivity or specificity and blood cultures may not reveal the offending agent. While CT, MRI, or ultrasonography may help differentiate liver abscesses from neoplastic processes, they are unable to identify a specific organism. Drainage of the abscess remains the mainstay of diagnosis. For immunocompromised patients, laboratory analysis of drainage fluid should include evaluation for atypical organisms as well as Gram stain, culture, and ova and parasites. In these patients, mycobacterial and fungal organisms may form abscesses in addition to typical granulomatous lesions when they invade the liver. LEARNING OBJECTIVES: 1. Acromegaly can present in younger patients 2. The role of insulin-like growth factor 1 in diagnosis and therapy 3. The importance of early diagnosis CASE INFORMATION: 22 y/o latino male presented to our urgent care clinic with a history of headache for over 2 years. The headache was intermittent in nature, mostly frontal and without exacerbating, relieving factors or associated symptoms and with minimal relief from analgesics. On systems review he admitted to an increase in the size of his hands and feet. His family also reported a gradual change in his appearance. On examination, the patient was normotensive. He had prominent supraorbital ridges and a large lower jaw with poor occlusion of teeth and lower teeth overbite. His facial wrinkles were exaggerated and lips full. The nose tongue and ears were enlarged, the hands were large, doughy spade like and the skin over them thickened. Neurological exam showed grossly intact bitemporal visual fields. His appearance was consistant with the diagnosis of acromegaly. A work up consisting of growth hormone (GH), insulin-like growth factor 1 (IGF-1), TSH and random glucose was initiated with MRI imaging of the brain. The patient was also referred to ophthomology for visual field testing. IMPLICATIONS/DISCUSSION: Acromegaly is a disease caused by secretion of excessive amounts of growth hormone, almost always occurring as a result of a benign adenoma of the anterior pituitary gland. The disease occurs most frequently in middle age with an incidence of 3±4 per million. Somatotroph adenomas of the pituitary may be large enough to cause visual impairment or headaches as in our patient. Acromegaly leads to decreased life expectancy with a 2±3 fold increase in mortality from cardiovascular causes, cancer and all causes. The diagnosis is made clinically and confirmed by finding a high levels of GH and IGF-1. Growth hormone binds to receptors resulting in the stimulation of production of IGF-1 which mediates most of the actions of GH. Growth hormone release is pulsitile with concentrations varying throughout the day. Random GH is therefore not a reliable diagnostic marker and is only useful when correlated with the glucose tolerance test. IGF-I is present at more steady concentrations and is a reliable marker of disease and its control. Most patients are treated with transphenoidal surgery which usually results in clinical improvement with decreased GH and serum IGF-I concentrations. Other options include radiation therapy, medical therapy with dopamine agonists or somatostatin analogues. Pegvisomant is a newly developed GH receptor antagonist that leads to decreased IGF-I levels and clinical improvement. Reducing growth hormone levels improves symptoms and complications of the disease and increases life expectancy.\nJUST ANOTHER CASE OF UNSTABLE ANGINA? G.J. Van Londen 1 , J. Hefner 1 ; 1 University of Pittsburgh, Pittsburgh, PA (Tracking ID #76750) LEARNING OBJECTIVES: 1.) Distinguish between Type A and B aortic dissections; 2.) Recognize an uncommon clinical presentation of an aortic dissection; 3.) Manage and treat a dissecting aorta. CASE INFORMATION: A 56-year-old male with a history of a 3-vessel coronary artery bypass graft in 1991, HTN and hyperlipidemia presented to the Emergency Room (ER) with bilateral jaw and retrosternal chest pain accompanied by an occipital headache and dyspnea on exertion. The chest pain was waxing and waning in intensity, non-radiating with a maximum pain score of 5/10. There was diaphoresis but no nausea or vomiting. The night prior, he noted some parathesias in his fingers and blurred vision that resolved within two hours. On exam, the patient's pulse was 110 with a BP of 130/80 on the left and 124/70 on the right. Room air pulse ox was 96%. No pulsus paradoxus, JVD or bruit was appreciated. Lung exam revealed decreased breath sounds in the left lower lobe. Cardiac exam was regular rhythm with a 2/6 diastolic murmur. Exam of the upper and lower extremities revealed equal and symmetric pulses. EKG showed sinus tachycardia with no ischemic changes. His first set of cardiac enzymes was negative. Portable chest x-ray revealed a left pleural effusion and a widening of the mediastinum with the patient rotated to the right. The patient was diagnosed in the ER with unstable angina and admitted. Based on the diastolic murmur and left pleural effusion on chest x-ray, an aortic dissection was suspected and a stat MRI was performed. This revealed a Type A dissection. Emergent surgery with replacement of the ascending aorta, valve and evacuation of a left hemothorax was performed. Cardiac arrests and a CVA complicated the perioperative course. The patient expired nine days after admission. IMPLICATIONS/DISCUSSION: A dissecting aorta must be recognized quickly as an acute Type A dissection (involves the ascending aorta) is a surgical emergency, whereas an uncomplicated Type B dissection (all others) can be treated with pharmacotherapy. Acute management consists of blood pressure control while confirming the diagnosis and type. Aortic dissection is relatively common with over 2000 cases reported each year. A recent study showed that 100% of dissections were diagnosed when all of the following clinical features were present: 1.) Acute onset of tearing or ripping-like chest or back pain; 2.) Mediastinal widening on chest x-ray and 3.) Variation in pulse and/or pressure in the upper extremities. A diastolic murmur, EKG without ischemic changes and a left-sided pleural effusion (hemothorax) are uncommon presentations of a dissection. Transesophageal echocardiogram (TEE) is recommended for unstable patients with acute chest pain. MRI is preferred in stable patients with chronic chest pain. CT scan or aortography should be reserved for situations in which both TEE and MRI are unavailable.\nA. Venditto 1 , J. Wiese 1 , M. Landry 1 ; 1 Tulane University, New Orleans, LA (Tracking ID #77081) LEARNING OBJECTIVES: 1) Utilize effective diagnostic methods in cases of encephalitis. 2) Recognize the importance of early diagnosis of West Nile virus to control disease outbreaks. CASE INFORMATION: A 58-year-old man presented with two days of fever, headache, unsteady gait, and a macular rash. Although initially lucid, he deteriorated into a state of delerium within the first day of hospitalization. His vital signs were normal. He had meningismus with passive and active neck flexion; he had an ataxia gait and a macular rash over his extremities. There were 6 white blood cells /mm3 in the CSF and a normal protein and glucose. Owing to the degree of impairment, a serum immunofluorescent assay for flavivirus IgM was ordered. The titer was 1:32, increasing to 1:64 over the next five days. An assay for West Nile virus was sent to the CDC for public health monitoring. IMPLICATIONS/DISCUSSION: Most non-herpetic viral encephalitities are due to arboviruses that cycle with the lifecyle of the tick or mosquito that is the primary vector. While all are endemic to warm climates, viruses such as the Eastern Equine Virus, West Nile and St. Louis encephalitis can become epidemic when the mosquito vector greatly exceeds its primary host. For all three viruses, there is little more than supportive care to be offered. Contrary to most medical interventions, however, testing for a definitive diagnosis has merit in providing community outbreaks. The best test for distinguishing St. Louis from West Nile is an enzyme-linked immunosorbent assay (ELISA). A positive serum ELISA indicates probable infection with West Nile virus, whereas a positive cerebrospinal fluid ELISA confirms the diagnosis. Early identification of West Nile virus is important for mosquito population control and public education to prevent further disease spread. LEARNING OBJECTIVES: To Illustrate potential clinical presentations that must be considered in a patient with sarcoidosis CASE INFORMATION: History: 49 year old African American female with history of obesity, asthma and possible fibromyalgia presented with worsening fatigue, SOB, DOE over 2 months. She has been tobacco-free for the past 4 years and compliant with her asthma regimen. Over the ensuing 6 months she continued to have worsening symptoms which required episodic reinstitution of systemic steroids for presumptive asthma exacerbation. Further review revealed a past medical history of a 2 cm right upper lobe nodule with bilateral hilar adenopathy and interstitial lung disease 2 years ago. CT guided-biopsy of the nodule yielded non-caseating granuloma. Serum labs, such as CBC, Chem 10 (including calcium), TSH, ESR and ACE levels, were normal. LFTs were mildly elevated. Subsequent viral hepatitis serologies were negative. PFTS were consistent with combined obstructive and restrictive lung disease with mildly reduced DLco. The patient returned for an urgent care visit complaining of worsening DOE, bilateral lower extremity edema, polyuria, nocturia, blurry vision and severe persistent daily headache and stiff neck for the past 2 months. Prednisone 60 mg was started empirically for pulmonary sarcoidosis exacerbation and possible sarcoidosis involvement of the eyes, CNS, liver and heart. Repeat of the above serum labs were normal. Fasting glucose level was not diagnostic for diabetes mellitus or glucose intolerance. Urinalysis and urine C&S were normal. Urine osmolality was ordered but not performed. A water deprivation test was deferred until other test results were known. PT (INR) and albumin did not suggest cirrhosis. PFTs were unchanged from the prior study. PPD test was <5 cm. Gallium scan showed no uptake in the lung, salivary and lacrimal glands (e.g.``panda bear'' sign) or any other part of the body. ECG and echocardiography were also normal. MRI of CNS showed dural enhancement but no pituitary or other parenchymal lesions. CSF fluid analysis revealed only a lymphocytic pleocytosis and negative cytology for malignancy. Ophthalmology referral showed no uveitis, cataracts or retinal abnormalities. The patient's symptoms improved but she complained of increasing weight, generalized edema, low back pain and bilateral knee pain after 6 months of Prednisone therapy. The patient reported no further polyuria, headaches, stiff neck, and blurry vision but still had residual DOE and fatigue. Repeat MRI showed no dural enhancement. Prednisone was tapered off and the resultant decrease in weight and edema lead to resolution of her low back and knee pain. IMPLICATIONS/DISCUSSION: Discussion: Sarcoidosis can have numerous extrapulmonary involvement. CNS and cardiac involvement portend worse prognosis than hepatic, pulmonary or arthritic involvement. SOB and DOE is most likely due to pulmonary involvement (i.e., due to endstage parenchymal fibrosis with or without acute inflammation) but concomitant restrictive cardiomyopathy due to sarcoidosis may exist synchronously. Echocardiography is essential in ruling out this possibility. Hepatic sarcoidosis involvement can progress from mild elevations in LFTs to frank cirrhosis, which fortunately was not present in this patient. The LFTs elevation may, however, be simply due to fatty liver of obesity. Uveitis due to sarcoidosis must be a consideration in patients with vision complaints, but retinitis, episcleritis, glaucoma and premature cataracts, as well as the sicca syndrome due to involvement of the lacrimal and salivary glands, can occur. Symptomatic CNS sarcoidosis classically manifests as diabetes insipidus due to posterior pituitary infiltration. Other CNS syndromes include aseptic meningitis and diffuse white matter disease. Diabetes insipidus is a distinct possibility in this patient. Although comparison of urine and serum osmolality and water deprivation test were not done to confirm diabetes insipidus, the patient's polyuria readily responded to glucocorticoids therapy. The MRI and CSF findings clearly support an aseptic meningitis picture and her headache symptoms responded to glucocorticoids therapy. LEARNING OBJECTIVES: To recognize a hypersensitivity reaction to dialyzers in a patient on hemodialysis CASE INFORMATION: A 67 year old female with end stage renal disease secondary to chronic uncontrolled hypertension was admitted to the hospital with fevers and chills. On admission, the potential etiologies of her fever were line sepsis or lower extremity cellulitis. She was begun on appropriate antibiotic therapy. Within 5 minutes on her routine hemodialysis, she suddenly became bradycardic, hypotensive and unresponsive. On examination before dialysis, vitals were HR-80/min, BP-215/110 mm Hg, and RR-16/min. Lungs were clear to auscultation. Cardiovascular exam revealed a normal S1 and S2 and a 3/6 holosystolic murmur at the apex. After 5 minutes on dialysis, her vitals were HR-48/min, BP-64 mm Hg systolic; respirations became agonal, followed by apnea. The patient became obtunded and unresponsive, with twitching of her lips. She sustained cardio-respiratory arrest and required CPR with endotracheal intubation and mechanical ventilation and oxygenation. Dialysis was aborted during this episode. Her EKG revealed pulseless electrical activity followed by junctional rhythm. Once dialysis was stopped, it converted to normal sinus rhythm with normalization of hemodynamic parameters. Her WBC was 8000, H/H 8.2/ 25.1, electrolytes were normal, BUN 72 and Cr 14.6, BNP-1300, CPK 20, Troponin I-0.21. Chest X-ray showed mild congestion and her subclavian catheter tip was in right atrium. Echocardiogram revealed concentric left verntricular hypertrophy with normal LV function. 72 hours later, at her next routine hemodialysis, a similar episode occurred. Heparin was discontinued from her dialysis regimen due to possibility of heparin sensitivity as she was thrombocytopenic with presence of heparin associated antibodies. However, a third episode occurred on the next dialysis. At this time, the patient's dialyzer was changed from B3-1.3 to F-8. Thereafter, no further episodes occurred and she tolerated subsequent hemodialysis without complication. IMPLICATIONS/DISCUSSION: Dialyzer-induced acute hypersensitivity reaction is a rare entity (5/100,000 dialysis cases), but is a potentially life threatening condition. Presentation varies from mild urticarial reaction to cardiorespiratory failure. The onset of symptoms due to dialyzer-induced hypersensitivity reaction is usually 5±30 minutes after initiation of hemodialysis. Possible causes are bio-incompatible membrane (i.e. cellulose), ethylene oxide use for sterilization, use of ANP69 membrane in patients on ACE inhibitors, bacterial contamination of dialysate in high flux dialyzer, heparin allergy, acetate bath (acetate is a vasodilator and myocardial depressant), and inappropriate cytokine and complement activation. Differential includes severe hypotension, acute MI, arrhythmias, air or pulmonary embolism, or severe pulmonary edema. A high index of suspicion is necessary to recognize this entity. Treatment is standard care for any anaphylactic reaction and includes intravenous steroids, antihistamines and supportive care. Removal and avoidance of the culprit inciting agent is of paramount importance. A 67 year-old Filipino male presented to the ED with right facial erythema, edema, and subjective fever. The lesion progressed from a pimple 2 days prior. His past medical history was significant for Lepromatous Leprosy diagnosed in 1998. He had been treated with rifampin and ofloxacin since then. In the ED, he was treated with amoxicillinclavulanate for presumed cellulitis. Nevertheless, his facial lesion worsened, and he developed diffuse lymphadenopathy and a reddish rash on his upper extremities. Upon admission, the patient was afebrile with normal vital signs. His exam demonstrated left facial erythema, edema, and induration with central ulcertation extending from the maxillary area to the left ear and mandible and exhibited normal sensation. He had diffuse lymphadenopathy of the head and neck, and erythematous plaques on both upper extremities. His chronic findings of a left hand claw deformity and decreased sensation in the lower extremities were unchanged. Lab studies were notable for a WBC of 13 and blood and wound cultures were negative. Antibiotics were changed to vancomycin and imipenem without improvement. Subsequent skin biopsy revealed numerous acid-fast rods within macrophages and a dense neutrophilic infiltrate. His clinical presentation and biopsy findings were consistent with a Type II lepromatous reaction, or Erythema Nodosum Leprosum (ENL). He was treated with prednisone and thalidomide with ultimate resolution of his symptoms within days of treatment. IMPLICATIONS/DISCUSSION: ENL is characterized by fever with multiple erythematous tender nodules, sometimes accompanied by neuritis, edema, arthralgias, leukocytosis, iridocyclitis, pretibial periostitis, orchitis, and/or nephritis. It is associated with Borderline and Lepromatous forms of Leprosy and occurs in up to 25% of patients. ENL is most common during treatment, but can also occur before or after therapy. It is thought to be an immunecomplex disorder with tumor necrosis factor alpha playing a role in its pathogenesis. Acute treatment consists of steroids and/or thalidomide; the latter is highly effective, generally controlling the reaction within 48 hours. Clofazimine may be effective for chronic reactions. Prompt detection and treatment of ENL has decreased the number of patients with resultant chronic disabilities. Our patient had a clinical picture consistent with ENL which was initially difficult to distinguish from cellulitis. He responded to treatment with prednisone and thalidomide. As international travel and immigration continue to increase, it is important to recognize the reactions associated with Mycobacterium Leprae. year-old man was admitted for elective T12-L2 posterior spinal fusion. His past medical history included CAD, s/p CABG, and idiopathic scoliosis with prior spinal surgeries. Six hours post-operatively, the patient developed hypotension, fever and hy-poxemic respiratory failure. Intra-operatively, he received multiple blood products including 6 units of whole blood, 5 units of PRBC, 12 units of FFP, and 3 6-packs of platelets. On exam in the ICU post-operatively, he had a temperature of 398C and a BP of 70/ 40. He had a normal cardiac exam including a normal JVP, lungs had coarse breath sounds, and his extremities were warm without a rash. Chest x-ray revealed diffuse bilateral infiltrates and a chest CT was negative for PE. Echo showed no focal wall motion abnormalities. Labs were notable for a new low WBC count (0.4). Successive blood and sputum cultures were negative. The blood bank was notified to investigate a possible transfusion reaction. There was no evidence of hemolysis and a direct Coombs' test was negative. The patient was treated with vasopressors, including epinephrine, and broad-spectrum antibiotics. On post-operative day 7, the patient suffered a cardiac arrest and expired despite full resuscitative efforts. Post-mortem analysis revealed that 1 of the 32 total blood product donors was a multiparous female who donated a platelet pheresis. She carried an HLA-I antibody against one of the patient's neutrophil antigens (Bw4 public antigen). This confirmed the cause of death as transfusionrelated acute lung injury (TRALI). IMPLICATIONS/DISCUSSION: TRALI is a clinical syndrome characterized by fever, severe hypoxemia, non-cardiogenic pulmonary edema, and hypotension occurring one to six hours post transfusion. Case reports have identified transient leukopenia as another manifestation. 80% of patients recover within 48-96 hours with little permanent sequelae. Despite being the third most common cause of death from transfusions in the developed world (estimated incidence of 1/5000 transfusions, with a mortality rate of 5±14%), it is probably significantly under diagnosed. The majority of cases are associated with transfused complement-activating antibodies, either HLA (class I or II) or granulocyte specific. All blood components containing plasma have been associated with the injury. Risk factors include multiparous donors, underlying recipient conditions such as recent surgery, cytokine treatments, massive blood transfusions, active infection, and prolonged storage of transfused products. Current treatment is limited to respiratory and hemodynamic supportive measures. Diuretics and steroids have been used with variable efficacy and have not been tested in clinical trials. Our patient had an unusual course for TRALI in that his symptoms developed relatively late post-operatively and he did not recover despite maximal support. TRALI should be considered in all patients with hypoxemia following transfusions. Previously, the patient was playing golf 3 times a week without difficulty. His hypoxia began post surgery to relieve a small bowel obstruction. He was treated with antibiotics for pneumonia and ruled out for PE with 2 low probability V/Q scans. The patient was discharged home on oxygen, but his symptoms worsened. He had increasing malaise with shortness of breath upon minimal exertion, such as sitting up in bed. Eventually, he was bed bound. He denied cough, fever, wheezing, chest pain, or leg swelling. Upon admission the patient was afebrile, BP 130/70, RR 18 and supine O2 saturation of 93% on 10 liters of oxygen. Upon sitting, the patient was dyspneic with RR 40 and O2 saturation 70% on 10 liters of oxygen. His exam was normal including a normal cardiac exam including JVP, slightly decreased breath sounds at the right base, and his extremities were warm without edema. Laboratory exam revealed a normal EKG, CXR with unchanged right opacity, chest CT negative for PEor A-V fistula, electrolytes, CBC, and cardiac enzymes were also normal. PA02 on 4 liters of oxygen was 54 mm Hg and on 100% oxygen 84 mm Hg. Transthoracic echo showed normal left ventricular size and function with an inter-atrial septum hypermobility and a large patent foramen ovale with normal right sided pressures. Injection of agitated saline revealed a large amount of immediate right to left shunting. Transesophageal echo confirmed the PFO with right to left shunting. The patient underwent transcateter closure of his defect. One day post procedure, the patient was markedly improved with O2 saturation on room air of 95% supine and sitting. The patient continued to improve as an outpatient and was playing golf within 2 weeks after discharge. IMPLICATIONS/DISCUSSION: Our patient exhibited the platypnea-orthodeoxia syndrome. This is defined as dyspnea and arterial desaturation in the upright position and improved by recumbency. This syndrome occurs with an intracardiac or intrapulmonary shunt. Our patient had the most common etiology, a patent foramen ovale, with a right-to-left shunt. Shunting in the face of normal right sided pressures, as in this case, is unusual but described in the literature. Diagnosis is made by visualization of the PFO with shunting on transesophageal echocardiogram with agitated saline (``bubble study''). Percutaneous closure of the PFO is safe and effective. Cardiac shunts should be suspected in all patients with persistent hypoxia, especially in those who exhibit worsening symptoms and hypoxia in an upright position.\nSMOKING CHINESE BROCCOLI? J.L. Yuh 1 , E. Yee 2 , A.G. Gomez 3 ; 1 Olive View±UCLA SFVP, Sylmar, CA; 2 VA Albuquerque±University of New Mexico, Albuquerque, NM; 3 University of California, Los Angeles, Sepulveda, CA (Tracking ID #75461) LEARNING OBJECTIVES: 1. Recognize acute nicotine in toxication 2. Discuss the management and treatment of acute nicotine intoxication CASE INFORMATION: A 56 year old Thai nun, non-smoker, with no known medical history was brought in for an acute onset of confusion, lethargy, and diffuse body weakness. Following breakfast, the patient was found to be minimally responsive next to a bowl of soup. By report, this soup contained many leaves of Chinese broccoli self-grown at the temple. In the ED, the patient's presenting GCS was 9, T 37.1F, pulse 49, BP 91/39, respirations 16, and room air O2 sat 94%. She was minimally responsive and diffusely flaccid on motor exam. IV fluids, NG charcoal lavage, and sorbitol were administered. CBC, CHEM 10, LFTs, and UA were all normal; serum/urine toxicology screen, acetaminophen and alcohol levels were negative. CXR was clear, and head CT did not show any shifts, masses, or bleeds. EKG showed only sinus bradycardia at 50 bpm. On repeat exam, pt was slightly more responsive since presentation, with pupils dilated and sluggish, but not following commands in English or Thai. She was admitted to the ICU for monitoring. Several leaves of the Chinese broccoli used in the soup were brought in later. Upon calling poison control and researching botanical atlases, it was determined to be a tobacco plant. Her serum nicotine level was low, while her serum cotinine level was high, indicating the metabolism of nicotine to cotinine, and confirming an ingestion of 15+ tobacco leaves. The patient regained her strength and alertness gradually and was discharged in normal health on day 3. The temple members subsequently removed all remaining`Chinese broccoli' plants! IMPLICATIONS/DISCUSSION: Nicotine overdose can result from overuse of cigarettes, nicotine gum or patches, and plant ingestion. It is seen most commonly in patients smoking while on a patch. Patients can exhibit weakness, convulsions, coma, respiratory distress/apnea, pupillary dilatation, abdominal cramps, vomiting, initial tachycardia/hypertension, followed by bradycardia/hypotension. Diagnosis is by careful history and physical exam as there are no set serum toxicity levels. The half-life of nicotine is 0.5±2 hours while that of cotinine (a better marker due to its slower clearance) is 12±30 hours. Management includes airway protection, charcoal lavage, sorbitol, hydration, and circulatory support. Symptoms can last several hours, and survival after 4 hours of intoxication usually prognosticates complete recovery. Lethargy may remain for several days. Caution needs to be emphasized for patients ingesting unknown plants and to remind patients of the dangers of using nicotine replacements while continuing to smoke.\nPREECLAMPSIA AND PSEUDO POSTPARTUM CARDIOMYOPATHY. H. Zakariya 1 , R.D. Hobbs 1 ; 1 Oakwood Healthcare System, Dearborn, MI (Tracking ID #76568) LEARNING OBJECTIVES: To recognize that volume overload in the preeclamptic patient can lead to pulmonary edema and the misdiagnosis of postpartum cardiomyopathy. CASE INFORMATION: A 29 year-old, G5P3AB2 woman presented with severe dyspnea, orthopnea and peripheral edema five days after delivering a baby. Her pregnancy had been complicated by preeclampsia but managed without difficulty. Vital signs were: BP 157/96, P 110, R 22, T afebrile. Oxygen saturation was 94% on 100% O2. Bilateral rales, jugular venous distention, an S3 and S4 with 3+ pitting edema and a mitral regurgitation murmur were noted on physical examination. Cardiac enzymes were normal. Hgb was 9.7 g/dl. An EKG showed sinus tachycardia. Spiral CT was negative for pulmonary embolus. She was admitted with a diagnosis of postpartum cardiomyopathy. An echocardiogram showed severe mitral regurgitation, trivial tricuspid regurgitation, a small pericardial effusion and an ejection fraction of 50-55%. She was treated with furosemide and rapidly improved. Repeat echocardiography, done two weeks later when she was asymptomatic and off medications, showed that the mitral regurgitation had cleared, there was no pericardial effusion and her ejection fraction was unchanged. Further history revealed that in the last months of pregnancy and immediately postpartum she had been advised to``drink a lot of fluids.'' IMPLICATIONS/DISCUSSION: In preeclampsia the expected fall in colloid oncotic pressure is greater than that seen in normal pregnancies. Cardiac output increases 40% during normal pregnancy and extravascular volume is expanded. Following delivery, significant amounts of extravascular fluid move into the intravascular space and are cleared. In our patient, increased fluid intake led to over expansion of the extravascular volume with pulmonary edema occurring as the fluid was mobilized. Notably, this is not a case of postpartum cardiomyopathy. Her ejection fraction was normal on both initial and repeat echocardiograms and does not show primary muscular dysfunction. The mitral regurgitation cleared with diuretics and is not suggestive of a structural lesion. Small pericardial effusions may occur as a normal variant during pregnancy. Instead, her condition is explained by extreme fluid shifts in a preeclamptic woman. Diagnostic confusion occurs if the physician interprets the findings in terms of the non-pregnant state. year old female with no PMH except appendectomy, admitted with a five days of abdominal pain. It began as``indigestion'' in the epigastrum for several days but progressed to a sharp, constant, right-sided abdominal pain. Associated symptoms included anorexia, three days of constipation, with no bowel movement but some flaitus. She had no dysuria, normal menses, and no bloody bowel movements. She had one episode of``sweats'' and chills two days prior. On exam, she was afebrile and in moderate distress. Her vital signs were normal and abomen was soft with hypoactive bowel sounds and voluntary guarding in the right mid abdomen. Rectal exam was guiac negative. The remainder of the exam was normal. UA, CBC, LFTS, amylase, and lipase were normal. CT of the abdomen revealed a 5.3 cm mass( with a small air pocket) adjacent to the hepatic flexure with mesenteric stranding and sigmoid diverticulosis. Bowel rest and ampicillin -sulbactam were implemented. By the third day, her exam markedly improved. She was discharged on amoxicillin-clavulanate with follow up colectomy planned. IMPLICATIONS/DISCUSSION: The frequency of diverticulosis in younger patients is thought to be increasing. Most people will be asymptomatic. Overall, 15% will be right sided in western countries. Symptoms include bloating and abnormal bowel habits. About 20% develop clinical diverticulitis and of those, 10 to 20% will require surgery. Complications of diverticula include simple inflammation, abscess, bleeding, and fistula formation. Abscess may cause abdominal pain for several days duration, nausea, constipation and fever. WBC can be normal in up to 45% of patients. Treatment of complicated diverticulitis often requires combined medical and surgical approach. Evidence suggests more virulent disease in young people. Often these patients are treated with early colectomy. They have a higher incidence of right sided disease that can be mistaken for appendicitis. Longer lifespans lead to increased potential for morbidity. With CT guided drainage, the two part surgeries (with colostomy and then take down) decreased dramatically. Contained perforation is managed with antibiotics followed by partial colectomy. Offering colectomy after the second attack of uncomplicated diverticulitis has recently been challenged. Conservative treatment after the second attack is now considered a reasonable option in older patients. However, for younger individuals, with no medical comorbidities, early surgery may be the preferred option. The patient is a 33 year old female, gravida two para one, at eight weeks gestation who presents to the office. Her first pregnancy was complicated by a deep venous thrombosis at fifteen weeks gestation. At that time hypercoaguability work up was performed due to a positive family history. The patient had elevated protein C resistance and she was placed on full dose heparin through the duration of her pregnancy. Her third trimester she developed hypertension. She delivered at thirty-five weeks and remained on anticoagulation six weeks following delivery. Factor V leiden mutation was confirmed by gene testing following delivery. She had a seizure disorder as a child and took dilantin and multivitamin prior to pregnancy. Her father had a pulmonary embolus and has antithrombin III deficiency. She is a pharmacist and does not smoke or drink alcohol. Comprehensive physical exam is normal. The patient was placed on continuous subcutaneous infusion of heparin because she declined lovenox therapy. IMPLICATIONS/DISCUSSION: Factor V Leiden is the leading cause of thomboembolism in pregnancy (44%). Pulmonary embolus contributes greatly to maternal mortality rates. Venous thromboembolism occurs in one in 1500 pregnancies. The most prothrombotic time is immediately post partum. In addition factor V Leiden has been associated with HELLP, intrauterine growth retardation, miscarriage, and pregnancy induced hypertension. The activated protein C resistance test may be falsely positive because of changes that take place in the coagulation system during pregnancy. Diagnosis must be confirmed with gene testing. If factor V positive, patients have a three to five times greater risk of stillbirth. Factor V Leiden is present in 20±40% of abruptions. There have been no randomized control trials to demonstrate that full dose anticoagulation will reduce the risk of any of these complications. Treatment is based on extrapolation of data from nonpregnant patients. There is controversy over treatment of pregnant patients with factor V and a prior DVT. At present, patients are offered prophylactic dose heparin or full dose heparin or lovenox based on individual risk stratification (i.e homozygous vs. heterozygous). For individuals with factor V but no prior event, a variety of options exist: prophylactic subcutaneous heparin throughout the pregnancy with full dose anticoagulation postpartum for six weeks versus postpartum treatment only versus clinical surveillance alone. Lovenox will likely replace heparin because of its favorable side effect profile. Risks include a 2% major bleeding risk, heparin induced thrombocytopenia, and osteoporosis. (2) To review the presentation, treatment, and prognosis of CMV colitis among immunocompetent adults. CASE INFORMATION: A 17-year-old female was admitted to the hospital for a complaint of bloody diarrhea. She had been in good health until 4 months earlier when she noted intermittent passage of bright red blood and reddish tissue with her stool. Ten days before admission, she developed cramping periumbilical pain, soon followed by fever and watery diarrhea with hematochezia. Pertinent findings on examination included a temperature of 100.4 F, slightly pale conjunctivae, a soft abdomen with normoactive bowel sounds and minimal tenderness periumbilically, and bright red blood on digital rectal exam. Her white cell count was 8,200/mm3 with 37% neutrophils and 55% lymphocytes. Atypical lymphocytes were present. Stool samples were negative for ova, parasites, C. difficile toxin, and other bacterial enteropathogens. An abdominal CT scan showed thickening of the wall of the entire large bowel. On colonoscopy, there were diffuse inflammation in the colon, mucosal nodularities, and diminished haustral markings. Colonic biopsies demonstrated active colitis with CMV infection, but no architectural distortion to suggest an underlying chronic inflammatory bowel disease (IBD). A CMV antigenemia test came back positive, and anti-CMV IgM and IgG titers were both elevated. HIV serology, however, was negative and CD4 count was 354. Markers for IBD, including p-ANCA and ASCA, were likewise negative. The patient was treated with valganciclovir for 21 days." }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

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[ { "from": "human", "value": "Full Text: LEARNING OBJECTIVES: 1) Raise index of suspicion for severe metabolic derangement in elderly patients. 2) Recognize clinical signs of hypernatremia. 3) Recognize limitations of using concentrated supplements as meal replacement. CASE INFORMATION: A 79 year old woman with advanced Alzheimer's disease was brought to the E.D. due to a 4-week progressive decline in her overall level of function. She had been hospitalized 8 weeks prior for an aspiration pneumonia. Her daily fluid intake since that time consisted of six 8oz cans of a``balanced nutrition shake'', along with some soup. 4 days prior she was given antibiotics by her primary physician. She showed no improvement and developed diarrhea. Physical exam revealed an obtunded woman. Her pulse was 120bpm and rectal temperature was 38.9 Celsius. She was normotensive. She had dry oral mucosa and skin tenting. Her neurological exam was nonfocal. Lab studies were significant for sodium of 185mEq/L, chloride of 148mEq/L, BUN of 124mg/dL and a creatinine of 3.0mg/dL. Urine osmolality was 819mOsmol per kg with urine sodium of 22mMol/L. Her WBC was 13K. Labs from her prior admission revealed sodium of 143mEq/L; chloride of 106mEq/L; BUN and creatinine were 7mg/dL and .7mg/dL respectively. Her free water deficit was calculated at 9 liters, and she was placed on D5W1/2NS at 150cc/hr. Her tachycardia and fever resolved. She began keeping her eyes open and speaking some words. On the sixth hospital day serum sodium was 146 with normal BUN and creatinine. The patient was discharged on a diet of purees and thickened liquids. DISCUSSION: Patients with dementia are at a risk for hypernatremia due to their inability to verbalize a need for water or to obtain it for themselves. Hypernatremic dehydration can lead to mental status changes that are easily missed in a severely demented patient. While canned high calorie shakes are an attractive supplement for patients with poor oral intake, they are highly concentrated and contain little water. This renders them a poor substitute for total meal replacement.\nA. Aggarwal 1 , J. Brainard 1 , D. Brotman 1 ; 1 Cleveland Clinic Foundation, Cleveland, OH LEARNING OBJECTIVES: 1. Recognize that glucagonomas can present with spinal metastases. 2. Appreciate that the absence of cutaneous, endocrinologic and gastrointestinal manifestations does not rule out the presence of a glucagonoma. 3. Know that tissue staining for neuroendocrine peptides can confirm that a tumor is of pancreatic origin, even when there is no radiographically identifiable pancreatic mass. CASE INFORMATION: A 38 year-old African-American woman presented to the emergency department with low back pain of three months duration. The pain involved both flanks and radiated to the buttocks and posterior thighs bilaterally, as well as to the right groin and medial right thigh. By exam, she was thin and appeared uncomfortable. Shotty nodes were palpable in multiple regions, but none were fixed or enlarged. There was percussion tenderness of the lumbar spine, but motor function and reflexes were normal in the lower extremities. Serum chemistries were notable for an elevated alkaline phosphatase but otherwise normal liver enzymes. Serum calcium and glucose were normal. CT of the abdomen and pelvis showed peripancreatic lymphadenopathy and numerous hepatic lesions suggesting metastases. MRI of the spine demonstrated a destructive lesion in the second lumbar vertebra with soft-tissue extension compressing the spinal cord (figure). Whole body bone scan showed diffuse osseous metastases to the upper and lower extremities as well as the axial skeleton. Ultrasound-guided biopsy of the peri-pancreatic adenopathy revealed a low-grade neuroendocrine tumor that stained positive for glucagon, but stained negative for serotonin, pancreatic polypeptide and gastrin. The serum glucagon level was normal. Due to widespread metastases, the patient was deemed a poor surgical candidate, however she did undergo radiation therapy to the spine and to painful lesions in the right humerus and left femur. Subsequently she was treated with systemic chemotherapy consisting of adriamycin and streptozocin with no initial reduction in tumor mass. DISCUSSION: Glucagonomas have previously been reported to metastasize to bone, but to our knowledge there is only one other reported case of bony metastases as the initial manifestation of the tumor. This may be because most glucagonomas come to early clinical attention via the effects of high serum levels of glucagon. It has previously been reported that systemic and endocrine manifestations of glucagonomas are more common in advanced disease and are directly related to tumor size. This is clearly not the case in our patient. Notably absent were the typical skin rash (necrolytic migratory erythema), diarrhea, hyperglycemia, stomatitis and chelosis associated with the classic glucagonoma syndrome. Another important clinical feature in our patient was the absence of a radiographically identifiable pancreatic mass despite widespread metastases. While the absence of a clear primary source is not an uncommon situation in cancer patients, the identification of the primary tumor carries particular importance for neuroendocrine malignancies. Metastatic small-cell carcinoma of the lung has a more rapid progression than both carcinoid and islet cell tumors, and the treatment is different for these three types of malignancies. In our patient, tissue staining for neuroendocrine markers allowed a specific diagnosis to be made and appropriate treatment to be instituted. We conclude that the absence of clinical evidence of hormonal activity should not dissuade the physician from considering the possibility of a pancreatic islet cell primary when a patient presents with metastatic neuroendocrine malignancy, even when there is no radiographic evidence of pancreatic mass. Stains for neuroendocrine peptides are of crucial importance to making the diagnosis in such patients.\nDISCUSSION: Lyme disease involves multisystems with dermatological, rheumatological, neurological and cardiac manifestations. It is caused by Borrelia burgdorferi and transmitted by Ixodes. Several neurological syndromes had been described including lymphatic meningitis, cranial neuropathy (commonly unilateral or bilateral Bell's palsy), painful radiculoneuritis, optic neuritis, mononeuritis multiplex, Guillain-Barre syndrome, encephalomyelitis, peripheral neuropathy and encephalopathy. Myelitis has recently been reported as an unusual complication of Lyme disease. Our case represents one of the few cases of this type. The unique feature in our case is that Lyme disease was first presented as myelitis.\nCEREBRAL AUTOSOMAL DOMINANT ARTERIOPATHY WITH SUBCORTICAL INFARCT AND LEUCOENCEPHALOPATHY (CADASIL) SYNDROME. A. Alghadhan 1 , A. Hamad 2 ; 1 SUNY at Stony Brook, Port Jefferson, NY; 2 NUMC, Eastmeadow, NY LEARNING OBJECTIVES: -recognize cerebral autosomal dominant arteriopathy with subcortical infarct and leucoencephalopathy (CADASIL) in the differential diagnosis for normal pressure hydrocephalus. CASE INFORMATION: 53 years old white female presented with progressive cognitive dysfunction and dementia, gait disturbance, urinary incontinence over the last 3 years. She has long standing history of migraine. 3 years ago, she had a computed tomography (CT) of the brain that showed dilated ventricles and labeled as normal pressure hydrocephalus and the patient refused treatment. Family history was significant for early dementia and migraine in the mother. Her physical exam showed severe dementia, severe ataxia on standing or walking despite normal muscular strength. Sensory exam was normal. Laboratory tests were unremarkable. Head CT and MRI showed severe atrophy with severe dilated ventricles and severe periventricular white matter disease. Skin biopsy showed characteristics PAS positive granular material in the media and narrowing of the lumen of the vessels which is typically seen in cerebral autosomal dominant arteriopathy with subcortical infarct and leucoencephalopathy (CADASIL). The patient was placed in a nursing home after explaining the nature of the disease to the family. DISCUSSION: CADASIL can be considered as a new disease that had been described recently (1996) in few families. It predominantly affects the small vessels of the brain. The transmission is autosomal dominant linked to chromosome 19. The clinical features include migraine with or without aura (the gene for familial type of migraine is located on the same chromosome), strokes or stroke-like episodes, psychiatric symptoms and dementia. In our case the presentation was similar to the normal pressure hydrocephalus symptoms. We want physicians to be aware of this similarity as the management is completely different. year-old women presented to the hospital with nonhealing ulcers on her breast, thighs and toes. She developed systemic lupus at a young age that ultimately caused complete renal failure. She underwent renal transplant; however, she reinitiated hemodialysis after failure of the transplant. Her examination revealed necrotic ulcers in the left upper breast region. Radiologic evaluation of the breast, hands and chest revealed significant calcifications throughout the body. This pattern revealed systemic calciphylaxis. She underwent debridement and nerve block for pain control. She was dismissed; however, she eventually died three months after dismissal. DISCUSSION: Nonhealing ulcers are a common source of morbidity in hospitalized patients. Common etiologies include ischemia from vascular causes, chronic edema, and small vessel disease. Uncommon etiologies like calciphylaxis differ from traditional chronic ulcers. The skin appears violaceous with a plaque-like or a nodular appearance. The ulcers cause extreme pain independent of size or appearance. The pathogenesis of calciphylaxis remains unknown. Biochemical tests often reveal abnormal calcium or phosphorus levels. Treatment options often include subtotal parathyroidectomy in some patients. Normalization of calcium and phosphorus remains the primary focus of medical treatment. The chronic wounds are often aggressively de Âbrided and antibiotics are used in cases with infections. The use of immunosuppression for calciphylaxis has had mixed results. Despite aggressive wound care and calcium management, the prognosis of calciphylaxis remains poor at 60% morality. Further research needs to be performed on this uncommon yet potentially lethal illness.\nA. Almahameed 1 , S. Baghdasarian 2 , S. Frost 2 ; 1 Cleveland Clinic Foundation, Shaker Heights, OH; 2 The Cleveland Clinic Foundation, Cleveland, OH LEARNING OBJECTIVES: 1) Recognize the potential for severe complications in diabetic patients with urinary tract infection. 2) Diagnose renal emphysema. 3) Recognize that prompt medical and surgical treatment is essential to decrease the high rate of mortality associated with emphysematous pyelonephritis and pyelitis. CASE INFORMATION: A 48 yo black woman with multiple sclerosis and diabetes mellitus presented with abdominal pain, fever, and dysuria 3 days after starting ciprofloxacin for presumed urinary tract infection (UTI) . Physical exam demonstrated T=39C, HR=130, BP=150/80, and tenderness in the left lower quadrant without peritoneal signs. Laboratory investigation revealed WBC=9.91, Cr=1.2, and BUN=25. Urinalysis showed trace leukocyte esterase, WBC=6-10, 2+ blood, and RBC=0-3. Ampicillin and gentamicin were administered. CT scan of the abdomen was obtained demonstrating slight enlargement of the left kidney with stranding of the perinephric fat, and a dilated left renal pelvis and proximal ureter containing pockets of gas. Hydronephrosis, pyelonephritis, and emphysematous pyelitis were diagnosed and the patient promptly underwent intraoperative cystoscopy to evaluate for ureteral obstruction. Debris occluding the left ureter was visualized and a ureteral stent was deployed. Urine and blood cultures revealed Proteus Mirabilis and Aerococcus Urinae. The patient improved quickly and was discharged on the fourth day of hospitalization to complete antibiotic treatment with Augmentin. DISCUSSION: UTI is a significant problem in diabetic patients, and complications such as pyelonephritis, renal or perirenal abscess, focal or multifocal bacterial nephritis, xanthogranulomatous pyelonephritis, and renal papillary necrosis can occur. Emphysematous pyelonephritis (air in the renal parenchyma), and emphysematous pyelitis (air in the collecting system) are complications that occur more frequently in diabetic than in non-diabetic patients. Early diagnosis of emphysematous complications is crucial due to the high rate of mortality in patients whose diagnosis is delayed. Radiographic confirmation is essential (preferably by abdominal CT scan) as the history, physical exam and laboratory findings are rarely diagnostic. Emphysematous pyelonephritis responds poorly to antibiotics alone, with mortality rates as high as 80%. Surgical intervention is virtually mandatory, with nephrectomy lowering mortality to 20% or less. Emphysematous pyelitis is less critical, with an overall mortality of 20%. It is often associated with urinary tract obstruction, and responds well to intravenous antibiotics and obstruction relief.\nA 55-YEAR-OLD PHYSICIAN WITH LYMPHOCYTOSIS, FEVER, ABDOMINAL PAIN, AND PROFOUND THROMBOCYTOPENIA. A. Almahameed 1 , A. Absi 2 , D. Farray-berges 2 , S. Rehm 3 ; 1 Cleveland Clinic Foundation, Shaker Heights, Ohio; 2 The Cleveland Clinic Foundation, shaker hts, Ohio; 3 The Cleveland Clinic Foundation, Cleveland, Ohio LEARNING OBJECTIVES: 1-Recognize infectious mononucleosis as a rare cause of severe thrombocytopenia. 2-Utilize EBV-specific antigens to diagnose infectious mononucleosis in monospot-negative patients. 3-Treat EBV-induced severe thrombocytopenia. CASE INFORMATION: A previously healthy 55-year-old male physician reported 2 weeks of fatigue, malaise, and mild abdominal discomfort. On initial evaluation, a right submandibular lymph node was moderately enlarged and the CBC was normal. Several days later, he became febrile (39 C). He had a normal WBC count with lymphocytosis (50%) and atypical lymphocytes (25%), as well as transaminitis (ALT 239 U/L and AST 198 U/L). Monospot tests, CMV titers, HIV antibodies, and blood and urine cultures were all negative. Self-prescribed oral therapy with empiric clarithromycin and ciprofloxacin produced little improvement. One week later, his platelet count was 90,000/mm 3 . He was admitted three days later with petechial rash involving the upper extremities and the upper part of his trunk, and a platelet count of 8,000/ mm 3 . He was afebrile, had diffuse non-palpable, non-blanching petechiae on his upper extremities, ecchymoses on his buccal mucosa, 3 mildly enlarged non-tender lymph nodes in the right submandibular area, and a palpable spleen tip. The WBC count was 10,000/mm 3 with 26% polymorphs and 63% lymphocytes, of which 20% were atypical. His hemoglobin was 13.4 g/dl, and his platelet count was less than 1,000/mm 3 . Blood chemistries and coagulation parameters were normal. Occasional atypical, large lymphocytes and rare platelets were evident on a peripheral blood smear. Bone marrow biopsy showed normocellular composition with severe thrombocytopenia and adequate numbers of megakaryocytes. Flow cytometry of the blood found no malignant cells. Serologies were negative for toxoplasmosis, hepatitis C, hepatitis B, herpes simplex, and herpes virus 6, but the patient had elevated levels of IgM and IgG antibodies to Epstein-Barr virus viral capsid antigen (VCA) (IgG VCA: 152 IU/ml, normal < 18, and IgM VCA 71 TV (normal < 18). The patient was treated with high-dose prednisone and IVIG 1 g/kg/day for 2 days, and his platelet count rose gradually (11,000/mm 3 on discharge and 180,000 mm 3 3 weeks later). DISCUSSION: Infectious mononucleosis (IM) should be included in the differential diagnosis of severe thrombocytopenia in adults even when the initial monospot test is negative. Lowgrade neutropenia and thrombocytopenia are common during the first month of illness. Heterophile antibodies are useful in diagnosing disease in many cases but are often not detectable in children under age 5, in the elderly, and in patients presenting with symptoms not typical of IM. An alternative is EBV-specific antibody testing. Titers of IgM and IgG antibodies to viral capsid antigen are elevated in the serum of more than 90% of patients at the onset of disease. IgM antibody to VCA is useful for the diagnosis of acute IM because levels are elevated only during the first 2 months of the disease. We can not entirely exclude the possibility that ciprofloxacin or clarithromycin therapy caused the thrombocytopenia. Given the clinical illness, the results of serologic testing, and the rarity of profound antibioticassociated thrombocytopenia, we believe that acute Epstein Barr virus infection is the more likely cause of the abnormalities. DISCUSSION: Vitamin B12 comes from the diet and is present in all food products of animal origin. Liver stores of B12 can last for several years after dietary ingestion ceases. Although dietary B12 deficiency is extremely rare, it can occur in vegans who abstain from all such foods for prolonged periods of time.\nWHEN A PATIENT PRESENTS WITH WEAKNESS: IT MAYBE HELPFUL TO REVIEW YOUR PRESCRIPTION. A. Arshad 1 , R. Keating 1 ; 1 Fairview Hospital, Cleveland, OH LEARNING OBJECTIVES: 1) Diagnose drug-induced myositis from its presentation. 2) Recognize how adding multi drug antihyperlipidemic therapy increases the incidence of myositis. CASE INFORMATION: A 72 year old white female with past medical history of diabetes mellitus, hypertension and hyperlipidemia who was in relatively good health presents with progressive weakness for the last one week. A week ago she had a yeast infection and took two tablets of diflucan. Shortly after she started feeling weak and the weakness progressed gradually. She is now unable to stand or walk has trouble feeding due to weakness in her arms and also occasionally gets short of breath. She denies any headache, double vision, blurred vision, fevers, chills, pain in her muscles or joints or any recent falls. She is currently on cerivastatin, insulin, lansoprazole, gemfibrozil, atenolol, aspirin, nortriptyline, sertraline, and lisinopril. On physical examination she is afebrile with stable vital signs. The muscle strength was severely decreased in all extremities, upper greater than lower and especially in the shoulder and pelvic girdles. No skin rash or mouth ulcers were seen. Laboratory work shows a creatine kinase of 4132 U/L, myoglobin titer of 3634. Liver enzymes were mildly elevated. Patient was started on steroids and she adequately recovered. DISCUSSION: The onset of generalized progressive myopathy in a patient taking cholesterol lowering drugs should alert us to the diagnosis of drug induced myopathy. Several antihyperlipidemic drugs have caused myopathy including the statins, gemfibrozil, nicotinic acid and clofibrate. Lovastatin has been reported several times in literature to induce myositis. The concomitant use of gemfibrozil in patients taking statins increases the incidence of myositis to five percent. The creatine kinase and sedimentation rate is markedly elevated in these patients. Creatine kinase elevations are very specific and consist of only the MM fraction. Discontinuation of statin therapy in these patients is advisable because of the potential development of rhabdomyolysis and acute renal failure. Cautious use of statins is recommended in patients with prior history of liver disease, those with active liver disease and in heavy alcohol users. Hepatic and muscle enzyme levels should be tested periodically during statin treatment. Other medications known to increase the incidence of myopathy in patients taking statins are cyclosporine, nicotinic acid, prednisone and azathioprine. Treatment includes discontinuation of the medication and use of steroids. The use of diflucan was most likely incidental as there have not been cases of myopathy attributed to diflucan. 2) Recognize Propionibacterium acnes as a cause of intra-cranial abscess.\n3) Manage Propionibacterium abscess with surgical drainage and antibiotic monotherapy. CASE INFORMATION: A fifty-two year old white male presented with a lump on the head for over a year. Magnetic resonance imaging (MRI) showed a mass lesion in the parietal lobe. He subsequently underwent craniotomy for an epidural neoplasm and left parietal reconstruction with duragen. He then received two doses of radiotherapy. About three months later he presents with right sided weakness, focal seizures in the right hand and a fight facial palsy. After recurrent seizures on anticonvulsant therapy a repeat computed tomography scan and MRI scan was done which showed an epidural lesion with mass effect. The patient was afebrile and had a normal peripheral blood count. The patient was taken to surgery and an epidural abscess was drained. The abscess aspirate on gram stain revealed pleomorphic gram positive bacteria. Aerobic cultures were negative and so were the anaerobic cultures initially but after several days of incubation they grew out a pure culture of a pleomorphic gram positive organism which was identified as Propionibacterium acnes. He was started on intravenous ceftriaxone for 6 weeks and he recovered fully after completion of therapy. DISCUSSION: This case illustrates a case of post-surgical epidural abscess caused by Propionibacterium acnes. Propionibacterium acne is a corynebacterium usually isolated from the human skin where it plays a role in the pathogenesis of acne vulgaris. There have been reports in literature of clinically significant infections related to CNS shunts, intracranial abscesses related to dental sepsis, sepsis related to indwelling vascular catheters, and also a few cases of intracranial abscesses unrelated to foreign bodies. The above case is an addition to these rare cases of intracranial abscesses caused by this unusual pathogen. They are slow growing organisms and as their identification presents problems, they have been frequently overlooked as contaminants. It has been suggested that grain positive rods on gram stain and negative aerobic cultures of suspected infected fluids without organisms on gram stain should be cultured anaerobically. Most infections reported in literature were treated with a combination of antibiotics, though there have been reports of successful treatment with penicillin and surgical drainage alone. The above case also shows how ceftriaxone monotherapy is an effective treatment for such infections along with surgical evacuation of the abscess. year-old male with HIV (CD4 of 343, undetectable viral load) was admitted to the hospital with shortness of breath. He was diagnosed with pneumocystis pneumonia vs cocci pneumonia and treated with Bactrim and Fluconazole. One week later he returned with continuing dyspnea. A VQ scan showed high probability for pulmonary embolism. He was treated with heparin then coumadin and discharged. Three days later he returned with new left lower extremity swelling and pain. He had been taking Coumadin with an INR of 5.1. He denied shortness of breath, chest pain, IV drug use, smoking, ETOH, or recent travel. His medications included Abacavir, Efavirenz, Nelfinavir, Fluconazole, and Bactrim. Physical examination revealed a tender, swollen left lower extremity with a positive Homan's sign. An ultrasound showed thrombosis in the left popliteal vein. The swelling and pain resolved with heparin and he was discharged on Lovenox. A hypercoagulable evaluation revealed normal levels of protein S, activated protein C resistance, anti-thrombin III, and homocysteine. Protein C was slightly elevated. Lupus anticoagulant and anti-cardiolipin antibody were negative. An abdominal CT revealed no mass lesions. DISCUSSION: Thrombosis is an uncommon complication of HIV infection that many primary care providers may not be aware of. Anecdotal evidence in the literature relates thrombosis and HIV, especially in patients with IV drug use, Kaposi's, cytomegalovirus (CMV) disease, megestrol use, protease inhibitor use (including nelfinavir), and hypercoagulable states such as anticardiolipin antibody and protein S deficiency. A recent epidemiologic study showed that HIV patients with thrombotic events had the following characteristics: age greater than 45 years, CMV disease, AIDS-defining opportunistic infection, hospitalization, megestrol use, and indinavir use. The only risk factors for thrombosis that this patient had were hospitalization and nelfinavir use. HIV itself may be a risk factor for thrombotic events and should be considered in cases where shortness of breath remains unresolved despite maximal medical therapy. The mechanism by which HIV causes thrombosis remains unknown but several theories have been proposed. This case illustrates that the link of HIV as a possible cause of thrombosis may have implications for prophylaxis in HIV patients. There are no consensus studies and prophylaxis remains at the discretion of the clinician.\nLEARNING OBJECTIVES: 1. To illustrate clinical features of hypoglycemia from inadvertent administration of a sulfonylurea compared to an insulinoma. 2. To demonstrate the importance of carefully reviewing medications that patients with hypoglycemia are taking. CASE INFORMATION: Two patients were referred for recurrent episodes of hypoglycemia thought to be due an insulinoma. The clinical presentations were characterized by precipitous drops in blood glucose (BG) associated with marked adrenergic symptoms. The first patient was a 69 yo man with a coronary artery disease, heart failure, hypertension, chronic renal insufficiency, and peripheral vascular disease. He had recurrent acute hypoglycemic reactions (including 3 hospital admissions) accompanied by adrenergic symptoms. He also had a recent myocardial infarction. Medications included warfarin, metoprolol, nifedipine, isosorbide dinitrate (IsoD), furosemide, famotidine, lisinopril, potassium, pentoxyphylline, and colchicine. His chemistry panel was normal. Prior workup included normal cortrosyn stimulation test and abdominal CT scan, and negative serum sulfonylurea levels. A 36 hour fast ( the patient refused to go beyond 36 hours) showed a BG of 59 mg/dl, a corresponding cpeptide of 3.5 ng/ml (0.7±3.0) , and absent insulin antibody. Inspection of his medications revealed a bottle labeled for IsoD contained two types of light green pills: IsoD and glyburide 5 mg. The second patient was an 87 yo woman who was referred for surgical treatment for a presumed insulinoma. Over a 2 mo period she has had several episodes consisting of confusion, diaphoresis, and syncope with confirmed hypoglycemia(BG levels < 40mg/dl). Medications included cardizem, ranitidine, acetaminophen, and diazepam. Evaluation included a normal chemistry panels, somatomedin, and cortrosyn stimulation test. Serum insulin and c-peptide levels at the time of hypoglycemia (BG= 37 mg/dl) were 36.38 pmol/L and 1600 pmol/L respectively. Serum and urine SFU screens were negative. CT and octreotide scans of the abdomen showed no abnormalities. Identification of the patient's home medications was done. In a bottle labeled diazepam were light blue-green tablets identified as generic glyburide 5mg. To further confirm SFU induced hypoglycemia, a repeat 72 hour fasting was done. At a glucose level of 94mg/dl, the insulin and insulin antibody levels were 3.2uU/ml (4.0±24.0) and < 5 % respectively, and at a glucose level of 58mg/dl, the levels were < 2 uU/ml and < 5% respectively. An exploratory laparotomy was canceled. In each case, no further hypoglycemic episodes occured following discontinuation of the SFU. DISCUSSION: The biochemical features of hypoglycemia due to sulfonylureas and insulinomas are similar. Insulinomas are commonly associated neuroglycopenic symptoms that are insidious in onset, and recipitous hypoglycemia with adrenergic symptoms is uncommon. Presence of such symptoms mandates a search for other causes. SFU-induced hypoglycemia may mimic an insulinoma biochemically. Our two cases illustrate the importance of careful attention to history, and a thorough inspection of all medications in patients presenting with hypoglycemia before extensive, diagnostic tests, including invasive procedures or surgery are undertaken LEARNING OBJECTIVES: 1) Diagnose chronic lung disease in patients with sickle cell disease.\n2) Recognize the risk of pulmonary hypertension during pregnancy. CASE INFORMATION: A 30-year-old G2P1 with a history of sickle cell disease presented to our institution at 26 weeks gestation with the complaint of preterm contractions. She was admitted and placed on magnesium as a tocolytic. She then began to complain of back and leg pain leading to a diagnosis of a sickle crisis. Her medical history was notable for 3 to 4 inpatient admissions for sickle crises a year. She had a port-a-cath placed 11 months prior to the admission. Her gall bladder was removed two years prior to admission. Her evaluation included room air PaO2 of 68 mmHg and pulse oximetry of 93%, and a chest xray revealed bilateral pulmonary infiltrates in an alveolar pattern. Given a normal urinalysis and urine culture, and no other identifiable trigger for the crisis or the preterm contractions, she was treated for pneumonia. Magnesium was discontinued. The improvement in her oxygenation seemed slower than expected prompting the high-risk team to order an echocardiogram. Her left ventricle and valves were normal, but the right-sided chambers were enlarged and her estimated pulmonary artery systolic pressure was 55mmHg. V/Q scan was normal. This is the first time she was diagnosed as having pulmonary hypertension. Review of prior chest x-rays from another institution revealed that the pulmonary infiltrates had been present for at least 18 months. DISCUSSION: A review of the literature revealed many series of sickle cell patients with chronic lung disease. These patients may be asymptomatic, but abnormalities on pulmonary function testing are an early and reliable marker of this complication. Patients with abnormal chest radiography, pulmonary function testing,x or hypoxia during stable periods have an average life expectancy of 2.6 years. They are at risk for sudden death from subendocardial infarction without epicardial arterial disease. We are surprised that more pregnant patients with long standing sickle cell disease do not have routine screening for lung disease by their physicians. This is particularly important during pregnancy because severe pulmonary hypertension (pressures > 80mmg) from any cause has a post-partum mortality rate as high as 50%. The precise risk with less severe elevations is not known. Given the prevalence of sickle cell disease in the general medical population we recommend that all patients with long standing sickle disease have pulmonary function testing and/or echocardiography to evaluate pulmonary arterial pressures. If chronic pulmonary disease is diagnosed in a woman considering pregnancy then consultation with experienced internists, pulmonary specialists, and perinatologists is warranted.`H complaints or symptoms suggestive of cardiopulmonary diseases. On a follow up visit he reported dyspnea on minimal exertion of two months duration. He also reported chest pressure, dizziness and nausea with the dyspnea. He told his doctor that he has dyspnea even after speaking few sentences. His exam revealed BP= 146/90 Pulse 80 regular. Heart auscultation revealed a normal S1, split S2, and I-II/VI systolic murmur. Lungs were clear to auscultation. Abdomen was soft, no hepatosplenomegaly. No pedal edema. EKG was done in the clinic showed left ventricular hypertrophy. Chest X-ray was normal. Stress echocardiogram revealed a small apical wall motion abnormality worsening with stress. A cardiac catheterization revealed normal coronaries, no wall motion abnormality and left ventricular end diastolic pressure of 35 mmHg. The aortic root was bicuspid with severe coarctation of the aorta at the isthmus. DISCUSSION: Coarctation of the aorta is an unusual cause of hypertension in young adults and is often unrecognized by general practitioners. Although the left ventricle is usually hypertrophied because of the hypertension, the heart is usually normal in size or only slightly enlarged until cardiac decompensation occurs late in the course of the disease. Our patient left ventricular end diastolic pressure was 35 mmHg, which might account to his dyspnea. In retrospect knowing the diagnosis of coarctation our patient has the body habitus of a chronic coarctation of the aorta with a large head and upper extremities and is otherwise short. He had diminished leg pulses. His blood pressure was comparable in upper extremities; however, there was a difference of 50 mmHg between upper and lower extremities. Coarctation of the aorta is a potentially lethal condition at all ages. Many patients first come to medical attention because of signs and symptoms related to hypertension. The diagnosis should be considered in patients with hypertension and diminished leg pulses, and we recommend the routine examination of leg pulses in young adults with hypertension.\nHEMOLYSIS IN A RECENTLY HOSPITALIZED PATIENT. L. Chang 1 ; 1 UCLA/San Fernando Valley Program, Sepulveda, CA LEARNING OBJECTIVES: 1) To recognize drug-induced hemolysis as a common cause of hemolytic anemia 2) To recognize that 2nd and 3rd generation cephalosporins are the most common cause of drug-induced hemolytic anemia 3) To recognize that cephalosporin-induced hemolytic anemia is often serious, and can be fatal. CASE INFORMATION: A 62-year-old female discharged 7 days ago after being treated for Streptococcus pharyngitis presents to a follow-up clinic complaining of a 5-day history of nausea, nonbilious and nonbloody vomiting, and anorexia. She also complains of a generalized headache and subjective fevers. In her previous hospitalization, she presented to the emergency room complaining of fever, sore throat, and epigastric pain. At that time, she was found to have a WBC of 16 with 89%neutrophils, a Hct of 44, and a clean urinalysis. A CT abdomen was done revealing diverticulosis only. Her temperature was 38.4. She was given one dose of Cefotetan in the ER and admitted for further workup. On the medicine wards, further evaluation revealed an exudative tonsillitis with anterior cervical lymphadenopathy. She admitted to a history of recurrent pharyngitis. She was treated with penicillin VK, improved clinically, and sent home on penicillin. She had no known drug allergies. Physical examination: Temp 38.5, BP 141/70, pulse 97. She was moderately ill appearing. Eyes were icteric. Oropharynx revealed tonsillar swelling, but was significantly improved from her previous hospitalization exam. Neck was supple. Heart and lung exams were normal. Abdomen revealed mild epigastric tenderness without peritoneal signs. Neurological exam was normal. Laboratory studies: WBC 18.2, Hgb 11, Hct 30.6, Plt 300, MCV 89. Total bilirubin 13.2, direct bilirubin 0.5, AST 34, ALT 28, alkaline phosphatase 93. PTT 23, INR 1.04. LDH 1578, corrected reticulocyte count 1.8, haptoglobin < 50. Direct Coombs was markedly positive. DISCUSSION: The positive direct Coombs test was further investigated by the blood bank that, along with assistance from the Red Cross, determined that this patient had developed a Cefotetan-induced autoimmune hemolytic anemia (HA). It should be recognized that 1stgeneration cephalosporins rarely cause HA, but 2nd and 3rd generation cephalosporins are the most common cause of drug-induced HA. Most cases of cephalosporin-induced HA revealed moderate-to-severe hemolysis with fatalities reported. Most patients required treatment with blood transfusion and steroids. Because of the common use of 2nd and 3rd generation cephalosporins by physicians, and the severity of the hemolysis that can be induced by them, recognition of this entity is of significant importance to the practicing clinician.\nA CASE OF HEMOPTYSIS FOLLOWING PERCUTANEOUS TRANSLUMINAL CORONARY ANGIOPLASTY. S. Chebrolu 1 , J. Addagatla 1 , F. Ali 1 ; 1 St. Francis Hospital, Evanston, IL LEARNING OBJECTIVES: 1) Recognize the occurrence of bleeding complications including pulmonary alveolar hemorrhage following the use of Group IIb-IIIa receptor inhibitors in acute coronary syndromes. 2) Diagnose and manage pulmonary alveolar hemorrhage. CASE INFORMATION: A 68 year-old woman with a past medical history of diabetes mellitus and hypertension presented to the emergency room with chest pain. She was a non-smoker and had no known lung disease. Physical examination was normal. EKG showed an acute anteroseptal myocardial infarction. Emergent angiogram revealed a 100% proximal left anterior descending artery lesion. Percutaneous transluminal coronary angioplasty with placement of a multilink stent of the left anterior descending artery lesion was done. During the procedure, she was given 10,000U of heparin and 0.25 mg/kg of abciximab boluses followed by infusion of heparin at 700U/hr and abciximab at 0.125 mcg/kg/min after the procedure. An intra-aortic balloon pump was also placed. One hour later, she had hematemesis and hemoptysis and developed respiratory distress with hypoxia. Heparin and abciximab were discontinued and emergent intubation was done. A copious amount of blood was aspirated from the endotracheal tube. CXR revealed diffuse bilateral interstitial infiltrates. Hemoglobin dropped from 12.6g/dl to 10g/dl. A diagnosis of pulmonary alveolar hemorrhage was made. Bright red blood was suctioned from the endotracheal tube for a few hours. Hypoxemia improved rapidly with complete clearing of the CXR over the next two days. She was extubated on day three and was discharged home on day seven on aspirin, clopidogrel, metprolol and ramipril. She received two units of packed red blood corpuscles during her hospital course. DISCUSSION: Pulmonary alveolar hemorrhage is an infrequent and rarely reported hemorrhagic complication of the use of Group IIb-IIIa receptor inhibitors in acute coronary syndromes. It should be considered in the differential diagnosis of a patient who receives these agents and presents with respiratory distress, pulmonary infiltrates and a drop in hemoglobin, with or without hemoptysis. It is usually seen in older patients and in those with increased pulmonary capillary wedge pressure and evidence of pulmonary edema. Management is mainly supportive with discontinuation of all anticoagulants and with transfusions of packed red blood corpuscles and platelets as needed. An 82 year-old man was admitted with complaints of dysphagia and constipation for one week. It was associated with anorexia and significant weight loss over one month duration. He denied any history of abdominal pain, hematemesis or melena. Past medical history included COPD and coronary artery disease. Past surgical history included CABG, appendectomy and bilateral inguinal hernia repair. Abdominal examination was benign but he was found to be fecal occult blood positive. Laboratory work up revealed evidence of iron deficiency anemia and a gastrointestinal malignancy was suspected. He underwent gastroduodenoscopy that revealed hiatal hernia and a colonoscopy was planned. A few hours after the procedure, he complained of severe lower abdominal pain when he was noticed to have right lower quadrant tenderness and fullness but with no guarding or rigidity. CT scan of the abdomen was done which revealed a mass in the terminal ileum and a 5-cm mass in the left lobe of liver. It also revealed superior MVT with ischemic changes in the small bowel. He underwent emergent angiography that confirmed the presence of MVT and was given local t-PA for dissolution of the MVT. Following this, his abdominal pain worsened with development of peritoneal signs and he underwent exploratory laparotomy with the resection of terminal ileum, right hemicolectomy and biopsy of the liver mass. Histopathology of the mass and liver biopsy revealed malignant carcinoid tumor with mesenteric node and liver involvement. DISCUSSION: MVT is characterized by pain out of proportion to physical findings, nausea, vomiting, constipation and occasional bloody diarrhea. Previous abdominal surgery, hypercoagulable states, cirrhosis and gastrointestinal malignancies are the most common conditions associated with MVT. Abdominal distention, though nonspecific, is the most frequent sign. Leukocytosis may be found in half of the patients but laboratory tests are not helpful in the diagnosis. CT is the diagnostic test of choice and demonstrates thrombus in the superior mesenteric vein with evidence of bowel ischemia. Anticoagulation with heparin followed by warfarin in the absence of contraindications is the mainstay of medical management. All patients with localized or diffuse peritonitis should undergo immediate exploratory laparotomy with resection of nonviable bowel. MVT has not been reported previously as a presenting feature of malignant carcinoid tumor as seen in our patient here.\nMYELOMA IN A HYPERCALCEMIC PATIENT. S. Chebrolu 1 , F. Zar 1 ; 1 St. Francis Hospital, Evanston, IL LEARNING OBJECTIVES: 1) Recognize that Primary Hyperparathyroidism (PHP) and Multiple Myeloma (MM) are the most common causes of hypercalcemia in the elderly population and diagnose them. 2) Recognize that more than one independent cause of hypercalcemia is very rare and should be considered in a patient unresponsive to treatment. CASE INFORMATION: A 76 year-old male was admitted with weakness, lethargy and occasional confusion of three months duration. His past medical history included sick sinus syndrome and valve replacement for aortic incompetence. His primary doctor discontinued chlorthalidone four weeks prior to this admission after he was found to be hypercalcemic (13.9mg/dl). Examination was unremarkable. Initial studies revealed calcium 11.8mg/dl (normal 8.8±10.4mg/dl), ionized calcium 6.77 mg/dl (normal 4.6±5.3mg/dl), and phosphorus 2.3 mg/dl (normal 2.4±4.5mg/dl). CT scan of head was normal. Patient was hydrated with normal saline and later diuresed with furosemide. An intact parathyroid hormone level by immunochemiluminescent assay was 12.1 pmol/l. C-terminal peptide was 323 pmol/l. Serum osteocalcin was 21U/l. A CT scan of the neck showed a mass suggestive of a parathyroid adenoma, confirmed by Technetium-99m sestamibi scan showing a persistent crescentric area of increased activity in the left lower neck in both immediate and delayed films. In addition, urine protein electrophoresis showed monoclonal kappa light chains. Serum protein electrophoresis revealed a monoclonal spike and IgG kappa monoclonal protein (2770mg/dl). Beta-2 microglobulin was 6.4mg/dl. A skeletal survey revealed multiple lytic defects throughout the skeletal system. Bone marrow aspiration showed focal increase in plasma cells consistent with MM. The patient was given pamidronate and started on chemotherapy with melphalan and he responded well. DISCUSSION: Although a variety of conditions can give rise to hypercalcemia, nearly 90% of the cases can be attributed to either PHP or MM. PHP was diagnosed in our patient by finding elevated intact parathyroid hormone levels associated with elevated C-terminal peptide levels. Technetium-99m-sestamibi scan confirmed its presence. IgG kappa monoclonal paraprotein on serum and urinary protein electrophoresis associated with multiple lytic defects on the skeletal survey was highly suggestive of MM which was confirmed by bone marrow biopsy. However, more than one independent cause of hypercalcemia in a patient is rarely seen. Only eighteen cases of simultaneous occurrence of both PHP and MM were reported and our patient will be the nineteenth such case. Considering the rarity, it appears that this is a chance association. Persistent and unresponsive hypercalcemia may occur if only one of causes is recognized in such rare instances and in a MM patient, this may falsely lead to a higher tumor grading.\nMETASTASIS-INDUCED ACUTE PANCREATITIS. S. Chebrolu 1 , A. Naidu 1 , F. Zar 1 ; 1 St. Francis Hospital, Evanston, IL LEARNING OBJECTIVES: 1) Recognize that metastatic tumors to pancreas may rarely induce acute pancreatitis referred to as Metastasis-Induced Acute Pancreatitis (MIAP). 2) Diagnose and manage MIAP. CASE INFORMATION: A 37-year-old woman presented with a one-day history of epigastric pain radiating to the back associated with nausea and decreased appetite. One and half years ago, she had a missed abortion followed by a Placental Site Trophoblastic Tumor (PSST) which was treated with total abdominal hysterectomy and chemotherapy with methotrexate. Her beta-HCG was less than 2 IU/l six months prior to this presentation. She had no history of alcoholism. On examination, she was found to have epigastric tenderness. Laboratory investigations revealed amylase 309 IU/l, lipase 154 IU/l, lactate dehydrogenase 544 IU/l, calcium 8.5mg/dl, glucose 221mg/dl and hemoglobin 13.6g/dl. She was diagnosed as acute pancreatitis and treated medically. Her pain persisted and she had CT scan of the abdomen that showed a swollen and enlarged pancreas with multiple rounded lesions of variable sizes. There was also a lobulated 3x4cm mass behind the sigmoid colon with a surgical clip adjacent to it associated with retroperitoneal and inguinal lymphadenopathy. Beta-HCG was found to be 2320.6 IU. ERCP was unremarkable. Exploratory laparotomy with bilateral salpingoopherectemy and tumor dissection was done showing histologic evidence of recurrent PSTT. A diagnosis of MIAP secondary to metastatic PSST was made and chemotherapy with etoposide, methotrexate and adriamycin was started with resolution of her symptoms. DISCUSSION: MIAP refers to non-pancreatic tumors metastasizing to the pancreas and resulting in acute pancreatitis. It is a very rare cause of acute pancreatitis and only 42 such cases have been reported in the literature. Most of them were associated with bronchogenic carcinoma and lymphoma. Mechanisms implicated in the causation of MIAP include mechanical ductal obstruction and rupture with direct parenchymal tumor invasion or ischemia secondary to vascular occlusion and encasement of pancreatic vessels. The clinical and laboratory presentation is similar to the other causes of pancreatitis and MIAP remains a diagnosis of exclusion. An accurate history, supportive clinical evidence and radiological findings of pancreatic and peri-pancreatic metastatic lesions in the presence of a known primary tumor aid in the diagnosis. MIAP is managed conservatively with nasogastric tube suction, intravenous fluids and analgesics and this approach is helpful in most cases. If it persists, systemic chemotherapy for the metastatic tumor or palliative abdominal radiation may help. Overall, MIAP portends a bad prognosis and many patients succumb to the metastatic tumor sooner or later. PSST, a rare form of Gestational Trophoblastic Disease, has never previously been reported as a cause of MIAP and this is the first such instance. year old female Stanford graduate student presented with three days of fever and headache with associated nausea, neck stiffness, and photophobia. Past medical history significant for migraine headache and menstrual irregularity which responded well to oral contraceptives. Current medication includes three days of leuprolide injections for commercial egg donation. On exam, patient was febrile to 39.6 degrees Celsius with otherwise normal and stable vital signs. Patient was slightly lethargic and had slight photophobia and mild posterior neck tenderness on palpation, but otherwise completely normal exam. Ophthalmologic and neurologic exams were completely normal. Labs were normal except for Sodium 126 and Potassium 3.3. Lumbar puncture revealed opening pressure of 13cm. CSF studies revealed Glucose 44, Protein 51, WBC 27, RBC 510, and negative microscopic evaluation. Bacterial, viral and fungal cultures were eventually all negative. Empiric antibiotics of ceftriaxone, vancomycin and acyclovir were started. Patient continued to have headache, nausea, and fever with no clinical improvement or worsening except for Sodium continue to drop to 115 over next three days. On hospital day three, patient developed seizure and head MRI revealed 2.3cm  1.8cm sellar mass with hemorrhage and compression on optic chiasm. Surgical decompression of mass was complicated by right ACA/MCA territory infarct secondary to vasospasm and great sodium fluctuation due to SIADH and adrenal insufficiency. Patient eventully was discharged on thyroxine, cortisone, and DDAVP. She has made incredible recovery and will be resuming her PhD studies. DISCUSSION: The risk of pituitary apoplexy with use of leuprolide has been described in the literature but not well screened. Detailed clinical history, exam, and laboratory investigation might identify patients at higher risk of pituitary apoplexy for selective imaging evaluation. In this patient, the history of menstral irregularity controlled by oral contraceptives, the hyponatremia and loss of body temperature control could have been the starting point of investigation for pituitary adenoma and apoplexy. With increasing number of patients receiving elective leuprolide for fertility purpose, the medical community should be aware of potential risk in order to assess and advise patients accordingly. CASE INFORMATION: A 63-year-old male with a history of diabetes mellitus, hypertension, heavy alcohol consumption, and a T6 vertebral fracture was admitted to hospital with a one year history of progressive lower extremity proximal muscle weakness. Concurrent with his worsening weakness, the patient noticed increasing central obesity and easy bruising. Physical examination revealed a classic cushingoid appearance. The neurological examination demonstrated symmetric polyneuropathy as well as lower extremity proximal muscle weakness and wasting. A T6 sensory level was present. The reflexes were 1+ and the plantar responses were flexor bilaterally. Laboratory investigations confirmed Cushing's syndrome and suggested a pituitary source of hypercortisolism. An MRI of the spine showed multiple vertebral fractures and epidural lipomatosis in the posterior epidural space, from T2 to T8, compressing the spinal cord maximally at T6. Given the fragility of this patient's spine and the ongoing investigations to determine the cause of hypercortisolism, the patient did not undergo emergent epidural fat debulking. A pituitary microadenoma was diagnosed and resected by a transphenoidal approach. Although repeat imaging was not performed to document regression of the spinal lipomatosis, there was clinical and laboratory evidence of resolution of the hypercortisolemia. The post-operative course was complicated by pulmonary emboli and recurrent sepsis; the patient suffered a fatal cardiac arrest 96 days after surgery. DISCUSSION: Epidural lipomatosis has been described in steroid use and obesity, and more rarely in association with endogenous hypercortisolism such as Cushing's disease. This case emphasizes the difficulty in suspecting epidural lipomatosis in a patient with Cushing's syndrome in the setting of other neurologic processes. The diagnosis of epidural lipomatosis needs to be systematically considered in any patient with Cushing's syndrome who presents with back pain, lower extremity weakness, radiculopathy, or subtle signs of myelopathy. MRI of the spine is the diagnostic test of choice. Treatment modality (surgical debulking of epidural fat versus correction of the hypercortisolic state or weight loss) should be individualized based on the severity of neurological signs, the potential reversibility of the causative factor, and the estimated operative morbidity.\nS. Christopoulos 1 , A. Szilagyi 1 , S.R. Kahn 1 ; 1 SMBD Jewish General Hospital, McGill University, Montreal, Quebec LEARNING OBJECTIVES: 1) To recognize the importance of a careful medication history and a thorough review of side effects; 2) To recognize ergotism as a cause of mesenteric ischemia. CASE INFORMATION: Over a two-year period, a 43-year-old woman presented to the emergency department on five separate occasions with complaints of severe abdominal pain. Extensive investigations including gastroscopy, colonoscopy, two CT scans and ultrasounds of the abdomen, small bowel follow-through contrast study, enteroclysis, as well as a complete gynecological work-up were all negative. Over this period of time, the patient's treating psychiatrist diagnosed her as having Somatic Pain Disorder. The patient was admitted to the medical service of our institution with severe malnutrition. Her past medical history included a longstanding history of migraine headaches treated with caffeine/ergotamine tartrate enemas, weekly or more often, since the age of 18. The patient described her abdominal pain as sharp, located primarily in the epigastric region, and radiating to the back. The episodes occurred one hour postprandially, and were associated with nausea and vomiting. She reported a 60 lb weight loss over a one-year period. Physical examination revealed cachexia. Serial abdominal examinations were unremarkable. Stool occult blood testing was negative. Tube feeding was initiated. A CT angiogram was requested, which revealed significant narrowing of both the celiac and superior mesenteric arteries with the presence of collaterals. A diagnosis of mesenteric ischemia was established. An angiogram was requested prior to intestinal bypass surgery, however the patient developed worsening abdominal pain, significant leucocytosis, a lactate level three times normal, and evidence of peritoneal irritation. She underwent urgent laparotomy, which revealed extensive ischemia of the small bowel and diffuse arterial disease of the abdominal aorta, the iliac, superior mesenteric, and celiac arteries. An aorto-superior mesenteric bypass was performed. The patient underwent a second look laparotomy within 24 hours and 30 cm of dusky small bowel were resected. The post-operative course was uneventful. Following the discontinuation of ergotamine use, the patient no longer experienced abdominal pain and gained a significant amount of weight. DISCUSSION: Despite new modalities to treat migraine headaches, ergotamine is still used chronically by some migraine sufferers. The most recognized complication of ergotism is arterial vasospasm of the upper and lower extremities. More rarely, vasospasm is recognized in the carotid, retinal, renal, and mesenteric arteries. In our patient, failure to suspect this unusual cause of mesenteric ischemia led to delayed diagnosis, severe weight loss, and advanced intestinal infarction. The importance of a careful drug history cannot be overemphasized.\nMRI demonstrating thoracic epidoral lipomatosis with cord compression.\nLEARNING OBJECTIVES: 1. Diagnose polycystic ovary syndrome (PCOS). 2. Restore fertility in the setting of PCOS and subclinical hypothyroidism. 3. Recognize and manage the potential long-term sequelae of PCOS. CASE INFORMATION: A 28 year-old G0P0 Asian woman presented for a new patient appointment concerned about 7 months of amenorrhea. She recalled that her menstrual cycles since menarche at age 13 had often been irregular-varying in length from 28 to 45 days-but she had never gone more than two months without a period. Her last menstrual period was shortly before an extended and stressful trip back to her family in Taiwan. After several months without a period, a basic workup in Taiwan revealed a negative pregnancy test, elevated blood pressure, and a slightly increased TSH. She returned to the U.S. and had no subsequent followup. She made her appointment when she and her husband had become increasingly concerned about her fertility. Her past medical history was remarkable only for elevated triglycerides several years ago. She took no medications and denied any use of tobacco, alcohol, or drugs. Born in Taiwan, she emigrated to the U.S. as a child. She lived with her husband and worked as a psychiatric nurse. Her family history was unremarkable. Review of systems was notable for weight gain of about 15 pounds over the last year, intermittent fatigue and mood swings, frequent frontal headaches and lower abdominal cramping, bilateral hand numbness at night, and increased hair loss. She denied heat or cold intolerance, fevers, night sweats, nausea, vomiting, diarrhea, dysuria, vocal or visual changes. On exam, she was mildly obese and well-appearing. Compared with her driver's license photo from two years before, she had apparently gained some weight but showed no obvious changes in her facial structure. Her blood pressure was 110/70 and her heart rate was 80. Her BMI was 30. Her thyroid gland was slightly enlarged. There were terminal hairs present on her upper back but no other evidence of hirsutism. Her abdomen was slightly obese without striae. Bimanual exam revealed slight enlargement of her ovaries and associated mild tenderness. Initial laboratory data included normal CBC and renal panel, undetectable serum beta-HCG, TSH 5.02, free T4 14, FSH 6.4, LH 11.5, prolactin 9.3, fasting glucose 103, total cholesterol 167, triglycerides 314, LDL 66, and HDL 38. Pelvic ultrasound revealed bilateral polycystic ovaries. Additional blood tests were obtained. Daily administration of 10mg of medroxyprogesterone acetate yielded a normal menstrual period on day nine. She was also started on thyroxine and a program of weight reduction. DISCUSSION: PCOS is the most common endocrinopathy among women of reproductive age with an estimated prevalence of 4±7%. Though a clear consensus about diagnostic criteria for PCOS has not been achieved, the clinical hallmarks include evidence of hyperandrogenism, menstrual dysfunction, and polycystic ovaries. The diagnosis also requires the exclusion of other functional and neoplastic disorders with similar clinical features. Fertility may be restored in obese patients with PCOS through weight reduction alone. Metformin and clomiphene are other therapeutic options. In the unusual circumstance in which subclinical hypothyroidism may be contributing to menstrual disturbances, treatment with thyroxine has been demonstrated to restore normal function. Proper management of women with PCOS requires recognition of the long-term health risks associated with unopposed estrogen, insulin resistance, and lipid abnormalities including endometrial cancer, overt type 2 diabetes mellitus, and coronary artery disease.\nEPHEDRINE NEPHROLITHIASIS ASSOCIATED WITH EPHEDRINE USE. B.A. Costello 1 ; 1 Mayo Clinic, Rochester, MN LEARNING OBJECTIVES: 1) Awareness of nephrolithiasis as a potential complication of ephedrine overuse. 2) Recognition of importance of asking patients about over-the-counter (OTC) supplements. CASE INFORMATION: A 34 yo female was referred to our instution following a diagnosis of nephrolithiasis. She reported 2±3 similar episodes/yr over the past 20 years. Prior to transfer, she was found to have a 5mm stone in her left ureter. Upon admission, analgesia and hydration were provided; urine was strained for debris. 24h urine for citrate, oxalate, calcium, uric acid, and cystine was normal. She passed a stone which was analyzed and found to be composed of ephedrine. Upon further questioning, she reported long-term use of ephedrine and pseudoephedrine for nasal congestion. In addition, she had recently been using herbal supplements for weight loss. Although she was unaware, we found an additional ephedrine stone on this patient in our referral lab files. We advised her to discontinue all these agents. She has not had further episodes after 6 months of follow-up. DISCUSSION: Ephedrine nephrolithiasis has previously been reported in patients consuming ephedrine or ephedrine-containing herbal preparations (1, 2) . Our case suggests that chronic use of ephedrine can lead to recurrent nephrolithiasis. Herbal supplements used for dietary purposes commonly contain ephedrine and we suspect our patient was likely receiving additional ephedrine through the supplement. In 1997, the FDA did call into question the safety of supplements containing ephedrine (3), however, they remain readily available and are sometimes 3D Surface Shaded display showing high-grade stenosis of the celiac axis and superior mesenteric artery origins.\nwith hoarseness, dyspnea and sore throat. Since the morning of admission, the patient had drooling as he was unable to swallow saliva. His symptoms were aggravated when supine. He denied fever or cough. His vital signs were normal. Temperature was 36.8 C. Remainder of the examination was remarkable only for an erythematous posterior pharynx and stridor on auscultation of the neck. Laboratory evaluation revealed a white cell count of 21,700 with 87% neutrophils and a normal hematocrit, platelets and normal routine serum chemistries. Lateral view of the neck showed an edematous, thumb shaped epiglottis. Blood cultures revealed no bacterial growth, but throat culture grew Haemophilus influenza type b. The patient received intravenous methylprednisone and cefuroxime. His symptoms improved and he was discharged on the fifth hospital day. DISCUSSION: Acute epiglottis has increasingly become an adult disease, probably as result of immunization of children against Haemophilus influenza type b. A high index of suspicion is required in any patient with sudden onset of \" 4 Ds\" dyspahgia, dysphonia, dyspnea and drooling. The frequency of positive blood culture is lower in adults than in children. A lateral cervical soft tissue x-ray is a useful test with sensitivity of 79±90% and specificity of 86±90%. Indirect laryngoscopy is the confirmatory test needed only if diagnosis remains in doubt after initial evaluation. Conservative medical management consisting of antibiotics and corticosteroids with vigilant monitoring are the mainstay of therapy in adults. Prophylactic intubation is not recommended. Prognosis of adults with acute epiglottitis is good with early recognition and prompt treatment.\nTOO WARM FOR COMFORT±HEAT STROKE IN MINNESOTA. S. Dubey 1 , J.W. Leatherman 2 ; 1 Hennepin County Medical Center, Minneapolis, MN; 2 University of Minnesota LEARNING OBJECTIVES: 1) To recognize the morbidity and mortality from heat stroke 2) To emphasize that heat stroke is preventable and screening should be incorporated into seasonal health care maintenance. CASE INFORMATION: An 80 year old healthy male was found unresponsive by his wife in their sauna at home when he failed to come out in four hours time. Paramedics rushed him to the hospital where his core temperature was found to be 103 degrees F. He developed multiorgan damage including, respiratory failure requiring ventilatory support, myocardial injury, impaired hepatic and renal function, rhabdomyolysis and disseminated intravascular coagulation. With supportive treatment he made remarkable recovery and was discharged form the hospital on day six with a diagnosis of heat stroke. DISCUSSION: Heat stroke is a medical emergency manifesting with core temperature greater than 106 degrees F and multiorgan injury. Heat stroke can be fatal with almost 200 deaths occurring during an average summer in the United States alone. Risk factors include advanced age, lower socioeconomic status, chronic illness, dehydration, strenuous exercise and hot weather. Prevention is of utmost importance. Treatment consists of cooling, which must begin in the field, and supportive care. The mortality rate is as high as 10% in spite of aggressive management. Since it is easily preventable, screening and counseling need to be integrated into health care maintenance during high risk seasons to reduce the burden inflicted by this illness.\nIT QUACKS LIKE A DUCK, BUT IT ISNT A DUCK. S. Dubey 1 , B. Marinelli 1 ; 1 Hennepin County Medical Center, Minneapolis, MN LEARNING OBJECTIVES: 1) To recognize precordial changes on EKG as a manifestation of massive pulmonary embolism 2) To emphasize the importance of screening for major alternative diagnoses in patients who present with apparent acute coronary syndromes CASE INFORMATION: A 44-year-old man, with history of coronary stent placement in the left anterior descending artery five months previously, presented with sudden chest pain and dyspnea. Relevant past medical history included a motor vehicle accident several months ago, which resulted in multiple lower extremity procedures and infections leading to reduced ambulation. EKG on admission was significant for precordial T wave inversion, and preliminary trans thoracic echocardiogram done in the emergency department showed normal left ventricular function. Considering stent closure, a coronary angiogram was done which showed patent coronaries. At that time, the formal reading from the already done echo was obtained and showed right ventricular enlargement and reduced right ventricular function. Immediate pulmonary angiogram was done and revealed extensive thrombus in the left pulmonary arterial system. Thrombolysis was initiated with t-PA. DISCUSSION: This is an example of acute pulmonary embolus very convincingly masquerading as coronary stent closure. An important clue, namely, reduced ambulation due to recent leg injuries, lay hidden in the past medical history. Precordial T wave inversions on EKG in the setting of pulmonary embolism, has been well described in medical literature. Various theories, including true coronary insufficiency and catecholamine mediated global T wave inversions have been proposed. The presence of such T wave inversions correlates with increased severity of pulmonary embolism, and resolution over a few days correlates with improved prognosis. Treatment includes anticoagulation with or without thrombolysis. Such EKG changes warrant at least a detailed history and physical exam focusing on deep vein thrombosis and pulmonary embolism risk factors.\nEVALUATION AND DIAGNOSIS OF APPENDICITIS. S. Elad 1 ; 1 UCLA San Fernando Valley/ Olive View Medical Center, Los Angeles, CA LEARNING OBJECTIVES: 1. Recognize the clinical feautures of early appendicitis. 2. Identify atypical presentation of appendicitis. 3. Review common signs and symptoms of appendicitis. CASE INFORMATION: A 30 year old African American male with no significant past medical history presents with a one day history of right lower quadrant pain and constipation. Pt denied nausea, vomiting and anorexia. Pt was able to eat and tolerate both breakfast and lunch without incident. He denied fevers, chills. He was not taking any medications. Family history was noncontributory. He denied allergies and did not use tobacco, alcohol or drugs. On exam the patient was afebrile, pulse rate was 80 and blood pressure was 120/70. The cardiovascular exam was normal and his lungs were clear bilaterally. Abdominal exam revealed normoactive bowel sounds and mild right lower quadrant tenderness to palpation. He had no psoas and no murphys sign. No rebound, no guarding. His stool was heme negative. Neurological exam was within normal limits. Abdominal x-ray revealed right colon full of stool with no air fluid levels and no fecalith. Lab studies: NA 138 K 3.8 Cre 1.2 ALT 29 WBC 8 Hgb 12 Hct 39 Alk P 73 AST 36 TBili 0.5 Pt was sent home with diagnosis of constipation and given milk of magnesium and fleets enema. Pt returned to ER the following morning with complaints of worsening abdominal pain and anorexia. CT of abdomen revealed para appendiceal stranding. Pt was taken to the operating room for emergent appendectomy. Pt was found to have a gangrenous appendix with micro-perforations. Pt required a five day hospitalization and was discharged on oral antibiotics. DISCUSSION: The diagnosis of early appendicitis is often difficult to make. Classically appendicitis presents as a pentad of signs and symptoms consisting of RLQ pain, anorexia, vomitting, low grade fever and leukocytosis. Typically, there is a history of pain beginning in the peri-umbilical region that migrates to the RLQ. However, this presentation occurs in only 50% of patients. The location of the appendix may also alter the presentation of pain. A retrocecal appendix may cause flank pain whereas an appendix in the pelvis may elicit tenderness on rectal exam. Although the WBC is elevated in 80% of patients, patients with early appendicitis may have a normal WBC. The diagnosis of acute appendicitis remains a challenge to clinicians with up to a 20% missed diagnosis rate. Recently, studies have suggested that the introduction of focused appendiceal CT would reduce the cost of caring for patients with suspected appendicitis and aid in diagnosis. Prompt diagnosis of appendicitis can avert the development of complications such as perforation and subsequent septicemia. Physicians should rely on both their clinical judgement and the aid of radiological evaluation in the diagnosis of acute appendicitis. year-old male presented in clinic requesting genetic testing for Huntington's Disease (HD). Several of his family members had developed late-onset choreiform disorders. His nearest affected relative was an uncle who developed symptoms after age 60, progressively worsening until his death 5 years later. The patient's parents died of other causes and his siblings were well. The patient's physical exam was unremarkable. He demonstrated no involuntary movements; no abnormalities in sensation, motor strength, coordination, or gait; and no deficits in concentration, memory, or intellect. The patient was counseled as to the risks and benefits of testing. In particular, he was informed that no treatment exists to cure or forestall the course of the disease nor could the advent of symptoms be predicted. The patient continued to express a desire for testing, primarily to alleviate his anxiety with possibly harboring a genetic predisposition to HD. Laboratory testing revealed alleles with approximately 28 and 40 CAG repeats, consistent with high genetic burden for HD. 28 CAG repeats indicated a normal mutable HD allele which may expand upon transmission to offspring (27±35) while 40 CAG repeats was consistent with individuals who typically show features of HD ( > 39). The patient was informed of the results and their interpretation and referred to a neurologist for further discussion and counseling. DISCUSSION: Huntington's disease is a degenerative brain disorder with autosomal dominant inheritance manifested by chorea and dementia. Initial involuntary movements may not be noticed by the patient, but symptoms progress to irregular, sudden movements of the limbs and trunk, which can be disabling. Deficits in attention, memory, and judgment can be present as well as depression, social withdrawal, and disinhibition. Genetic testing for HD carries with it several important clinical and ethical implications. A positive test result implies an inherited mutation that will lead to the disaese at some unknown future date, with no available cure or even effective treatment. Protocols for pre-and post-test counseling have been proposed to address the potential psychological consequences, including anxiety, depression, family and marital stress, survivor's guilt, and even suicide. Anonymous testing has also been suggested to alleviate fears of discrimination in employment and insurance. year-old female presented with several days of increasing cough, wheezing, and dyspnea. She was in good health until 18 months prior when she first developed a non-productive cough. Six months later, she noted intermittent wheezing that increased in frequency and intensity. Recently she developed dyspnea at night when lying down. Inhaled steroids and bronchodilators gave little improvement. She denied fevers, chills, abdominal pain, and symptoms of acid reflux. Her past medical history was significant only for hypothyroidism treated with thyroxine. She had never smoked and had no pets. On examination, the patient was afebrile and not tachypneic. She was able to speak in complete sentences and coughed frequently. A loud, monophasic, inspiratory wheeze was audible without a stethoscope. Auscultation of the lungs revealed stridor. Prior pulmonary functions tests were reported as normal. Inspiratory and expiratory PA and lateral radiographs of the chest revealed a 2 cm diameter soft tissue mass in the tracheal air column. This mass moved from the carina at inspiration to the level of the clavicular heads at expiration. A subsequent CT scan of the thorax confirmed a distal tracheal mass. The patient was admitted for bronchoscopy to remove the tumor. Pathological examination found that the tumor did not fit with any known entity and was conjectured to be a hamartoma-like lesion of the seromucinous glandular tissue normally present in the distal trachea. DISCUSSION: Primary tracheal neoplasms are extremely rare, accounting for fewer than 0.1% of cancer deaths. They occur with equal frequency in men and women, most often between the ages of 30±50. The most common presenting symptoms relate to airway obstruction, notably cough, wheezing, dyspnea, and recurrent pneumonia. A \"tracheal syndrome\" of dyspnea, wheezing, stridor, hemoptysis, and voice change is present in up to 85 percent of patients with primary tracheal neoplasms. As this case illustrates, the diagnosis of primary tracheal neoplasms is typically marked by significant delay between the onset of symptoms and proper diagnosis. One study reported an average delay of eight months for malignant lesions and 25 months for benign tumors. Most patients are unsuccessfully treated for presumptive diagnoses of asthma or chronic bronchitis, with little improvement, before a diagnosis of a tracheal neoplasm is made. Other diagnoses, including tracheal neoplasms, should be considered when the usual treatment for asthma does not improve the symptoms.\nDIFFICULT DYSURIA. S.B. Fazio 1 ; 1 Harvard University, Brookline, MA LEARNING OBJECTIVES: 1. Characterize the differential diagnosis of dysuria 2. Distinguish prostatitis from other causes of rectal pain 3. Recognize atypical presentations of appendicitis CASE INFORMATION: A 21 year old male presented to urgent care complaining of three weeks of dysuria and rectal pain. The pain was sharp and intermittent, brought on by urination or defecation, and radiated into the right inguinal area. He denied fever, nausea, vomiting, abdominal pain or tenesmus. Past medical history was unremarkable; he was on no LEARNING OBJECTIVES: (1) Recognize thyroid disease as an extrahepatic manifestation of chronic hepatitis C. (2) Manage thyroiditis associated with hepatitis C when the disease is either subclinical or mild. (3) Realize that thyroiditis can occur in patients with hepatitis C with or without the use of interferon therapy. CASE INFORMATION: A 29 year old woman with a 15 year history of hepatitis C (HCV) presented to general internal medicine clinic to establish primary care. She had undergone initial evaluation of her HCV disease 3 years earlier but had declined liver biopsy or interferon therapy at that time and discontinued followup. She had recently returned to hepatology and had proceeded with liver biopsy. On questioning and complete review of systems, the patient had no complaints. Notable findings on exam included a symmetric, smooth, nontender goiter, 6 cm with no palpable nodules. Exam of the liver was normal. Studies from 3 years and 6 months earlier were reviewed. At both time points, TSH was below normal, ALT was moderately elevated and HCV viral quantitative measurements were low. Genotype was 1A. Liver biopsy revealed minimal disease with Grade I lobular inflammation and Stage 0 fibrosis. Updated studies were ordered and revealed a normal TSH, mildly elevated total T3, normal free T4 and elevated anti-thyroid antibodies. No treatment was prescribed. At followup 1 month later, she remained asymptomatic and her goiter had markedly resolved to a minimally palpable and visible gland. Repeat laboratories revealed an isolated mildly elevated total T3. DISCUSSION: Chronic HCV infection has been associated with several extrahepatic conditions including thyroiditis. The mechanism may be related to HCV infection of mononuclear cells but the exact mechanism is still largely unknown. Recent case-control studies have found the prevalence of thyroid abnormalities and anti-thyroid antibodies significantly higher in patients than controls. Most commonly, there is no associated thyroid enlargement. Approximately 50% of those with anti-thyroid antibodies have subclinical thyroid disease and 50% have overt symptoms. Thyroid dysfunction has also been noted during interferon-alpha therapy in up to 12% of HCV patients but studies have found disease in both interferon-treated and untreated patients. Prospective studies show thyroid dysfunction may develop in patients on interferon therapy with or without antibodies. Those with antibodies do not show a significantly higher propensity to develop thyroid dysfunction during interferon therapy. In summary, as in the general population, management includes timely recognition of signs and symptoms of thyroid disease. In the HCV population, these symptoms can often be subtle or may be attributed to HCV itself. HCV patients with thyroiditis should be followed closely; however, they may not necessarily require intervention if thyroid disease is subclinical and the thyroiditis or goiter may remit spontaneously. Finally, pre-existing thyroiditis is not a contraindication to interferon therapy.\nSPACE OCCUPYING LIVER LESIONS WITH FAMILIAR OCCUPANTS. F. Francois 1 , L. Yatskar 2 ; 1 New York University, Brooklyn, NY; 2 New York University, New York, NY LEARNING OBJECTIVES: 1. Develop a high index of suspicion for tuberculosis in refugees and immigrants 2. Recognize immunocompromised hosts as being at risk for extrapulmonary tuberculosis 3. Assess space occupying hepatic lesions in the setting of possible tuberculosis CASE INFORMATION: A 35 year old Tibetan male with the Acquired Immune Deficiency Syndrome (AIDS), CD4 = 32/mm 3 and viral load = 102,000 copies/ml, presented complaining of fever, chills, abdominal pain, diarrhea, and malaise of increasing severity over five days. Six years prior to this clinic visit the patient was treated with Isoniazid for 4 months after a positive purified protein derivative skin test. Two years prior to the visit he was diagnosed with AIDS and completed treatment for multiple episodes of infectious diarrhea. Two weeks prior to this current evaluation he began to experience abdominal discomfort associated with loose non bloody stools. A course of Metronidazole did not improve his symptoms. He had never been on antiretroviral therapy, and on examination was noted to be a thin febrile male in no acute distress with bitemporal wasting, oral thrush, nonicteric sclerae, moderate right upper quadrant tenderness to deep palpation, and hyperactive bowel sounds. The patient was admitted to the hospital for evaluation, at which time laboratory analysis was notable for a white blood count of 5.8, an hematocrit of 26.6, and an INR of 1.59. An abdominal CT revealed low attenuated lesions in the liver, the largest measuring 6cm in diameter, with a smaller lesion in the porta hepatis extending into the head of the pancreas. Percutaneous drainage of one of the lesions revealed acid fast organisms subsequently identified as Mycobacterium Tuberculosis (MTB) which was pan sensitive. A bronchoscopy did confirm concurrent pulmonary TB. There was radiographic evidence of resolution of the hepatic lesions over several months after initiating treatment with Isoniazid, Rifampin, Pyrazinamide, and Ethambutol. DISCUSSION: One in three individuals worldwide is infected with MTB. In the U.S., 40% of cases occur among foreign born persons. Tuberculosis most commonly presents as pulmonary or lymph node disease, but can also affect other organs. Immunosuppressive therapy, malignancy, and AIDS are known risk factors for disseminated complications with the organism. Extrapulmonary disease is seen in 60% of AIDS patients with lung disease, and of those patients, 7.5% have tuberculosis of the liver. Hepatic tubercular involvement is most commonly described as granulomatous, but can also be seen as discrete space occupying lesions such as tuberculomas, and abscesses. Patients typically present with fever, night sweats, abdominal discomfort, weight loss, and hepatomegaly. Diagnosis is made by aspirating a lesion, and treatment is based on drug sensitivities. her abdominal wall, breasts, thighs, and calves. The lesions were associated with central necrosis and peripheral mottling. She was alert, oriented, afebrile, and without leukocytosis. She had a calcium-phosphorus product of 75 and a parathyroid hormone level of 438. Despite aggressive wound care and antibiotics, her condition worsened precipitously over 72 hours. Her skin lesions became necrotic, ulcerative, and suppurative. She became febrile, hypotensive, and stuporous. Despite broadened antibiotics and aggressive fluid resuscitation, she became unresponsive, pulseless, apneic, and died. DISCUSSION: Calcific Uremic Arteriolopathy (CUA) is characterized by calcification of subcutaneous arterioles and infarction of skin and subcutaneous adipose tissue. It occurs in a minority of chronic renal failure patients. In CUA, calcification of the media of arteriolar vessels leads to regional ischemia and then infarction of skin and subcutaneous tissue, especially in areas of high adipose content. Skin necrosis and ulceration develop and patients usually die as a result of ischemic or infectious complications. While the true pathogenesis of CUA is unknown, several factors have been associated with a higher incidence. A high calcium-phosphorus product and secondary hyperparathyroidism are often associated with a increased incidence of CUA. Other features such as female gender, white race, obesity, recent significant weight loss, insulinrequiring diabetes mellitus, and use of calcium carbonate as a phosphorus binder have been reported as predisposing factors. It is likely that modification of these factors in our patient, such as management of high calcium-phosphorus product with non-calcium containing phosphorus binders, reduction of calcium content in dialysate fluid, and gradual weight loss may have prevented this deadly outcome.\nDISCUSSION: HMG-CoA reductase inhibitors are popular lipid lowering agents. The most common adverse drug reaction is a transient chemical hepatitis of little clinical significance. Clinically significant rhabdomyolysis is extremely rare with an incidence of < 0.5%. HMG-CoA reductase inhibitors require metabolism by P-450 enzymes before they become active. Inhibition of the P-450 system leads to decreased metabolism of the statin drugs and higher plasma levels. The resultant increased delivery of the HMG-CoA reductase inhibitors to muscle cells decreases the production of cholesterol and cytochrome Q10 and makes muscles susceptible to oxidative damage and rhabdomyolysis. Cerivastatin is a third-generation HMG-CoA reductase inhibitor that is marketed as a``safer'' drug because it is metabolized by two different P-450 isoenzymes, CYP3A4 and 2C8. Cases of rhabdomyolysis in patients treated with cerivastatin and gemfibrozil have been reported; however, there have been no published reports of cerivastatin alone causing rhabdomyolysis. The etiology of rhabdomyolysis in this patient is somewhat puzzling. He was on no potent inhibitors of CYP3A4 nor did he have pre-existing hepatic or renal dysfunction. It may be that he had an intrinsic deficiency of CYP2C8. The treatment of HMG-CoA reductase inhibitor induced rhabdomyolysis involves withdrawal of the drug and supportive care. The myositis usually resolves completely although renal failure has been reported.\nAN UNUSUAL CASE OF FLANK PAIN. F. Green 1 , R. Granieri 1 ; 1 University of Pittsburgh School of Medicine, Pittsburgh, PA LEARNING OBJECTIVE: Identify and treat acute renal infarction. CASE INFORMATION: J.C. is a 63 year old female with ischemic cardiomyopathy, mitral regurgitation, hypertension, and rheumatoid arthritis who developed spontaneous right flank pain that was constant, 8/10, sharp, and sufficient to wake her from sleep. The pain was associated with nausea, vomiting,and intermittent diaphoresis. There was no radiation to the groin, abdomen,or chest. There was no history of chest pain, palpitations, dysuria, abdominal trauma, vaginal discharge, or invasive vascular procedures. She has never had thromboembolic disease or atrial fibrillation. Medications included digoxin, losartan, prednisone, furosemide, hydroxychloroquine, aspirin, and methotrexate. Physical examination was notable only for compensated biventricular heart failure, mitral regurgitation, and right flank tenderness. Electrocardiogram revealed sinus tachycardia. Laboratory data was notable for BUN 35, Cr 1.2 WBC 14.1 (94% PMN), ALT 28, AST 37, GGT 180, Alk Phos 192, and LDH 386 . Urinalysis revealed 4 RBC, 0 WBC, and trace protein. CT scan of the abdomen and pelvis with contrast showed an acute infarct of the inferior pole of the right kidney. The patient was admitted and anticoagulated. Echocardiogram demonstrated an ejection fraction of 25% and severe mitral regurgitation with no evidence of mural thrombus. DISCUSSION: Renal infarction is an often overlooked clinical syndrome than can be mistaken for more common causes of flank pain. The etiology of most cases is embolic disease from underlying atrial fibrillation, valvular disease, or cardiomyopathy. No historical variable aside from risk factors for thromboembolic disease has been shown to be associated with renal infarction. Physical examination is non-specific although an incomplete infarction may cause a hyper-renin state with secondary hypertension. LDH is the best marker for renal infarction although initial measurements may be normal. Other lab abnormalities are inconsistently present. CT scans have replaced IVP and angiogram in making the diagnosis of renal infarct. While areas of infarction can be seen on non-contrasted CTs, contrast is necessary to increase sensitivity, better define the infarct, and distinguish acute from completed infarcts. Surgery, angioplasty, fibrinolysis, and conservative medical management are therapeutic options. Revascularization appears to be most successful in early infarction (first 3 hours). Conservative therapy with anticoagulation (and additional antihypertensive therapy if needed) is thought to have lower morbidity than invasive therapy in most cases. Although the mortality from the renal infarct itself is low, these patients have a dismal one year prognosis because of further embolic disease and underlying cardiac disease. year-old male roof repairman presented with shortness of breath for the past two weeks. He had no history of alcoholism, smoking or previous lung disease. He had been taking Azithromycin during the week prior to admission but had become worse. He had right-sided, pleuritic chest pain, fever, chills, yellow-greenish sputum, night sweats, and mild weight loss. Examination revealed moderate respiratory distress, a temperature of 101.1 8F and a pulse of 115. The mouth, teeth and oral pharynx were normal. The cardiac exam was normal except for tachycardia. Diminished breath sounds were noted on the right side of the chest. His WBC was 19.2 with 89% PMN's. A CXR and subsequent CT revealed a large loculated pleural effusion with right upper and middle lobe atelectasis. A sputum gram stain showed gram positive cocci in chains. A 2D echo showed a large peri-cardial effusion without evidence of tamponade. 1,200 cc of yellowish-greenish pus was drained following thoracentesis and placement of a chest tube. Cultures grew only Streptococcus constellatus which was sensitive to Clindamycin and Ampicillin. He recovered following these antibiotics with chest tube drainage. DISCUSSION: Streptococcus constellatus is a gram positive microaerophillic coccus that grows in chains or pairs and is a member of the Streptococcus intermedius group. It is usually present as a harmless commensal organism in the mouth and GI tract but like other members of the S. intermedius group has the propensity to form abscesses. Intrinsic virulence factors (hydrolytic enzymes, different adhesins on cell surfaces and a polysaccharide capsule) facilitate adherence, decrease phagocy-tosis and increase pathogenicity. S. constellatus is frequently associated with tho-racic infections and more prone to occur in males, alcoholics, people with previous pneumonia and in those at risk for aspiration. Mortality ranges between 15±30%. Although this patient had few risk factors for the development of this infection, the presence of a 3D Surface Shaded display showing high-grade stenosis of the celiac axis and superior dense empyema and pericardial effusion suggested S. costellatus or other organisms in the S. intermedius group as a possible cause.\nA. Hamad 1 , R. Clark 1 , J. Singh 1 ; 1 Nassau University Medical Center, Eastmeadow, New York LEARNING OBJECTIVES: 1-deep venous thrombosis (DVT) can cause of fever of unknown origin (FUO). 2-the importance of computed tomography of the abdomen during work up for FUO.3-AIDS is associated with high incidence of thrombosis. CASE INFORMATION: A 45 years old Haitian female presented with generalized weakness, weight loss, and low-grade fever. She had a history of AIDS with wasting syndrome and cerebral toxoplasmosis, and recurrent deep venous thrombosis in the lower extremities. She had a temperature of 100 0F, with no swelling in the lower extremities. Plain radiograph of the chest and Urinalysis were normal. Blood, stool, and urine cultures were negative. Stool for Clostridium difficile, ova and parasite, Cryptosporidium, acid-fast bacilli and Isospora belli was negative. Sputum for acid-fast bacilli smear and culture was negative. Hepatitis profile was negative. Sputum for acid-fast bacilli and Pneumocystis carinii were negative. Few days later, she complained of severe perineal pain, rectal exam was very painful and frank blood was found on a gloved finger. Sigmoidoscopy was not done because of the pain. She was treated for an anorectal fissure and the pain improved after few days but she continued to have fever. Computed tomographic scan of the head with contrast and cerebrospinal fluid analysis were unremarkable. Computed tomography of the abdomen was done as work up for FUO. It showed massive thrombosis extending from the right renal vein to the inferior vena cava up to the liver. Hematologic work up was significant for decreased total protein S (28%) with normal protein C (64%) and antithrombin III (102%). Anticoagulation therapy was started and fever improved after more than a week. DISCUSSION: Our patient has massive thrombosis, which we think caused her prolonged fever, as an extensive work up ruled out other causes of fever. Total protein S is normal in AIDS patients with decrease in free protein S level. We believe that our patient has two separate disorders predisposing her to thrombosis (HIV infection and primary protein S deficiency). This belief depends on the fact that her total protein S was < 50% of the normal, consistent with the classic type of the hereditary protein S deficiency. Although acute thrombosis can decrease antithrombin III, it is less likely to cause a decrease in protein S or protein C. The decrease of protein S in our patient is not secondary to the thrombosis, as her antithrombin III and protein C levels were normal. We found only few reports of patients with HIV infection with protein S deficiency who presented with significant DVT. Weakness started the night before presentation and deteriorated rapidly in the next day. He reported having polydipsia and polyuria in the last few days. The patient was afebrile with blood pressure of 110/70 and heart rate of 90 bpm. Physical exam was significant for short stature (155cm) and moderate weakness of all extremities. Laboratory tests showed severe hypokalemia (1.3 meq/dl) and hypophosphatemia (1meq/dl), low bicarbonate (9 meq/dl) and normal anion gap. Arterial blood gas showed acidosis (pH = 7.12) and pCO 2 of 28 mmHg. After receiving 280 meq of potassium supplements over 16 hours his potassium level improved to 2.7 meq/dl and he regained his power completely. The patient received replacement of potassium, phosphorus, and bicarbonate for 4 days period till his numbers became normal and was discharged home on potassium citrate supplements only. Work up during hospitalization revealed urine pH repeatedly > 6.5, urine specific gravity of 1.010, urine anion gap of +40, urine pCO 2 of 50 mmHg after correction of the acidosis, daily requirement of 20 to 40 meq of bicarbonate only to maintain normal serum level, and fractional excretion of bicarbonate of 2.5% and phosphorus of 22%. Renal ultrasound revealed echogenic shadows distinct between the medulla and the cortex, consistent with calcification. DISCUSSION: Distal and proximal renal tubular acidosis are uncommon diseases. The proximal type characterized by reduction in reabsorpsion of bicarbonate while the distal type results from inability to secrete hydrogen ion. This patient represents a typical case of type I RTA (distal) with all the manifestations and complications. Short stature, severe hypokalemia with secondary paralysis and polyuria (loss of urine concentrating ability), renal calcifications, positive urine anion gap, inability to acidify the urine despite severe acidosis (oppose to proximal type), decreased urine pCO 2 (which is usually higher than the plasma because of secretion of H+ in the distal tubule that binds the bicarbonate and become CO 2 ), fractional excretion of bicarbonate of 2.5% (in proximal RTA this number is > 15%) and minimal amount of bicarbonate replacement was enough to keep normal blood level. We want physicians to be aware of this rare disease that is very easy to treat but if left untreated can have detrimental consequences.\nQT PROLONGATION SECONDERY TO HYPOCALCEMIA AFTER CORRECTION FOR ELEVATED SERUM ALBUMIN. A. Hamad 1 , M. Zihlif 1 , M. Salameh 1 , A. Alghadban 2 , D. Feinfeld 1 ; 1 Nassau University Medical Center, Eastmeadow, New York; 2 SUNY at Stony Brook, Port Jefferson NY LEARNING OBJECTIVE: 1-recognize that correction of calcium level for albumin should be done also if albumin is high. CASE INFORMATION: 42 years old white male with no significant past medical history except alcoholism came to the emergency room with vomiting of small amount of blood associated with mild epigastric pain. He was not taking any medications at home. He was afebrile, blood pressure was 123/83, heart rate was 126 bpm. Physical exam was unremarkable except for mild dehydration and tachycardia with no added sounds or murmur. Laboratory tests were significant for normal calcium level (9 mg/dl), magnesium of 2 meq/dl, serum albumin of 5.3 mg/dl, potassium of 3.3 meq/dl. Arterial blood gas showed normal pH of 7.44. Electrocardiogram showed regular sinus rhythm with prolonged QT interval (QTc = 44 msec). Because of this prolongation, the patient was given calcium gluconate intravenously as corrected calcium with elevated albumin was low (7.9 mg/dl). Ionized calcium was low (1.04 meq/dl. Repeat EKG showed return of QT interval to normal limit. The patient was treated with vitamins and anxiolytics for his mild alcohol withdrawal and was discharged home after 2 days. DISCUSSION: QT prolongation can occur as a congenital or an acquired disorders. The acquired form is secondary to pharmacologic agents (mainly antiarrhythmatics, tricyclic antidepressants, antihistamines and erythromycine), and electrolytes disturbances (hypokalemia, hypomagnesemia and hypocalcemia). We are presenting this case to stress on fact of correcting calcium level to the serum albumin, this correction can be by adding 0.8 mg to each 1 mg of albumin below the normal level (4mg/dl) or by subtracting 0.8 mg to each 1 mg of albumin above the normal limit.\nNEUROMYELITIS OPTICA. I.P. Haque 1 ; 1 University of Virginia at Roanoke-Salem, Roanoke, VA LEARNING OBJECTIVE: Diagnose the Different Variants of Multiple Sclerosis including Neuromylelitis OpticaTreatment of Neuromyelitis Optica CASE INFORMATION: Neuromyelitis optica or Devic's syndrome is an uncommon neurological illness characterized by optic neuropathy and myelopathy. This case presents a 39 year old African-American female with new onset of visual changes, periocular pain, headache, malaise, lower extremity weakness, and back pain. The patient's symptoms began one week prior to admission. She previously presented with lower extremity pain and weakness six months prior and was suspected clinically to have multiple sclerosis and had resolution of her symptoms with corticosteroids. She had no changes on magnetic resonance imaging of brain and cerebral spinal fluid findings were nonspecific at the time. Her symptoms of visual changes and loss with periocular pain were not present upon this prior admission. On physical exam, she had papillitis, severe visual field defect, profoundly decreased lower extremity weakness and sensation, and Lhermitte's sign. On laboratory exam, cerebral spinal fluid showed increased protein and she had an elevated ESR. Magnetic resonance imaging of spine showed diffuse cervical cord swelling and cervical signal changes. This patient presented with bilateral optic neuritis and myelitis, known as Devic's syndrome or Neuromyelitis optica. She was treated with intravenous methylprednisolone and had gradual improvement of her symptoms. DISCUSSION: Devic's syndrome generally has a rapid onset of optic neuritis and myelitis. Patients with acute or subacute Devic's syndrome often respond to corticosteroids as this patient did. In Devic's syndrome, acute spinal cord lesions can demonstrate diffuse swelling extending over several levels or the entire cord in either a continuous or patchy distribution. The optic nerve and chiasm can show either demyelinating lesions and/or necrotizing lesions. Approximately 35 percent of patients have a monophasic illness and 55 percent develop relapses, and rarely patients have a fulminantly progressive course without relapses or a course typical of multiple sclerosis. (2) Identify the indications for valve replacement in IE. CASE INFORMATION: A 41-year-old white male with past medical history of migraine headaches presented to the emergency department for a seizure occurring in a restaurant. While sitting, the patient suddenly developed stiffness and ridgidity in the right arm then the left arm. He then collapsed and had a generalized seizure. According to an eyewitness the patient lost consciousness for 3±4 minutes, after which he was confused but without incontinence or focal weakness. Prior to this event the patient experienced low-grade fever as high as 1018F and myalgias for one week but no other symptoms. Physical examination revealed a young ill looking male; temperature was 378C. He was tachycardic and the blood preasure was 164/78 mmHg. Significant findings included abrasions over the left temple, evidence of an old infarct in the left parietal region thought to be embolic. Echocardiogram showed a dilated left ventricle with borderline left ventricular hypertrophy and good systolic function, severe aortic regurgitation and suspicion of an aortic valve vegetation. A transesophageal echocardiogram showed a bicuspid aortic valve with prolapse of one of the leaflets, 3+ aoritic incompetence and evidence of a vegetation 4±5 mm in size on one of the leaflets of the aortic valve. Blood cultures initially reveled Gram + Cocci in clusters later identified as coagulase negative Staphylococcus. All the cultures drawn later were negative for any growth. Because of evidence of embolic disease on the CT scan of the brain and the recent generalized seizure (representing multiple embolic events), aortic valve replacement was indicated. The patient had a St. Jude HB mechanical valve replacement. He had an uneventful post-operative course. He was started post-operatively on heparin and then later on Coumadin. Blood cultures and cultures from the aortic and mitral valves were negative for any growth. The culture from the aortic vegetation was also negative. The patient was continued on cefrtiaxone for 4 weeks to treat likely atypical organisms. DISCUSSION: When considering the differential diagnosis of a new onset seizure, IE is a relative uncommon cause, but should be considered when fever and/or a heart murmur is present. When IE is associated with seizures, the seizures usually occur later in the course of the disease rather than as part of the initial presentation as in this case. The bicuspid aortic valve in our patient was a risk factor of IE but it is important to recognize that one third of patients with IE have no identifiable predisposing cardiac lesions. Negative cultures occur in 2±5% patients with IE. The indication for valve replacement in this case was evidence of recurrent embolic events. Mechanical valve was chosen because of the patient's young age.\nDOCTOR, CAN YOU HELP ME DIE? J.M. Hauser 1 ; 1 University of Chicago, Chicago, IL LEARNING OBJECTIVES: 1. Explore reasons for a request for physician-assisted-suicide 2. Learn how to respond to a requests for physician-assisted-suicide CASE INFORMATION: Mr. Hayes [name changed] was a 57 yo man with advanced esophageal cancer who requested assistance in dying. He initially presented with dysphagia and underwent endoscopy which revealed esophageal cancer. After an abdominal CT scan showed multiple liver masses consistent with metastases, he was referred to hospice. His predominant symptoms were abdominal pain treated with morphine, anxiety treated with lorazepam and dysphagia which required he stop eating solid foods. Mr. Hayes initially resisted medications because of possible drowsiness. It soon became clear, however, that his refusal was because medication could not address his most significant suffering: as a relogious person, he told me, he was angry at God for not curing his illness and for letting him die without a wife. He did not know how to cope with this anger and did not think that either hospice or his own priest could help address it. At the end of our second visit, after discussing his symptoms and his continuing spiritual distress, I asked Mr. Hayes``Is there any other way I can help you?'' He answered:``You can get put a needle in my vein and help me end it.'' I asked what he meant. \"You can help me die. It's in your oath to stop suffering.\" I explored with him how he was suffering, whether there were things he looked forward to in his future and whether there were ways we could help him short of ending his life. After discussions with many members of the hospice team, aggressive treatment of his symptoms, and help from two of his brothers helping care for him, his desire that we help end his life waned but never completely disappeared. DISCUSSION: Requests for physician assisted suicide (PAS) are troubling, partly because they highlight intense and unrelieved suffering. This case illustrates how such a request can be a window into inadequately addressed suffering, can reveal misunderstandings about what physicians are legally permitted to do; and how palliation of many symptoms may not be adequate for some patients' suffering. Rather than a debate about the ethics of PAS, this case discussion will consider the reasons behind such requests and our possible responses to them.`I CEBERG LETTUCE AND CARROTS'': A MEAL IN SEARCH OF A DIAGNOSIS. R.D. Hobbs 1 ; 1 Oakwood Healthcare System, Dearborn, MI LEARNING OBJECTIVE: To recognize the varied presentations of Pica as well as its clinical significance. CASE INFORMATION: A 57 year-old woman presented with memory loss, a fear of becoming obese and a recent four-month history of cravings for``iceberg lettuce and carrots.'' She was eating two heads of lettuce and a pound of carrots daily. Her past history included hypothyroidism (1970) , treated with oral replacement; a hysterectomy (1970) ; severe osteoarthritis with chronic left hip pain; fibromyalgia treated with amitriptyline; a gastric exclusion procedure for weight loss (1994) ; and severe personal stress due to the recent suicide of her 27 year old son six months prior. Her weight loss procedure was a partial gastrectomy with creation on a six-ounce pouch and anastomosis of the distal duodenum to the terminal ileum. This resulted in a``four foot common channel.'' She lost 117 pounds without incident. Physical exam was unremarkable except for mild memory loss, obvious osteoarthritis with a left sided limp and well healed surgical scars. She weighed 170 pounds. DISCUSSION: Although there was obvious depression and a profound fear of weight gain, the cravings for``iceberg lettuce and carrots'' were of recent onset. Because of the history of the gastric bypass procedure and its expected result of intestinal mal-absorption, a Pica variant was suspected. Although Pica has been associated with such bizarre food cravings as laundry starch, clay, chalk, ice, and paint chips, it has been associated with normal foodstuffs such as apricots, almonds, chocolate, carrots, etc. Because of the association between Pica and iron deficiency, laboratory tests for anemia were ordered. The results were compatible with iron deficiency anemia. Additional studies revealed zinc deficiency, early vitamin B12 deficiency, and vitamin D deficiency with hypocalcemia and secondary hyperparathyroidsim. With these findings, the patient revealed that although she had taken nutritional supplements following her operation, she stopped them following the death of her son. With reinstitution of nutritional supplementation her behavior and laboratory studies have been improving. 1. Recognize (DI) diabetes insipidus clinically. 2. Confirm DI using standard diagnostic testing. 3 . Through thorough review of its possible causes, establish the etiologic lesion for DI in a patient. CASE INFORMATION: A 23 year old male came to the emergency room with a chief complaint of a nine day history of blood in his stool, lightheadedness, nausea, diarrhea, and profound weakness. He was admitted for dehydration, anemia, and bright red blood per rectum. On review of systems, it was noted that he had been drinking several gallons of water a day since 5 months prior to admission and had been urinating hourly. Diabetes insipidus (DI) was suspected when it was observed that he had a clear dilute urine despite his tachycardia and orthostatic hypotension. His urinalysis revealed a low specific gravity of 1.002 and no glucose; serum chemistries were normal. A water deprivation test was consistent with diabetes insipidus. Subsequent workup revealed panhypopituitarism and ultimately the causative lesion, a midline germinoma. The rectal bleeding, which might have lead us toward a different evaluation, was hemorrhoidal. The diarrhea was most likely secondary to his hypoadrenal state. DISCUSSION:\nCERVICAL SPINE OSTEOMYELITIS CAUSED BY STREPTOCOCCUS MILLERI. M.R. Huber 1 , P.S. Mueller 1 ; 1 Mayo Clinic, Rochester, Minnesota LEARNING OBJECTIVES: #1 Recognize that poor dental hygiene can lead to serious complications, such as cervical spine osteomyelitis.\n#2 Distinguish Streptococcus milleri (S. milleri) from other viridans streptococci by its tendency for suppurative infections. #3 Recognize S. milleri as a rare but known cause of vertebral osteomyelitis. CASE INFORMATION: A 61 year old male was found to have fractures of C5 and C6 with associated left sided radiculopathy after falling down stairs. The patient had no chronic medical conditions except for recurrent tonsillitis and tonsilloliths. The patient was admitted to an outside hospital and found to have a WBC of 13,300  10 9 /L and a WESR of 67 mm/hr. Surgery was performed to stabilize the fracture and revealed cervical osteomyelitis with associated phlegmon and epidural abscess. The patient underwent debridement and internal fixation. Five of six cultures grew only pan-sensitive S. milleri. With ampicillin, the patient's WBC had normalized and WESR had improved. However, he experienced two internal fixation rod failures and developed a mediastinal abscess with associated cutaneous fistula. He was then transferred to our hospital. Tissue cultures from the neck and mediastinal abscess grew several organisms, and as such, antibiotic coverage had to be broadened. The patient required three additional spine stabilizing operations before a satisfactory result was achieved. He had a number of complications including drug induced neutropenia, thrombocytopenia, rash, and atrial fibrillation. At the time of this writing, the patient has made a nearly full recovery. He has a WBC of 4,300  10 9 /L and WESR of 0 mm/hr. He has been maintained on gatifloxacin and minocycline while he has metal hardware in the previously infected region. DISCUSSION: Streptococcus milleri represents several biologically diverse, yet clinically homogenous, types of viridans streptococci. They are characterized by their tendency for suppurative infections. They can be found in abscesses of the mouth, brain, and liver as well as empyemas. Several cases of vertebral osteomyelitis have been previously reported. Of the two cases of cervical osteomyelitis, one involved an intradiscal steroid injection and the other had known severe periodontal disease. The patient in this case had known palatine tonsillar disease which may very well have led to his cervical spine osteomyelitis.\nM.T. Hughes 1 ; 1 Johns Hopkins University, Baltimore, Maryland LEARNING OBJECTIVES: 1. Assess the role of weak paternalism for patients lacking decision-making capacity, when no readily identifiable surrogate decision-maker is available. 2. Weigh the obligations of beneficence, respect for autonomy, and an ethic of care. 3. Determine the goals of care for cognitively impaired patients with a life-limiting illness. CASE INFORMATION: 82 year old male with multiple medical problems including coronary artery disease, severe chronic obstructive pulmonary disease, and moderate, multi-factorial dementia presented with poor appetite after being lost to medical follow up for six months. Previous physicians had prescribed megestrol for anorexia. Left renal mass had been detected five years earlier and``watchful waiting'' approach had been arranged with urologist two years prior to presentation. Six months prior to presentation, CT scan demonstrated enlargement of mass and findings consistent with renal cell carcinoma. Patient had been referred back to urology but appointment did not occur. Over course of current evaluation, when asked whether to pursue repeat CT scan, patient stated,``It don't bother me, so don't bother with it.'' Patient lived with his 84 year old wife, who likewise had moderate dementia and had recently undergone surgical resection of Stage III gastric carcinoma. Patient had lost contact with distant cousins in another state and denied any other family members who could help with decision-making. His day consisted of \"chewing the fat\" with friends at the local market. He enjoyed his life and wanted to stay at home with his wife as long as he could. CT scan and urology consultation were not pursued. One year later, the patient suffered a major stroke. Home hospice was arranged, but his wife was unable to care for him at home. On presentation to the emergency room, she had no recollection of his stroke and no recognition of his dense hemiplegia. The patient was transferred to an inpatient hospice, where he died two weks later. His wife underwent psychiatric hospitalization for her dementia and was subsequently transferred to an assisted living facility. DISCUSSION: Informed consent rests upon the principle of respect for autonomy and thus requires voluntariness and competence. This patient had impaired judgment, poor recall, and an inability to comprehend complex information. Informed consent was not possible. His wife lacked decision-making capacity. Together, both could live independently in the community, compensating for each other's deficits. Neither could weigh the benefits and burdens of a major surgery. In acting out of beneficence, the physician in this case needed to determine whether definitive treatment for the renal mass (nephrectomy with significant perioperative risk) was a benefit or a harm. Goal setting and understanding the patient as person resulted in a decision that did not strictly adhere to an autonomy model. An ethic of care, focusing on maintenance of the relationship between husband and wife, was judged to be more determinative than a principle-based ethic. This case demonstrates that in certain well-defined situations, the physician may be justified in invoking weak paternalism.`P ANCREATITIS, HEPATITIS, AND DERMATITIS'' IN A 70 YEAR OLD FEMALE. L. Humphreys 1 , E. Yee 1 , A. Gomez 1 ; 1 UCLA San Fernando Valley Program, Sepulveda, Ca LEARNING OBJECTIVES: 1) Recognize the clinical presentation of dermatomyositis, 2) Discuss the work up and treatment of dermatomyositis, 3) Discuss screening for occult malignancies associated with dermatomyositis. CASE INFORMATION: A 70 yo Latina female with a history of cholecystectomy presented with 3 days of nausea, vomiting, and epigastric pain. She denied fever, chills, diarrhea and constipation. She took no medications and denied ETOH use. She was a thin female with T 36.1 C, BP 96/52, P 68, R 18. A violaceous rash was present on her forehead, eyelids, and nose. She had epigastric tenderness with guaiac negative stool. Extremities had subcutaneous calcifications with diffuse weakness proximally. Lab work was remarkable for AST 842, ALT 467, T bili 1.3, D bili 0.3, Alk Phos 352, lipase 9,759, and serial CK's of 5640, 4811, 3835. EKG was normal and troponin negative x 2. An U/S showed no biliary dilation, gallbladder, or stones, and a poorly visualized pancreas. She was hospitalized with the presumptive diagnosis of \"pancreatitis, hepatitis, and dermatitis\". Subsequent workup found normal triglyceride and calcium levels, negative viral hepatitis serologies, and an unremarkable abdominal CT scan. An elevated aldolase of 24.9, EMG with myopathic changes, and muscle biopsy demonstrating myositis confirmed the diagnosis of dermatomyositis. She improved clinically with steroids. Work up for occult malignancy including CXR, PAP smear, mammography, CA-125 level, endoscopy, and colonoscopy were all normal. An etiology for her pancreatitis was never found. DISCUSSION: Dermatomyositis (DM) is an idiopathic inflammatory myopathy that usually presents with proximal muscle weakness. Distinctive skin manifestations include a psoriatic rash over the knuckles (Gottron's papules), periorbital discoloration (heliotrope rash), and subcutaneous calcifications. DM occurs in approximately 5/1,000,000 people. Diagnosis is confirmed by elevated muscle enzymes, myopathic EMG findings, and muscle inflammation on biopsy. Presence of anti-Mi-2 antibody is specific for DM, but is found in only 25% of cases. First line therapy is systemic corticosteroids, with methotrexate, azathioprine, or cyclophosphamide reserved for refractory cases. Malignancy occurs between 7% and 34% in DM patients, with ovarian and colon cancers found most often. Cancer associated disease is more common in older patients and is associated with a worse prognosis. There may be a delay of up to five years before any associated malignancy develops. Although this patient had an extensive workup because of her symptoms, asymptomatic patients with DM should routinely undergo age specific screening tests for occult malignancies. LEARNING OBJECTIVE: To recognize the early signs of cerebral aneurysms and make an early diagnosis CASE INFORMATION: 59-year-old male with history of well controlled hypertension and COPD presented with a severe headache 10/10 in intensity, throbbing, constant and gradual in onset which began 11 days ago in the occipital area and then extended to the right temporal/ posterior orbital area. Two days prior to admission he started having diplopia, ataxia and nausea/vomiting. The patient denied fevers, chills, photophobia, phonophobia, neck stiffness, numbness, weakness, vertigo or seeing flashing lights. Past surgical history includes splenectomy after gun shot wound in 1965. Patient also with history of polysubstance abuse, last used 2 years ago. Family history is non-contributory. On initial examination blood pressure was 167/90, temperature 36.5, heart rate 72 and respiratory rate 18. The patient was alert and oriented and in no acute distress but did report a 10/10 headache. Eye examination was significant for a right pupil which was 5 mm and sluggish to react as opposed to a left pupil which was 2 mm and reactive. There was ptosis of the right eyelid, a deficit in medial and upward gaze on the right and a deficit in accommodation on the right side. There was no papilledema and pressures were 12 bilaterally. Visual acuity was normal and equal bilaterally. The neck was supple and there were no carotid bruits. Neurologically there were no other cranial nerve deficits and no other neurological deficits. CBC and chem.-7 were normal and patient had an ESR of 10. The patient had an MRA which confirmed the diagnosis of posterior communicating artery aneurysm compressing the third cranial nerve. The patient was then transferred to neurosurgery and underwent clipping of the aneurysm. DISCUSSION: 90%of all intracranial aneurysms are congenital. Common locations include: anterior communicating artery, bifurcation of the middle cerebral artery, posterior communicating artery, internal carotid artery bifurcation and the basilar circulation. Unruptured aneurysms are usually < 6±7 mm and 20% of patients present with oculomotor nerve palsy. These patients usually have aneurysms of the internal carotid artery at the posterior communicating artery. Rupture most commonly causes subarachnoid hemorrhage. Symptoms of rupture include sudden severe headache, altered mental status, meningeal irritation, vomiting, diaphoresis, fever, focal neurological deficits, seizures and visual changes (diplopia, homonymous hemianopsia, miosis or mydriasis). The incidence of sudden death is greatest in the first week. Potential complications include rebleeding, cerebral swelling, hydrocephalus, and infarction secondary to vasospasm. The diagnosis can be confirmed with lumbar puncture where the CSF will show red blood cells, low glucose, elevated protein and a pressure usually > 150. A CT is also useful in detecting aneurysms > 5 mm, subarachnoid blood, infarction and edema. MRA is more sensitive and detects 80% or aneurysms. The treatment is usually surgical but medical therapy can be used to delay surgery and is surgery is contraindicated. Medical therapy includes lowering of blood pressure, bed rest, steroids, anticonvulsants and antifibrinolytic agents. year-old male presented with a one week history of abdominal pain, nausea, and vomiting. The night prior to presentation, he was noted to be disoriented and lethargic. Several hours later, he was febrile and diaphoretic and was taken to the hospital. On physical exam, he was alert and responsive. His BP was 70/36, heart rate 132, respiratory rate 40, and temperature 35 degrees celsius. Significant findings also included edematous sclerae with petechiae, diffuse rales, a distended abdomen, and diffuse purpura over his extremities. ABG revealed a pH of 7.08, pCO 2 of 71, pO 2 of 77, and bicarbonate of 20. His WBC was 25,600 (28% bands), platelet count 18,000, BUN 36, Cr 4.2, and INR 3.2 . Chest xray showed bilateral alveolar edema. Despite receiving several liters of saline, he continued to be hypotensive and became more hypoxemic, requiring intubation. His cardiac rhythm deteriorated to pulseless electrical activity from which he was successfully resuscitated. The diagnoses of septic shock, acute renal failure, disseminated intravascular coagulation, and adult respiratory distress syndrome (ARDS) were made. He was started on Penicillin G for suspected meningococcemia, dopamine, norepinephrine, continuous veno-venous hemodiafiltration (CVVHD), fresh frozen plasma and antithrombin III. His blood cultures grew Neisseria meningitidis. After 2 weeks, his renal function and coagulopathy resolved. His left lower extremity became ischemic and nonviable and was amputated. One week later he was discharged to a rehabilitation facility. DISCUSSION: Infections with Neisseria meningitidis range from mild upper respiratory infections to fulminant meningococcemia in 10±20% of cases. Few other diseases present as catastrophically as fulminant meningococcemia and prompt recognition and early treatment are crucial. Symptoms include sudden onset of fevers, chills, nausea, vomiting, and myalgias, progressing to shock and multisystem organ failure within hours. Purpura fulminans is present in 75% of patients, characterized as maculopapular or petechial lesions which can become hemorrhagic. In addition to antibiotics and coagulation factors, patients may also require ventilatory support. In order to remove toxic substances and cytokines, CVVHD is beneficial. The mortality rate with fulminant meningococcemia is as high as 50%, but with prompt recognition and treatment, up to 80% of patients can have favorable outcomes.\nHEPATIC ENCEPHALOPATHY MASKING A DEEPER ABNORMALITY. H. Jasti 1 , R. Granieri 1 ; 1 University of Pittsburgh, Pittsburgh, PA LEARNING OBJECTIVES: 1) Identify the clinical presentation of hemochromatosis (HC). 2) Recognize the diagnosis and management of this condition. CASE INFORMATION: A 53-year-old female with hypertension, diabetes, and cirrhosis presented with worsening lethargy and confusion. Social history was remarkable for alcohol abuse, which ended two years before presentation. On physical, she had scleral icterus, hepatosplenomegaly, generalized jaundice with palmar erythema and spider angiomatas. Neurologically, she was alert but intermittently confused. Asterixis was present. Laboratory studies were significant for elevated transaminases and ammonia levels. Subsequent lab work revealed a ferritin of 2000, Fe of 250, and transferrin saturation of 89%. A liver biopsy demonstrated increased iron deposition in the hepatocytes, confirming the diagnosis of HC. A consequent family discussion revealed that her younger brother (age 41) and nephew (age 36) both complained of arthralgias. Genetic testing confirmed HC in both of them. DISCUSSION: Hemochromatosis is the most common inherited disorder in the U.S. It is an autosomal recessive condition in which there is increased absorption of iron that causes damage to the liver, heart, pancreas, joints, and other organs. The mean age of presentation is in the fifth decade. The classic triad of cirrhosis, diabetes, and skin hyperpigmentation is not usually the presenting sign; rather it represents late-stage disease. Arthropathy, especially in non-weight bearing joints, can be a common presenting complaint, as it is prevalent in 60% of symptomatic patients. Other early signs and symptoms include erectile dysfunction, amenorrhea, depression, and fatigue. Diabetes and cardiomyopathy may also be presenting signs in younger individuals. The liver is the most affected organ with iron deposition leading to functional abnormalities, fibrosis, and eventually cirrhosis. Liver failure is the most common cause of death. A combination of increased transferrin saturation ( > 50%) and an elevated ferritin is about 95% accurate for the diagnosis. Confirmation is made with a liver biopsy. Genetic testing has also shown promise, especially to test the first-degree relatives of patients with HC. Treatment involves phlebotomy, which depletes iron stores. Given the high prevalence of the condition and effectiveness of early intervention, early recognition of the disease is critical for successful treatment and prevention of premature morbidity and mortality. Therefore, one should consider the diagnosis in those cases presenting with abnormal transaminases, idiopathic diabetes, or unusual presentations of arthritis.\nAN UNWANTED FAMILY INHERITANCE: THE CASE OF THE RAPIDLY PROGRESSIVE DEMENTIA. H. Jasti 1 , R. Granieri 1 ; 1 University of Pittsburgh, Pittsburgh, PA LEARNING OBJECTIVES: 1) Recognize the clinical presentation of Creutzfeldt-Jakob disease (CJD). 2) Recognize the diagnosis of this condition. CASE INFORMATION: A 44-year-old white female with no significant past medical history was in her usual state of health until two months prior to presentation when she began to experience an unsteady gait and problems with memory. One month prior to presentation, she experienced a seizure and then, two weeks later, she developed a rapid decline in her function, starting with short-term memory loss and progressing to unresponsiveness. Family history was significant for a paternal aunt who, at the age of 49 years, had a similar decline in cognitive function over 14 months. Physical exam revealed an awake woman who was unresponsive to commands. Vital signs were within normal limits. Neurological exam revealed dystonic posturing of the head with spontaneous conjugate eye movements, myoclonic activity of the upper extremities, and increased motor tone bilaterally. DTR's were normal bilaterally in the upper and lower extremities, with downgoing plantar reflexes. Bilateral grasp and glabellar signs were elicited. ANA, ESR, RPR, and Lyme titers were negative. An LP and a MRI were non-revealing. An EEG revealed periodic sharp-wave complexes. CSF was positive for a mutation of the prion protein gene, confirming familial CJD. DISCUSSION: The prion diseases constitute a family of subacute, neurodegenerative diseases referred to as transmissible spongioform encephalopathies. Incubation periods last from months to years and symptoms gradually increase in severity and result in death over a period of months. CJD is the major transmissible spongioform encephalopathy in humans. While a sporadic form tends to occur after the sixth decade and has a rapidly progressive course of under a year, an inherited form usually manifests at a younger age and has a more protracted course. Cardinal clinical features include confusion and memory loss progressing to severe cortical dementia, ataxia, and myoclonus. The EEG typically demonstrates bilateral periodic discharges. Neuroimaging is helpful to exclude other etiologies, since there are usually no abnormalities. Elevated levels of the 14-3±3 protein in the CSF, which has a 96% sensitivity and specificity for CJD, are also helpful for the diagnosis. The defining pathology is the presence of \"spongy\" degeneration of cortical gray matter. At present, there is no effective treatment for CJD. The diagnosis of prion disease should be considered as part of the differential in cases presenting with dementia and an atypical movement disorder, especially if the rate of disease progression is rapid.\nBEYOND THE OBVIOUS ABNORMAL LABORATORY VALUE. H. Jasti 1 , R. Granieri 1 ; 1 University of Pittsburgh, Pittsburgh, Pennsylvania LEARNING OBJECTIVES: 1) Recognize secondary causes of hypercholesterolemia. 2) Recognize the association between hypercholesterolemia and hypothyroidism. CASE INFORMATION: A 47-year-old male with hypertension and diabetes presented for a new visit. He endorsed occasional myalgias and constipation. He denied fatigue, weakness, cold intolerance, or dry skin. On physical, blood pressure was 150/86 and pulse was 74. The remaining exam was unremarkable, including the absence of tendinous xanthomas and delayed DTRs. Old records revealed a diagnosis of hypercholesterolemia (295 mg/dL) and hypertriglyceridemia (1200 mg/dL), with subsequent initiation of gemfibrozil. His lipid levels continued to be elevated 6 months after initiation of the medication. Subsequent testing revealed an elevated TSH and abnormal TFTs, consistent with a picture of hypothyroidism. Levothyroxine was started. Upon follow-up in 2 months, the TSH levels had decreased; cholesterol levels had also declined modestly. Further follow-up 4 months later demonstrated normal TSH levels and significantly decreased cholesterol (220 mg/dL) and triglyceride (480 mg/dL) levels. DISCUSSION: Significant hyperlipidemia may be present in the absence of clinical features; thus, secondary causes need to be considered before the initiation of lipid-lowering therapy. These include Type II diabetes, chronic renal insufficiency, nephrotic syndrome, cholestatic liver disease, obesity, smoking, alcohol, steroid, and oral contraceptive use. Hypothyroid patients also display a variety of patterns of lipid abnormalities, including elevated cholesterol, triglyceride, LDL levels. The main mechanism is a decrease in the number of LDL cell receptors, resulting in an increase in LDL levels. Other mechanisms include a diminished secretion of cholesterol into bile and reduced lipoprotein lipase activity, resulting in hypertriglyceridemia. The severity of the hypothyroidism may play a role in treatment, since many studies show a more significant improvement in the lipid levels in those with a more severe form of hypothyroidism. The evidence is less clear regarding the significance of the relationship between subclinical hypothyroidism and elevated cholesterol levels. In general, those with overt hypothyroidism seem to respond best to levothyroxine treatment and subsequently show a decline in their cholesterol levels, whereas those with subclinical hypothyroidism seem to need additional lipid-lowering agents. In conclusion, this case underscores the importance of identifying secondary causes of hypercholesterolemia before initiating treatment for an apparent lipid abnormality.\nITS NOT ALWAYS DKA. A. Tabas 1 , G. Tabas 1 ; 1 University of Pittsburgh Medical Center Shadyside, Pittsburgh, PA LEARNING OBJECTIVES: 1.Recognize lactic acidosis as a complication of metformin use. 2.Differentiate between diabetic ketoacidosis and metformin induced lactic acidosis. CASE INFORMATION: A 22 year old black female was admitted with three days history of nausea, vomiting, headache and abdominal pain, preceded by two days of rhinorrhea, and diarrhea. She was diagnosed with insulin requiring diabetes three years ago. There was no history of pre-existing renal insufficiency, chronic pulmonary disease or heart disease. Her medication was changed from insulin to metformin 500 mg bid a week prior to hospitalization. Her physical exam revealed tachycardia, and tachypnea. Pulse oximetry revealed 100% saturation on room air. The rest of the exam was unremarkable. Laboratory data were significant for ABG on room air - . Sodium-139 meq/L, Potassium-4.3 meq/L, Chloride-106 meq/L, CO 2 -7 meq/L, BUN-5 mg/dl, Creatinine-0.8 mg/dl, Glucose-276mg/dl, serum acetone-positive. The anion gap was 26. CBC & differential, and chest x-ray were within normal limits. Lactate and metformin levels were not checked. The patient was treated with normal saline, insulin drip, sodium bicarbonate and metformin was held. The metabolic acidosis resolved and the patient was discharged on long acting insulin. DISCUSSION: We do not know whether our patient had atypical diabetic ketoacidosis or metformin induced lactic acidosis. The presenting serum glucose in patients with diabetic ketoacidosis is usually between 450 mg/dl and 750 mg/dl. This patient's initial serum glucose of 276 mg/dl is somewhat uncommon for diabetic ketoacidosis, and suggests that other causes of high anion gap acidosis must be considered. The incidence of lactic acidosis is about 9 per 100,000 person-years in patients taking metformin. It usually occurs in patients who have preexisting cardiac disease, congestive heart failure, chronic pulmonary disease with hypoxia, sepsis or old age. Metformin induced lactic acidosis in patients such as ours, with no pre-existing risk factors, is rare but has been reported in literature. The mechanism is unknown. In patients taking metformin, presenting with ketoacidosis, metformin induced lactic acidosis should be considered. Assessing risk factors, appropriate laboratory workup (arterial lactate level, metformin level), withdrawing metformin and supportive care are key components of management.\nA. Jha 1 , K. Bernard 1 , D. Wilson 1 , R. Quinlin 1 ; 1 University of Pittsburgh Medical Center Shadyside, Pittsburgh, PA LEARNING OBJECTIVES: 1) To recognize rhabdomyolysis due to morphine. 2) To consider rhabdomyolysis in the differential diagnosis of post-operative renal failure. CASE INFORMATION: A 33-yr. old female underwent a laparoscopic`Roux-en-Y' gastroplasty under general anesthesia for morbid obesity. The anesthesia was induced by Midazolam, Fentanyl, Rocuronium and Succinylcholine. There was no masseter rigidity, or hyperthermia noted. Maintenance of anesthesia was done by Nitrous oxide, Propofol and Fentanyl. The patient had an uneventful perioperative course. There was no hematoma, or muscle tenderness following surgery. Morphine PCA was started. She has a past medical history of sleep apnea, hypertension, tubal ligation and hysterectomy without any complications. Her medications include Sertraline, Hydrochlorothiazide, Enalapril, and Albuterol inhaler. Her family history was unremarkable. The next morning she was found unresponsive and hypoventilating. An ABG revealed acute respiratory acidosis. She received Naloxone and the ABG normalized. Lab data revealed BUN -4 mg/dl, creatinine-2.0mg/dl (baseline -0.8), calcium -6.7 mg/dl, phosphorus-5.7mg/dl, magnesium-1.6mg/dl, sodium-138meq/L, potassium-4.1 meq/L, chloride-103 meq/L, C02-21meq/L, CPK-658 IU/L, CPK-MB-5.8 IU/L, Urine myoglobin-374( < 27mcg/L). EKG showed sinus tachycardia and an echocardiogram was normal. EEG, thyroid function, and serum Cortisol were normal. CPK levels increased to around 9400 IU/L and CPK-MB increased to 26 IU/L within two days. A diagnosis of morphine-induced rhabdomyolysis was made and patient was treated with D5Wand NaHCO3. Her Creatinine normalized the following day, and CPK, CPK-MB became normal on the 7th day. DISCUSSION: Prolonged surgery, electrolyte disturbances, infection and drugs are common causes of rhabdomyolysis and myoglobin-induced renal failure in the post-operative period. Morphine infrequently causes rhabdomyolysis. Proposed mechanism for morphine-induced rhabdomyolysis is myohypoxic necrosis and myocompression due to sedation related immobility. Reversal of narcotic effect with naloxone, withdrawing morphine, and hydration are the key components of management.\nPARKINSONS TREMOR MIMICKING MULTIPLE ARRYTHMIAS. M.F. Jhandir 1 , D. Brotman 1 ; 1 Cleveland Clinic Foundation, Clevland, OH LEARNING OBJECTIVES: 1. Rule out Parkinson's tremor first when pateints with Parkinson's disease present with symptoms or electrocardiogram changes suggestive of tachyarrythmia. 2. Appreciate that holding anti-Parkinon's medications can be a simple provocative manuever to determine whether the electrocardiogram abnormalitites are of noncardiac origin in such patients. CASE INFORMATION: A 75 year old nursing home resident with a history of coronary artery disease and Parkinson's disease presented to the emergency department for evaluation of pre-syncopal episodes and hypotension. He had a three day history of feeling lightheaded and unsteady while standing but had no true syncope. He denied chest pain, dyspnea and palpitations. There was no prior history of documented arrhythmia. His medications included aspirin, donepezil, levodopa/carbidopa, and lisinopril. On physical examination, he was not in acute distress. Heart rate was 68 with occasional premature beats. Blood pressure was 65/45 bilaterally with orthostatic changes. Cardiac exam was without murmurs or gallops. Neck veins were flat, lungs were clear and there was no peripheral edema. He had cogwheel rigidity but no tremor. The admission ECG showed sinus rhythm with occasional premature ventricular contractions. After intravenous crystalloids were given, the blood pressure normalized, and he was admitted to a monitored hospital bed for further observation. The following morning, the intern was called to the patient's bedside because of an apparent supraventricular tachycardia (SVT)(figure 2). Despite his abnormal ECG, the patient was asymptomatic, with a pulse of 64, and a blood pressure of 160/90. He had developed a pronounced bilateral upper extremity resting tremor at about 5 Hz, but otherwise his examination was unchanged. After the patient received his morning anti-Parkinson medications, the tremor resolved and his ECG returned to baseline. In order to prove that the tremor was responsible for the ECG changes, the next dose of levodopa/carbidopa was held; his tremor returned as did his pseudo-atrial flutter. DISCUSSION: Because the classic Parkinson's tremor has a frequency of 4±5 Hz (260±300 cycles/min), it is not surprising that its electrical potentials on ECG can mimic atrial flutter, as in our case. Furthermore, Parkinson's disease is commonly associated with orthostatic hypotension and syncope, which may in turn prompt cardiac evaluations. Although there are previously documented cases of Parkinson's tremor leading to ECG artifacts resembling wide complex tachyarrhythmias such as ventricular tachycardia, we know of no other reports of Parkinson's tremor mimicking atrial flutter or SVT. Careful review of our patient's rhythm strip reveals that there are buried QRS complexes within the pseudo-SVT complexes (figure 2). The importance of correlating the ECG with the clinical exam cannot be overemphasized in this case, as the heart rate during the pseudo-SVT was normal, and the ECG artifacts were present only when the patient was tremulous. Finally, our case illustrates that if a patient with Parkinson's disease has transient ECG abnormalities suggestive of tremor-induced artifact, holding medicines can be a simple provocative maneuver to determine whether the ECG abnormalities are of non-cardiac origin. We conclude that Parkinson's tremor should be thoroughly ruled out when a patient with Parkinson's disease presents with symptoms and ECG changes suggestive of a tachyarrhythmia.\nSTRONGYLOIDIASIS AND THE HYPERINFECTION SYNDROME. H. Jneid 1 , N. Correia 1 , F. Michota 1 ; 1 Cleveland Clinic Foundation, Cleveland, Ohio LEARNING OBJECTIVES: 1. List the differential diagnoses for immunocompromised patients with chronic obstructive pulmonary disease and progressive dyspnea. 2. Recognize the risk factors and clinical features of strongyloidiasis. 3. Recognize the hyperinfection syndrome of strongyloidiasis. CASE INFORMATION: A 65-year-old retired coal miner presented with a 2-month history of progressive shortness of breath and nonproductive cough. He had a history of emphysema and coal worker' pneumoconiosis and had been using an inhaler and oxygen therapy. He had grown up in a rural Appalachian area of Ohio and stopped using tobacco 20 years ago. He had been started on cyclophosphamide and high-dose prednisone therapy for rapidly progressive glomerulonephritis 3 months previously. He was morbidly obese, with T 37.48C, BP 134/65 mm Hg, Pulse 100, RR 22/min, and SpO2 91% on 4 L of oxygen. Lung auscultation showed decreased air entry and diffuse wheezing. Initial chest x-ray showed no parenchymal lung disease. Review of his kidney biopsy revealed chronic interstitial nephritis, cyclophosphamide was stopped, and prednisone was gradually tapered. His dyspnea and oxygen requirement increased despite aggressive therapy with bronchodilators. Venous duplex ultrasound of the upper and lower extremities showed no thrombosis. V-Q scan revealed no mismatches. Computed tomography of the chest subsequently showed diffuse alveolar ground-glass appearance. The patient was started on broad-spectrum antibiotics, including intravenous Bactrim and amphotericin. Bronchoscopy was done, and Strongyloides stercoralis was diagnosed by transbronchial biopsy. The patient was started on intravenous thiabendazole. Prednisone was tapered quickly and stopped. His respiratory status deteriorated, and the patient eventually died. Autopsy found disseminated infection, with panlobar pulmonary infestation and patchy small and large bowel strongyloidiasis. DISCUSSION: Our immunocompromised patient had worsened respiratory symptoms despite inhaler therapy, which prompted us to seek other diagnoses. Initial work-up was negative for congestive heart failure and thromboembolism. His previous diagnosis of simple coal worker' pneumoconiosis was not supported by initial radiologic findings and would not have explained his rapid clinical deterioration and radiologic evolution. In this setting, opportunistic infections should be strongly suspected. Strongyloides is an intestinal parasite that is common in tropical and subtropical areas. Infection rates are highest among residents of the southern states and Appalachia, and among immigrants from endemic areas. Transmission occurs when infective larvae come in contact with the skin. The parasite has an autoinfection cycle that is usually contained by the host immune system; therefore, larval forms may persist in the body up to 50 years. Clinical manifestations of strongyloidiasis include abdominal upsets, lung manifestations such as wheezing and cough, and a pathognomonic skin eruption called larva currens. The autoinfection cycle accelerates inversely with the strength of the immune system and may yield to the highly lethal hyperinfection syndrome in immunosuppressed patients, characterized by massive dissemination to multiple organs and systems, such as the central nervous system, heart, liver, and endocrine glands. People at risk for Strongyloides infection should be screened before being given immunosuppressive therapy. Recognize the predisposing conditions and the treatment of cerebral arterial air embolism. CASE INFORMATION: A 50-year-old white woman presented with two transient 15-minute episodes of left upper extremity parasthesia, slurred speech, and diplopia. She had had inflammatory breast cancer 3 years previously, and had undergone right modified radical mastectomy, chemotherapy, and radiotherapy. Liver metastasis had recently been discovered. Neurologic exam on presentation was nonfocal. Computed tomography (CT) of the brain and lumbar puncture fluid analysis were normal. Magnetic resonance imaging found no leptomeningeal enhancement or focal lesions. Duplex ultrasound of her extracranial arteries showed no significant atherosclerotic stenoses, but 15 minutes of continuous Doppler monitoring showed active embolization (>1000 emboli /minute) to the brain. Venous duplex ultrasound of her upper and lower extremities showed no thrombosis, and CT scan of her abdomen showed no pelvic or inferior caval thrombosis. Holter monitoring showed no cardiac arrhythmias. Transesophageal echocardiography showed no valvular vegetations, intracardiac thrombi, or patent foramen ovale, but revealed spontaneous air bubbles in the left atrium. The patient' neurological status progressively worsened until she became comatose, entered respiratory arrest, and died. No autopsy was performed.\nDISCUSSION: Cerebral arterial air embolism has been described with cardiac surgery, mechanical ventilation, underwater diving and bronchoscopic laser operations. Arterial embolization results from direct passage of air into the arterial circulation or from paradoxical embolization. The effect of an air embolus depends on its size as well as its rate of entry into the circulation. Arterial embolization is dangerous in that it can cause ischemic damage to organs. Because absorption of arterial emboli is inversely related to the arterial pressure of nitrogen, treatment with hyperbaric oxygen has been effective. This treatment also helps reduce the air bubble size and increases the oxygen content of blood. In our patient, no bubbles were detected in the right heart chambers, which makes paradoxical embolization unlikely. A plausible biological mechanism is cerebral air embolism through bronchovenous fistula, with possible pathogenetic implication of either micrometastases to the lung or the prior thoracic radiation therapy, but this remains to be proven. Recognize the possibility of multiple opportunistic infections in immunocompromised patients. CASE INFORMATION: A 40-year-old man presented to a hospital with a 5-month history of progressive dyspnea on exertion, productive cough, and gradual weight loss. He had a 52 packyear history of smoking. He was treated with several courses of antimicrobials for bronchitis and was maintained on high-dose prednisone (60 mg daily) for several months. He presented to an outside hospital with respiratory distress, requiring 50% FiO2. Chest x-ray showed bilateral alveolar infiltrates, and on computed tomography (CT), the chest had a diffuse ground-glass appearance. Bronchoscopy was nondiagnostic, so he underwent an open lung biopsy, which showed pulmonary alveolar proteinosis. He was transferred to our tertiary care facility where he was given normal saline lavage (17 L per lung) under general anesthesia. His respiratory status improved and he was discharged home on 6 L/min of oxygen and tapering doses of prednisone. He returned few days later with worsening dyspnea, fever, and increased cough, now productive of copious brown rusty sputum. His vitals: T 101.08F, BP 95/56 mm Hg, Pulse 120, and RR 30/min. His lung auscultation revealed diffuse crackles. His PaO2 on 6 L of oxygen was 57 mm Hg. He was intubated and started on broad-spectrum antibiotics. Sputum staining showed branching gram-positive filaments with weak acid-fast stain, and intravenous Bactrim (trimethoprim and sulfamethoxazole) was added for treatment of Nocardia pneumonia. Ophthalmologic exam and CT of the abdomen and brain showed no evidence of nocardial dissemination. His sputum stain subsequently showed septate hyphae, which was targeted with amphotericin B. The fungus was identified as Trichosporon beigellii. The patient eventually died of septic shock. Autopsy showed panlobar bilateral necrotizing pneumonia and the presence of silver-positive organisms consistent with Nocardia, Trichosporon, and Aspergillus. DISCUSSION: Pulmonary alveolar proteinosis (PAP) is a diffuse lung disease characterized by dense accumulation of PAS-positive (periodic acid-Sciff) phospholipid material in the alveoli, with preservation of the lung architecture and absence of inflammation. Patients usually present with a subacute prodrome of cough and progressively decreasing exertional tolerance. Tissue biopsy, by open lung or thoracoscopy, is the gold standard for diagnosis, though transbronchial biopsy and fluid obtained by bronchoalveolar lavage may also be used. Whole lung lavage is the treatment of choice, and recently interest has grown about the potential therapeutic role of GM-CSF (granulocyte/macrophage colony stimulating factor) because defective alveolar macrophages are involved in the pathophysiology. There is no role for immunosuppressants in treatment, and, in fact, some suspect that corticosteroids may increase mortality. The association of pulmonary nocardiosis with PAP has been described frequently. Nocardia asteroides tends to disseminate from a pulmonary or cutaneous focus to any organ, notably the central nervous system, retina, heart, kidneys, and joints. The new onset of fever and change in the quality of sputum in our immunocompromised patient prompted us to think of superimposed opportunistic infections. A thirty-three year old Caucasian man presented with history of recurrent spells. He described episodes of palpitations and hot flushes, followed by tunnel vision, pre-syncopal feeling, and loss of consciousness. He had spontaneous recoveries, with no incontinence or postictal state. He reported loose stools, intermittent urticarial rash, and occasional wheezings associated with these episodes. He had an AICD (automatic implantable cardioverter-defibrillator)placed after the second spell for complete heart block that degenerated into ventricular fibrillation. Subsequent interrogation of the AICD showed no evidence of dysrrhytmias despite recurrent spells. 24-hour EEG monitoring showed no epileptiform activity. He underwent a cardiac catherization, which showed normal coronary arteries. CT scans of the head and the abdomen were non-revealing. Pulmonary angiography showed no evidence of pulmonary embolism. On presentation, his physical exam, including neurological exam, was completely normal, except for urticarial rash on the dorsum of his hands. Telemetry monitoring didn't reveal any arrhythmia. A tilt table test showed asymptomatic postural tachycardia. Urine metanephrine and 5-HIAA levels were within normal. Echocardiography showed no valvular lesions. Glucose levels and CBC were normal. A MIBG scan was also negative. The patient was discharged home on a beta-blocker. His spells recurred. Further work up included a twenty-four hour urine histamine collection, which was normal. Punch biopsy of his urticarial skin lesions, revealed urticaria pigmentosa, with perivascular and interstitial mast cells infiltration of the superficial dermis. Colonoscopy showed mast cells infiltration of the lamina propria of the ileum and right colon. A bone marrow aspirate revealed 15% mast cell cellularity. The diagnosis was consistent with systemic mastocytosis. The patient was switched to a combination of H1 and H2 blockers, and later had a leukotriene receptor antagonist added to his regimen. He remained spell-free afterwards. DISCUSSION: One needs to exclude other causes of spells, like cardiovascular, neurologic, endocrine, pharmacologic, and psychologic etiologies. Systemic mastocytosis is a disease characterized by mast cell infiltration of multiple organs. It presents with episodic flushing, diarrhea, abdominal pain, bronchospasm, alcohol intolerance, and syncope. Triggers include emotional stress, iodinated contrast agents, aspirin, and exercise. Urticaria pigmentosa is a characteristic skin lesion. Therapeutic armamentorium includes H1 and H2 blockers, cromolyn sodium and prostaglandin synthesis inhibitors. Epinephrine is used in severe cases associated with anaphylaxis, while interferon therapy is reserved for proliferative disease. Our patients had a dramatic presentation and a dramatic response after addition of a leukotriene antagonist. -old white woman presented with a 4-week history of progressive shortness of breath and persistent dull right-sided chest pain. She had never been a smoker. On admission, she was afebrile, RR 14, and had oxygen saturation of 95% on room air. Lung auscultation in the right lower lung fields found decreased breath sounds and decreased tactile fremitus. Chest x-ray confirmed the presence of right pleural effusion. Thoracentesis showed a milky fluid consistent with chylothorax (pH = 7.26, RBC = 79, WBC = 4.4, LDH = 398 u/l, protein = 1.6 g/dl, glucose = 51 mg/dl, triglyceride = 715 mg/dl, cholesterol = 26 mg/dl, and positive chylomicron screen). CT of the chest and abdomen revealed a large pleural mass emanating from the posterior hemithorax, with extension below the right diaphragm. Thoracoscopic biopsy of the mass revealed malignant mesothelioma. The patient had no occupational exposure to asbestos, but her father (who had no lung disease) had worked in the steel industry. The patient was switched to a medium-chain fatty acid diet, and her pleural drainage became serous, although it persisted. Eventually, a Pleurex catheter was inserted for self-administered therapeutic drainage as needed, and the patient was discharged home with hospice care. DISCUSSION: Malignant mesothelioma is an insidious neoplasm that usually afflicts patients in their fifth to seventh decades of life. Patients usually present with dyspnea, nonpleuritic chest pain, and unilateral pleural effusion. Exposure to asbestos is elicited in only 70% to 90% of cases. Domestic exposure to asbestos through family members is well documented and could have been the cause in our patient. Multimodality therapeutic approaches (surgery with chemotherapy +/-radiotherapy) produce a slight improvement in quality of life and survival. Our patient' tumor extended across the diaphragm and was therefore nonresectable (stage IV). Chylothorax is usually due to either trauma such as surgery or nontraumatic causes such as malignancies, with lymphoma accounting for the majority of cases. Prolonged chyle loss can lead to nutritional depletion and immunologic deficiency. A triglyceride level above 110 mg/dL in a pleural effusion is very suggestive of chylothorax, and the presence of chylomicrons confirms the diagnosis. The presentation of a young patient with no asbestos exposure, with malignant mesothelioma and a chylothorax is extremely rare.\nCOMBINED ADRENAL CRISIS AND MYXEDEMA COMA DUE TO HYPOTHYROIDISM. K. Kamjoo 1 , N. Michail 2 ; 1 University of California, Los Angeles, Sherman Oaks, Ca; 2 UCLA LEARNING OBJECTIVES: 1-To recognize the clinical presentation of severe hypopituitarism. 2-To understand the physiology of pituitary hormones. 3-Patient compliance as a cause of serious illness. CASE INFORMATION: A 37 years old female with a history of pituitary adenoma status post trans-sphenoid resection in 1990, was brought to Emergency Department (ED). Pt was not responsive and paramedics' report indicated that patient had gotten worse over the past month with progressive lethargy, vomiting, and altered mental status. Patient had stopped her medications approximately one year prior to admission. In ED patient was comatose and her initial vitals were: Rectal temperature 33.3 C, pulse 64, respiration rate 14, blood pressure 80/ 44, and blood sugar of 26. On physical exam patient appeared pale and had marked facial puffiness. She had no heart murmur but was bradycardiac. Patient's neuro exam showed bilateral hyperreflexia with a definite delay in the relaxation phase of her deep tendon reflexes. Patient's skin was very dry and she had no axillary or pubic hair. CXR only showed mild cardiomegaly. EKG showed sinus bradycardia with diffuse low voltage and flat T-waves. Patient was given 2L of intravenous (IV) D5W and 100 mg of IV hydrocortisone. After administration of IV steroids, patient's core temperature increased to 37.2 C and her blood pressure was stable at 89/50. She had an O2 saturation of 100% on room air and became conscious and alert. Subsequently, she was admitted to step-down unit with a diagnosis of adrenal crisis due to untreated hypopituitarism. Patient was started on DDAVP, and hydrocortisone. Serum TSH and free T4 were pending at this time and patient was started on 0.1 mg of oral Synthroid. Patient was doing well after the first day of hospitalization. However, on the second day her mental status deteriorated again and became obtunded. She had increased puffiness in her face and marked edema of her lower and upper extremities. Patient's blood gas showed CO2 retention. Patient was diagnosed with myxedema coma and tranferred to medical ICU. Patient was intubated and a single 300 mcg dose of IV Levothyroxine was administered, followed by 100 mcg of IV Levothyroxine daily. At this time serum TSH and free T4 drawn on admission showed TSH = 0.1 and free T4 < 0.40. The patient subsequently improved markedly and was discharged from hospital after 7 days. DISCUSSION: Myxedema coma is a very rare disease occuring primarily in elderly patients with primary thyroid failure. This case illustrates the importance of early recognition that myxedema coma can occur in combination with adrenal crisis in patients with decompensated hypopituitarism. Non-compliance in this disease could lead to fatal consequences. A 56 year old male presented for routine primary care evaluation. His past medical history was significant only for mild COPD and tobacco use. He specifically denied any history of coronary artery disease,diabetes,hypertension or dyslipidemia. Review of systems identified long standing complaints of erectile dysfunction and fatigue. Specifically, the patient noted a frequent inability to reach orgasm and lower extremity fatigue when climbing stairs. Physical examination was notable for a blood pressure of 110/65,an arm span greater than height,intact lower extremity pulses,sparse male pattern hair distribution,gynecomastia,small penis size and small,firm testes. Pulse volume recordings of the lower extremities and electrocardiogram were unremarkable. Laboratory evaluation found LDL=68, fasting glucose=104,Hct=48,TSH=1.08,free testoster-one=2.6,LH=14,FSH=26 and prolactin=3.5. Subsequent chromosomal karyotyping indicated the presence of an 82% XXY pattern consistent with Klinefelter Syndrome. The patient was treated with intramuscular testosterone. On follow-up, this patient reported improved energy levels and sexual performance. Indeed, the patient presented with new complaints consistent with bilateral epicondylitis. Apparently, following diagnosis and therapy, this patient was now hard at work buffing combines! DISCUSSION: Klinefelter syndrome,a primary form of testicular failure, is a common disorder that affects 1 in 500 male patients. It results from nondisjunction during parental gametogenesis and yields an individual with an XXY karyotype. Individuals with Klinefelter syndrome do not progress through puberty and ultimately develop the clinical features of eunuchoidism. These features of androgen deficiency include:arm span greater than height,long legs,sparse male pattern hair distribution,gynecomastia,small testes and penis and infertility. Endocrine evaluation of patients with this syndrome indicates low free testosterone together with elevated gonadotropin levels (ie LH and FSH). Klinfelter syndrome is treated with testosterone supplementation. This produces positive changes in mood,behavior,muscle mass and bone density. Once testosterone supplementation is begun, patients must be routinely screened for erythrocytosis and lipid abnormalities. year-old man with history of recurrent spontaneous pneumothorax (he underwent pleurodesis 5 years ago), presented to our service complaining of 2 months of progressive abdominal swelling, leg edema, jaundice, and worsening shortness of breath. The patient is a chronic smoker who rarely consumes alcohol. On exam the patient was afebrile with a regular pulse of 110, blood pressure of 118/78, respiratory rate of 18/min, distended neck veins, S4 gallop, massive ascites and +3 leg edema. Other than high total/direct bilirubin 2.2/3.6, the labs were unremarkable. Echocardiogram revealed a markedly dilated right ventricle with moderate pulmonary hypertension, severe tricuspid regurgitation and left ventricle of normal size and function. Spiral chest CT revealed pulmonary embolism of both left lower and upper divisions of the pulmonary artery. Doppler ultrasound of both legs was negative. The patient was anticoagulated (heparin, then warfarin) and once stable discharged home. DISCUSSION: CTEPH results from single or recurrent pulmonary emboli arising from sites of venous thrombosis. Most emboli completely resolve spontaneously, or they have minimal residual effects. In CTEPH, for unclear reasons, the emboli do not resolve completely. Rather, the emboli undergo abnormal organization and recanalization, leaving endothelium-covered residua that obstruct or significantly narrow major branches of the pulmonary artery. This is very different from primary pulmonary hypertension, where obstructive lesions appear in the small distal pulmonary arteries. Most patients with CTEPH have history of venous thrombosis with embolism; however this history may not be elicited unless actively sought for. After the initial embolic event the patients' condition gradually improves and they usually resume their normal activities. This period of improvement is however a temporary``honeymoon period' frequently followed by more subclinical thromboembolic events. Recurrent pulmonary embolism destroys the pulmonary vascular bed and causes pulmonary hypertension. With time patients deteriorate and become dyspneic at lower levels of exertion. Clinically, they present with a triad of dyspnea, syncope and right heart failure. Initial treatment includes chronic anticoagulation; consider embolectomy for massive embolism. and thrombotic and vasculitis work up. Transesophageal echocardiography (TEE) with agitated saline demonstrated a PFO, with significant right to left shunt, with transcranial Doppler demonstrating contrast reaching cerebral circulation. The patient underwent surgical closure of PFO by direct suturing. The patient now presented with the fourth episode of transient right-sided numbness and paresthesia in the last three years. TEE with agitated saline showed no residual shunt. Patient was anticoagulated (heparin, then warfarin) and once stable sent home. DISCUSSION: Stroke causes more than 100,000 deaths in USA each year, and leaves many others with major disabilities. In 25 to 40% of strokes in young adults, an extensive evaluation fails to identify the cause; these are classified as crypotogenic strokes. Notably, patients with crypotogenic strokes have a higher incidence of PFO than patients with stroke of determined cause, even after correcting for presence of recognized stroke risk factors. Potential mechanism for PFO related ischemic event includes paradoxical embolism in which a thrombus dislodges from a venous thrombotic site, traverses the interatrial septum and reaches the brain. Other mechanisms may include a thrombus formation secondary to atrial arrhythmias or atrial septal aneurysm. The incidence of stroke will be higher as the size of a PFO increases and if it is associated with an atrial septal aneurysm. Treatment options for PFO may range from no therapy, to medical therapy, to surgical closure of PFO. Choosing the type of treatment would depend on the patient's age, surgical and anticoagulation risks. The average annual risk of recurrence of cardiovascular events is 3.8% for medical therapy (aspirin 250 ± 500 mg/d, warfarin with target INR 2 ± 3, or both together) and 2.5 % after percutaneus transcatheter closure of the PFO. Closure done through open heart surgery by direct suture or patch closure is the gold standard of therapy and offers permanent closure of the defect with minimal risk and avoidance of long term anticoagulation. Recurrence of cerebrovascular ischemic events is still possible after successful surgery. Having multiple neurological events before surgery is the only significant risk factor of recurrence after the procedure. One should try to identify causes other than paradoxical embolism in patients who experience recurrent cerebrovascular events after surgical closure of PFO.\nA. Karcic 1 , J. Dell'orfano 1 ; 1 Departments of Internal Medicine and Cardiology, State University of New York at Stony Brook and Nassau University Medical Center, East Meadow, NY LEARNING OBJECTIVE: 1. To understand that ischemic myocardium has a decreased capability to conduct pacemaker beats. CASE INFORMATION: A 73 years-old hypertensive man, had one year ago a pacemaker inserted for symptomatic sinus bradycardia (fainting spells). Coronary angiography performed at that time showed only mild nonobstructive coronary disease, The pacemaker was a dual chamber device, with leads in the night atrium and night ventricle, and it was functioning appropriately until a week ago, when the patient noticed chest pressure while shoveling snow. At that time his pulse rate, on self-exam was 40 bpm, so the patient went to see his internist, knowing that``there is something wrong with the pacemaker, since it was programmed to keep the heart rate above 60 bpm.'' During hospitalization the patient had slightly positive cardiac enzymes. The ECG revealed sinus bradycardia and inferior wall ST depressions with regular vertical pacemaker spikes at 60 bpm, but failure of these paced stimuli to``capture'' the myocardium and lead to ventricular beats. Coronary angiography revealed a 100% obstructed night coronary artery (RCA) as the cause of ECG changes and ischemia. Angioplasty was performed on the RCA, and a stent inserted to keep the vessel open. Several hours after the procedure a repeat ECG revealed no ST depressions, with all the pacemaker spikes 'capturing' the myocardium, and pacing the heart at the set rate of 60 bpm. DISCUSSION: Our patient had a complete RCA obstruction, leading to ischemia of the inferior areas of the heart, as seen on the ECG. It also caused ischemia of the myocardium where the night ventricular pacemaker lead was inserted. Ischemic myocardium (or dead myocardium Ð as a scar from an old myocardial infarction) has a decreased ability to conduct electrical stimuli, as those received from a pacemaker. It effectively acts as an obstacle to the conduction of these pacemaker beats to the surrounding healthy nonischemic myocardium. For at least some paced beats to be conducted through the ischemic myocardium, impulses at much higher energy levels need to be delivered by the pacemaker. This not only leads to faster pacemaker battery depletion, but also usually does not guarantee that all beats delivered by the pacemaker will be conducted, which is needed. The solution to this problem is relieving the ischemia, if possible (like in our patient), or by changing the position of the night ventricular lead, if it was located in myocardial scar tissue. (8 years), risk factor HIV positive hemophiliac husband, with history of recurrent respiratory infections, was admitted for worsening dyspnea of several months duration, and two weeks of abdominal and leg swelling and oliguria. On exam she was tachypneic with a blood pressure 100/60 mm Hg and pulse rate of 100/ min. Lungs were clear to auscultation, while a right-sided gallop was audible. The jugular veins were distended, the liver painfully enlarged, and there was bipedal pitting edema. EKG revealed an incomplete RBBB. Chest radiograph showed mild cardiomegaly, and a prominent pulmonary artery, with clear lungs. Sonogram of the heart revealed large right-sided chambers, paradoxical septal motion and pulmonary hypertension. VQ scan was low probability for pulmonary embolism. Finally a right-sided cardiac catheterization excluded a left to right shunt and revealed severe pulmonary hypertension, with preserved left ventricular function.\nDISCUSSION: PPH is a diagnosis of exclusion. In order for it to be diagnosed, no cause for the pulmonary hypertension should be found. Frequently, in HIV positive patients, pulmonary hypertension develops secondary to HIV related cardiomyopathy and left ventricular failure. Early reports of PPH in HIV involved hemophiliacs receiving large amounts of factor VIII concentrates, which over a period of time can cause pulmonary vascular insults leading to PPH. Only later came reports of non-hemophiliac HIV positive patients with PPH. Intravenous drug users can develop pulmonary hypertension as tale emboli cause pulmonary fibrosis and vascular disease. Progressive pulmonary thromboembolism has also been reported to cause pulmonary hypertension in HlV positive patients. While mild and moderate pulmonary hypertension can develop in many cardiac and pulmonary diseases, it is proposed that a genetic predisposition (rare familial PPH), in concord with pulmonary vascular insults (shear stress, chronic hypoxia and chronic inflammation), leads to severe and fatal PPH. Patients with AIDS have a multitude of pulmonary vascular insults (infections, emboli, hypoxia etc.), but only 0.5% of them develop severe PPH. Our patient for example had frequent bronchitis. It appears that PPH in patients with HIV is multifactorial in origin, rather than being merely a coincidental finding. As a conclusion, one could mention that pulmonary hypertension in HIV which is labeled as primary may in fact be secondary to a multitude of factors that may remain unrecognized, year old woman with a history of smoking and hypertension was admitted to the hospital with syncope. She was found to have episodes of significant sinus bradycardia for which she needed a pacemaker. A dual chamber pacemaker was inserted, with pacemaker leads placed in the right atrium and right ventricular apex. ECG, fluoroscopy and a chest radiograph confirmed the correct position of the leads, while postoperative pacemaker interrogation revealed that the right atrial and right ventricular pacing and sensing thresholds were within normal limits. Initially after the procedure the patient was feeling fine, but the same night she started complaining of persistent, rhythmic, frequent and annoying contractions on the right side of her upper abdomen, feeling somewhat like hiccups. There was nothing the patient could do in order to bring upon or abort these symptoms. After having interrogated the pacemaker, the cardiologist abandoned right atrial pacing and the symptoms resolved completely. DISCUSSION: This patient had right hemidiaphragmatic contractions that were induced by pacemaker beats. The diaphragm is a muscle and hence can be stimulated by electrical stimuli from the pacemaker. In order to understand how a pacemaker induces diaphragmatic contractions, one should recall that the right phrenic nerve traverses the right atrium (atrial electrode location), and the left phrenic nerve the right ventricular apex (ventricular electrode location). If the pacemaker stimuli are of adequately high energy, not only the surrounding myocardium wall be paced, but the paced beats will also be conducted through the overlying right or left phrenic nerve to cause contractions of the right or left hemidiaphragm respectively. In our patient the right hemidiaphragm was paced, telling us that the right phrenic nerve was paced via the right atrial electrode. Frequently there is a therapeutic window that enables us to pace the myocardium without pacing the overlying phrenic nerve. This is due to the fact that usually lower energy levels are needed to pace the myocardium. Gradual decrease of pacemaker energy output below that needed to pace the phrenic nerve, but still high enough to pace the myocardium is the perfect solution for this problem. If such a therapeutic window does not exist, the culprit electrode must be abandoned (atrial), or a new one can be placed in a different location (ventricular).\nDELAYED DIAGNOSIS OF PERFORATED SIGMOID STERCORAL ULCER. M. Kasubhai 1 , M. Singh 1 , T. Saleem 1 , A. Singh 2 , V. Dimitrov 1 ; 1 Lincoln Medical and Mental Health Center., Bronx, NY; Cornell University LEARNING OBJECTIVES: 1) Recognize that stercoral ulcers result from pressure necrosis of the mucosa by the direct effect of a mass of retained feces. 2) Fecal Impaction is commonly diagnosed and rapid and gentle care can prevent this morbid complication CASE INFORMATION: 67 year old male presented with history of seizures, lower abdominal pain and constipation. Past history was relevant for seizure disorder, diabetes, stroke and bilateral above knee amputation. He was afebrile and hemodynamically stable. Left lower quadrant tenderness was noted. Rectal examination revealed fecal impaction and was manually disimpacted. The hemotocrit was 40 with leucocytosis. X-rays of chest and abdomen were normal. The patient subsequently developed fever and was treated for diverticulitis. Fever resolved with antibiotics. CT scan revealed thickening of the wall of the rectum and sigmoid, colonic dilatation and small bowel ileus. Colonoscopy performed two weeks later to evaluate the cause of constipation revealed an ulcer at the recto-sigmoid junction with biopsy revealing dysplastic changes. The other considerations of the ulcer being viral, bacterial, fungal or tuberculous were excluded by biopsy and culture. A stercoral ulcer was also considered and treated conservatively. After two weeks a repeat colonoscopy showed a persistent ulcer with acute and chronic inflammatory exudate. An exploratory laparotomy was performed because of persistent colonic dilatation and ileus. A sigmoid tear was noted over the ulcer, which was plugged by matted loops of small bowel. Sigmoid resection and colostomy was done. The patient recovered completely and was discharged home. DISCUSSION: Stercoral ulcers are usually found in the rectum and sigmoid. Their size is variable and margin usually irregular. When symptomatic, they usually reveal themselves by bleeding or perforation. Relieving the fecal impaction manually can result in perforation of the ulcer. Fever and leucocytosis, as in our patient, should suggest perforation. Our patient also had unexplained localised ileus and colonic dilatation, which should have led to an early exploratory laparotomy. Fecal impaction is commonly diagnosed and the rapid and gentle care can prevent its morbid complications HYPOKALEMIC PARALYSIS: PURSUING A DIAGNOSIS. J. Kent 1 , W.P. Moran 1 , L.B. Jones 1 , J. Stopyra 1 ; 1 Wake Forest University School of Medicine, Winston-Salem, NC LEARNING OBJECTIVES: 1) Recognize an uncommon presentation of SjÐgren's Syndrome. 2) Use a literature search as a diagnostic aid. 3) Demonstrate the importance of pursuing a diagnosis to the end. CASE INFORMATION: A 69 yo w female with past medical history of gastroesophageal reflux disease, hypertension, anxiety, arthritis and hypokalemic paralysis presented to the emergency department with a one-week history of progressive right-sided weakness. Her symptoms included difficulty using her right hand, impairment of gait, and a decrease in appetite. She had a history of right-sided paralysis due to hypokalemia nine months earlier requiring hospitalization, which had resolved with aggressive potassium replacement. She received neurologic and endocrine workups at that time without any etiology found, and she was discharged on oral potassium and spironolactone. With this second episode, her CT scan was negative for an acute CVA, and her potassium level was found to be 2.1. On exam, her oral mucous membranes were dry. Her lab values were suggestive of renal tubular acidosis. After doing a MEDLINE search with keywords including hypokalemia, paralysis, and acidosis, it was found that there have been over 20 case reports of patients with SjÐgren's syndrome presenting with a hypokalemic paresis or paralysis. Upon further questioning, the patient did note that her eyes and mouth had felt unusually dry for the past year. Diagnostic testing included a positive Schirmer's test and a minor salivary gland biopsy that was strongly suggestive of Sjo È gren's syndrome. The hemiparesis completely resolved with replacement of her potassium, and she was discharged on potassium supplementation with spironolactone, as well as artificial tears and lozenges for treatment of her Sjo È gren's symptoms. DISCUSSION: This patient's episode of hemiparesis/ paralysis due to hypokalemia was the second such episode within 9 months. During her first episode, it was also noted that she had symptomatic dryness of mouth and eyes and that her mucous membranes were dry. Sjo È gren's syndrome itself was not mentioned in a differential diagnosis. The main clinical problem appeared to be the hypokalemia and hemiparesis, which are not traditionally associated with Sjo È gren's. When the patient's hypokalemia and paralysis resolved, she was discharged with outpatient follow up. During this second episode, the patient's main symptoms were again relieved before a diagnostic etiology was found. However, the diligent search for a reason for her hypokalemia by the team resulted in the discovery of her underlying problem, Sjo È gren's Syndrome, which then allowed for more complete treatment. The use of a literature search and its finding of a series of case reports was a significant aid to the team in reaching the diagnosis. An 86 year old woman with no significant past medical history and taking no medications was admitted to the intensive care unit after a one day history of weakness, dizziness, nausea and vomiting. The patient was hypotensive with a blood pressure of 90/50 mmHg, without orthostatic changes. The pulse was 64; she was afebrile, and had dry oral mucosa. The remainder of the physical examination was normal. Laboratory workup was significant for pyuria and mild anemia (Hgb = 11.4 g/dL). Intravenous fluids were started and antibiotics were given for her urinary tract infection. During the hospital stay, the patient developed disorientation, hallucinations, and incoherent speech interpreted as delirium secondary to her infection and ICU psychosis. When her delirium did not resolve with hydration, antibiotics and transfer to a regular floor, a hepatic profile was ordered as part of the workup for metabolic causes for her delirium. This testing revealed an isolated elevated alkaline phosphatase (650 U/L). Abdominal ultrasound showed marked dilation of the common bile duct and subsequent abdominal CT scan showed no masses in the area of the gall bladder and liver. A 1.5 cm common bile duct stone was successfully extracted by endoscopic sphincterotomy and her mental status returned to baseline. DISCUSSION: The causes of delirium include medication side effects or withdrawal, dehydration, infections, hypoxia, hypoglycemia, and other metabolic abnormalities. Many systemic illnesses may produce delirium in elderly patients, especially in combination with new medications, fever, or sleep deprivation. In this patient, a comprehensive evaluation for metabolic causes of delirium (despite lack of localizing symptoms or signs on physical examination led to a diagnosis of unsuspected common bile duct stone and institution of appropriate treatment. Since delirium is associated with adverse outcomes, making the correct diagnosis is important. It is essential to do a complete workup for delirium in order to determine all treatable causes, particularly in hospitalized elderly patients.\nANTICONVULSANT HYPERSENSITIVITY SYNDROME: A CALL FOR INCREASING AWARENESS. N. Khan 1 , B. Cheong 1 , A. Jaffer 1 ; 1 Cleveland Clinic Foundation, Cleveland, OH LEARNING OBJECTIVES: 1. Diagnose the distinct entity of hypersensitivity reaction to anticonvulsants. 2. Recognize the prevelance of cross-reactivity between different anticonvulsant medications. CASE INFORMATION: A 27 year-old lady with a history of multiple sclerosis was initiated on oral phenytoin for tonic spasms of extremities. Two weeks later she noted a mild maculopapular erythematous rash on her extremities. Concerns for drug induced rash and minimal response to treatment, led to a change in regimen from phenytoin to carbamazepine. One week later the patient was hospitalized with worsening pruritic maculopapular rash, more prominent on the trunk and extremities, and recurrent fevers. Except for a temperature of 101.5 Å F, her vital signs were within normal limits. Notable changes on physical exam included mild facial edema, with 1.5 to 2cm bilateral, tender, mobile jugulodigastric and submandibular lympadenopathy (LAD). Laboratory abnormalities on admission included peripheral eosinophilia and elevated hepatic enzymes: AST=271 (7 ± 40 U/L), ALT=324 (5 ± 45 U/L). Other hepatic enzymes were normal. A CBC with differential revealed WBC=15.63 (4 ± 11.0 K/uL) with neutrophils 59% (40 ± 70%), lymphocytes 27% (22 ± 44%), eosinophils 11% (0 ± 4%), monocytes 2% (0 ± 7%), and atypical lymphocytes 1%. Carbamezepine level= 9.0 (8 ± 12 ug/mL) Blood and urine cultures were normal. Titers for Epstein-Barr virus and cytomegalovirus were normal. Given the rash, pyrexia, eosinophilia, lymphadenopathy and recent treatment with anticonvulsants the patient was diagnosed with anticonvulsant hypersensitivity syndrome (AHS). Progressive resolution of the dermatological changes and hepatitis was noted with discontinuation of anticonvulsants and treatment with corticosteroids for 1week. DISCUSSION: AHS is an adverse drug reaction associated with medications such as phenytoin, carbamezepine and phenobarbital. Originally cited prevelance of 15% is increasing with broader spectrum of use of anticonvulsants, such as for treatment of neuropathic pain. This patient exhibited a typical presentation including hepatitis and eosinophilia. The pathogenesis of AHS involves inadequate function of epoxide hydroxylase enzyme, leading to decreased inactivation of toxic metabolites of anticonvulsant drugs. Current data indicates allergic cross-reactivity between these medications to be as high as 80%. Treatment involves discontinuing the medication and initiating steroid therapy. With a mortality rate suggested at 40% (mostly due to hepatic necrosis), it is important for general practitioners to recognize AHS; its presentation, cross-reactivity of medications, and treatment options. A high index of suspicion based on the patient's history and exam will enable an astute primary care provider to make the diagnosis early.\nHYPERPARATHYROIDISM IN PREGNANCY. R. Khurana 1 ; 1 University of Alberta, Edmonton, AB LEARNING OBJECTIVES: 1) Recognize the changes in calcium metabolism that occur with pregnancy. 2) Manage hyperparathyroidism in pregnancy. CASE INFORMATION: A 38-year old pregnant woman, G4P1 at 21 weeks gestation was seen for assessment of hypertension. Her blood pressure had been normal at the first prenatal visit, but had started to rise and had been high for 4 weeks. She was asymptomatic. Past medical history was significant for two miscarriages and one full-term pregnancy, all in Nigeria. There was no prior history of hypertension. Her only medications were prenatal vitamins. Physical exam was normal other than a blood pressure of 168/94 and a grade II/VI ejection systolic flow murmur. Laboratory screening for preeclampsia was normal. Laboratory screening for secondary causes of hypertension revealed hypercalcemia at 2.79 mmol/L (11.16 mg/dl) with an albumin of 36 g/L (3.6 mg/dl). An elevated ionized calcium confirmed hypercalcemia. A 24 hour urine calcium was elevated at 9.0 mmol/24h (360 mg/24h). 25-OH Vitamin D levels were normal, but her Parathyroid hormone (PTH) level was elevated at 15.0 pmol/L (normal 1.1 ± 6.8 pmol/L). She was told to keep herself well hydrated with oral fluids and started on oral phosphate 500 mg bid. A 24 hour urine for metanephrines was ordered to rule out pheochromocytoma as part of a multiple endocrine neoplasia (MEN) syndrome in combination with the hyperparathyroidism. It was normal and she proceeded to exploratory surgery with removal of a right superior parathyroid adenoma. Post-operatively, her calcium levels normalized. DISCUSSION: Primary hyperparathyroidism is rare during pregnancy. It may be masked because total calcium levels decrease secondary to a decrease in albumin from the volume expansion that occurs with pregnancy. However, ionized calcium levels remain unchanged despite decreases in PTH levels. This is likely due to the effects of PTH related peptide (PTHrp) which is found to be increased during pregnancy. PTH-rp may also help regulate fetal calcium levels which are higher than maternal levels. Maternal hyperparathyroidism can cause significant morbidity for the fetus and neonate. There is an increased risk for stillbirth, intrauterine growth restriction, premature labor and neonatal hypocalcemic tetany. The diagnosis is often made on a retrospective basis postpartum after the development of neonatal tetany. Surgical management is usually preferred as it causes less morbidity than medical management. However, hyperparathyroidism can be associated with pheochromocytoma in Type 2 MEN and this should be ruled out prior to surgery.\nA. Kim 1 , Y. Braver 1 , A. Jaffer 1 ; 1 The Clevelend Clinic Foundation, Cleveland, OH LEARNING OBJECTIVES: 1) Recognize the clinical presentation of diabetic neuropathic arthropathy. 2) Review the management of acute-onset Charcot arthropathy. CASE INFORMATION: A 51-year old African American female insulin-dependent diabetic of 12 years presented with a 2 month history of right lower extremity (RLE) swelling. This was associated with erythema, warmth, and minimal pain. She also reported a 3-day history of lowgrade fever and generalized weakness. She had previously been treated with oral antibiotics for presumed cellulitis with no improvement. Diagnostic studies at that time revealed a normal WBC of 10 K/cmm with an elevated sedimentation rate (WSR) of 31 mm/hr. X-ray of the right foot/ankle showed soft tissue swelling without evidence of bony destruction. Lower extremity duplex revealed no deep vein thrombosis. CT scan of the extremity revealed only subcutaneous edema. A subsequent right ankle joint aspiration was performed which was negative for crystals and infection. The physical exam on admission was notable for an afebrile, obese female with 2+ pitting edema extending from the dorsal aspect of the right foot to the knee associated with warmth, erythema, and tenderness. There were no areas of skin breakdown or ulcers. Decreased sensation to pain, temperature, and proprioception was noted. The patient was admitted for cellulitis and started on IV antibiotics. Laboratory studies showed WBC of 8 K/cmm with an elevated WSR of 64 mm/hr and a CRP of 3.8 mg/dl. Blood cultures were negative. The patient remained afebrile with mild improvement of her symptoms. A MRI of the extremity revealed changes in the midfoot and forefoot consistent with an acute neuropathic arthropathy. Orthopedic consultation fitted the patient with a total-contact cast, instructed her on no weight bearing in the RLE with follow-up in 4 weeks. DISCUSSION: Diabetes mellitus is now the most common cause of neuropathic (Charcot) arthropathy. The foot and ankle are the most commonly affected joints. Unilateral swelling, warmth, and erythema are typical clinical findings and it is usually painless. The pathogenesis of this disease is unclear but may be related to a combination of peripheral neuropathy and neurovascular insufficiency allowing repetitive microtrauma. This can result in chronic destruction of bones and joints ultimately resulting in deformities and disabilities. In early disease, it may be difficult to differentiate from cellulitis, osteomyelitis, septic arthritis, crystalline arthritis, and osteoarthritis. Therefore, having a high clinical suspicion and obtaining studies such as MRI or In-111 labeled leukocyte scan can help make a prompt and accurate diagnosis so that aggressive immobilization can be initiated.\nNOT JUST A RASH. B. Kinder 1 , P. Basaviah 1 ; 1 University of California at San Francisco, San Francisco, CA LEARNING OBJECTIVES: 1) Recognize the clinical features of Anticonvulsant Hypersensitivity Syndrome (AHS). 2) Manage a potentially fatal adverse drug reaction. CASE INFORMATION: A 36 year old Caucasion female reported a four day history of a progressive maculopapular rash associated with a mouth ulcer. On day 2 of the rash, she developed mucosal sloughing and presented to an emergency room (ER). At that time she denied drug allergies or use of new detergents or skin products. Of note, three weeks prior to this presentation, the patient had been prescribed phenytoin by her psychiatrist for treatment of refractory bipolar affective disorder. In the ER she was treated with hydroxizine, prednisone, and analgesics and told to discontinue the phenytoin. Progression of her skin lesions to blisters over the next two days prompted a medical admission. Exam revealed a temperature of 38.2, pulse of 100, injected, desquamating conjunctivae, oral and vaginal erosions, a confluent maculopapular rash involving her face and trunk, and no hepatosplenomegaly or lymphadenopathy. Data were remarkable for a WBC of 2.4, AST of 70, and ALT of 81. Intravenous hydration, methylprednisolone, morphine, diphenhydramine, and local wound care were initiated. The rash progressed to bullae affecting her face and upper body,were limited to 10 ± 15 % of body surface area, and gradually improved over several days. Her elevated transaminases normalized within twenty-four hours of steroid initiation. Steroids were tapered. The patient was discharged with outpatient follow-up. DISCUSSION: AHS is a potentially fatal drug reaction induced by arene oxide-producing anticonvulsants, including phenytoin, carbamazepine, and phenobarbital. It occurs in 0.01%-0.1% of the population and is fatal in 5 ± 50% of cases if toxic epidermonecrolysis (TEN) or hepatitis develop. The mechanism apparently involves an inability to detoxify arene oxide metabolites which then bind to proteins and elicit an immune response. AHS may be genetically determined, conferring increased risk to siblings. The hallmark clinical features include fever, rash, and lymphadenopathy which can be associated with hepatitis, hematologic abnormalities, and interstitial nephritis. The skin eruption typically begins as a morbilliform rash involving the upper trunk, face and upper extremities. The cutaneous manifestations can be variable, including target lesions, bullae, and skin sloughing and mucosal involvement resembling Stevens-Johnson Syndrome and TEN. AHS usually occurs 2 ± 6 weeks after initial drug exposure, later than most other skin reactions. Steroid therapy is controversial and has not demonstrated benefit in randomized control trials. However, some experts recommend steroid use if there is evidence of hepatic or renal involvement and if skin desquamation is limited. Essentials of management include prompt discontinuation of the offending drug, supportive management, avoidance of cross-reactive drugs in the future, and counselling regarding potential genetic implications to family members. LEARNING OBJECTIVE: To recognize that in the face of renal dysfunction, azotemia may reflect non-renal causes. CASE INFORMATION: A 76 year-old white male presented with shortness of breath and hypoglycemia. He had vomited that morning and had nothing further to eat or drink. His history was significant for diabetes mellitus (27 years), COPD, CHF, and stable chronic renal insufficiency (BUN 54, creatinine 1.6). The patient' home medications included aspirin qd, acetominophen #3 prn pain, potassium 20 mg qd, glyburide 2.5 mg qd, furosemide 20 mg bid, prednisone 10 mg qd, and lisinopril/HCTZ 20/25 mg qd (started three weeks previously). Physical exam revealed bilateral wheezing. Lab studies showed BUN 93, creatinine 3.2, Hgb 9.4, U/A showed 2+ protein and no casts. On ultrasound, the kidneys were normal size with no hydronephrosis. The patient had good urine output. The patient was admitted with an exacerbation of COPD, hypoglycemia and renal failure. Hypoglycemia was considered to be secondary to inadequate clearance of glyburide and poor oral intake. Glyburide, furosemide, and lisinopril were held. The patient was treated with albuterol, ipratropium, fluticasone, salmeterol inhalers and IV solumedrol for the COPD exacerbation. Over several days the BUN increased to 115 while the creatinine declined to 1.9 . Because of persistent azotemia, other non-renal causes were considered. Colonoscopy revealed several large, hyperemic polyps which were removed. Eventually the BUN declined to 46 and the creatinine returned to baseline. DISCUSSION: This case illustrates the multifactorial nature of illnesses in the elderly and emphasizes the importance of considering alternate or confounding hypotheses when making a diagnosis. Although this patient had underlying renal failure, focusing on this alone would have missed such potentially life-threatening problems as dehydration, steroid-induced azotemia, and GI bleeding from colonic polyps, all of which could have contributed to this patient' condition.\nA HIGH RISK EXPOSURE TO MENINGOCOCCAL MENINGITIS. L.M. Kosseim 1 , C.M. Stoltz 1 ; 1 University of Pennsylvania, Philadelphia, PA LEARNING OBJECTIVES: 1. Recognize patients at high risk for meningococcal meningitis 2. Understand options for prophylaxis for meningococcal meningitis 3. Identify candidates for the meningococcal vaccine CASE INFORMATION: A 20 year-old male college student made an urgent appointment at his student health service for evaluation of possible meningococcal meningitis. He had returned the day before from a 14 day trip to Israel with other college students. A student with whom he had spent time in Israel became ill prior to boarding the flight home. This contact student died from meningococcal meningitis within hours of landing in the United States. The patient was advised by the airline company to see his physician for evaluation. History was remarkable for sharing a drinking glass at a party with the young man who later died. A thorough physical exam was unremarkable. Because of his potentially high risk exposure to meningococcus, the patient was given rifampin 600 mg PO bid for two days and counselled on the symptoms of meningococcal meningitis. He was also advised to receive the meningococcal meningitis vaccine. As this patient had been on a school trip, the other students from his group were also counselled and given prophylaxis. Additionally, the student health service used this as an opportunity to educate the student body on the availability of the meningococcal vaccine and on recognizing the signs and symptoms of meningitis. DISCUSSION: Meningococcal meningitis is a rare but often deadly infection affecting approximately 2,800 Americans annually. Chemoprophylaxis with antibiotics is recommended for all close contacts of an index case of meningococcal meningitis. Close contacts include household members, daycare workers, and anyone directly exposed to the case patient's oral secretions. Internists should be comfortable prescribing chemoprophylaxis for meningococcal meningitis and also know which patients are advised to get the meningococcal vaccine. The Centers for Disease Control has recently changed its guidelines, and now advises that all college students be aware of the increased risk of meningococcal meningitis in this population and in the availability of the meningococcal vaccine. CASE INFORMATION: A 32-year-old female hotel banquet manager with no signficant past medical or surgical history presented with nausea, diffuse myalgias, clear rhinorrhea, and frontal headache. She took oral contraceptives and just finished her menses. The patient was afebrile and had frontal sinus tenderness. Her physician prescribed trimethoprimsulfamethoxazole for sinusitis. The patient stopped the sulfa after five days because of worsening nausea with diarrhea. She also developed arthralgias of the knees, ankles, and elbows. Two days later she returned to the doctor with a maculopapular rash over her anterior shins with a few urticarial lesions. The elbows appeared discolored. The knees were warm, tender, and swollen. The tissue around the ankles was tender, but they were not swollen or warm. A serum sickness like reaction secondary to sulfa was suspected. A CBC and LFTs were normal, and an antihistamine was prescribed. Over the next week her condition worsened with fever, more extensive arthralgias (now including the hips and wrists) and the development of tender nodules along the anterior tibias and elbows characteristic of erythema nodosum. A chest radiograph showed right hilar and mediastinal lymph nodes. Prednisone 20 mg. daily was begun with rapid improvement of the arthritis, erythema nodosum, fatigue, and fever. The prednisone was tapered over the next month. A CXR three months later showed resolution of the hilar and mediastinal adenopathy. Nine months later the patient remains well without recurrence. DISCUSSION: This case illustrates that some episodes of``serum sickness\" are due to an underlying disease for which an antibiotic is used and then blamed. In retrospect, the symptoms of fatigue, myalgias, and URI that lead to the antibiotic prescription were actually the prodrome for acute erythema nodosum (EN). While half of cases of EN are idiopathic, it is important to rule out secondary causes. Infectious etiologies can include streptococcal pharyngitis, non-streptococcal URIs, TB, intestinal pathogens, and systemic fungi such as coccidiomycosis. Drugs such as sulfonamides and oral contraceptives can precipitate EN. One of the most common etiologies of secondary EN is Lo È fgren's syndrome, a triad of EN, bilateral hilar adenopathy, and arthritis. If EN is not clearly due to an infection, medication, pregnancy, or inflammatory bowel disease, a CXR and PPD should be performed. LÐfgrens is an acute, self-limited form of sarcoidosis, most common in young women. EN is a good prognostic sign, in which case an invasive diagnostic procedure for the hilar adenopathy is not necessary. NSAIDs are the treatment of choice; corticosteroids are indicated for severe, debilitating arthritis. A 51-year-old man was seen in the Emergency Department for one week of sore throat, fever, and hoarseness; he was diagnosed with a respiratory virus. Two days later, he was evaluated in a primary care clinic for persistent fever, fatigue, and a new, nonpruritic rash. He was ill-appearing and had a maculopapular rash over his upper back, chest, and thighs; anterior cervical lymphadenopathy was present. Laboratory data revealed WBC 4.2, and a negative monospot and throat culture. HIV testing was obtained two days later when risk factors were identified. Intial HIV antibody was negative, but his viral load was 300,000. His symptoms resolved after two weeks. One month later, his HIV antibody test became positive. CASE 2: A healthy, 35-year-old man was seen for urethral discharge. History revealed recent unprotected intercourse with a new partner. Urethral culture was negative but urine PCR was positive for Chlamydia trachomatis. He was treated with a one-time dose of azithromycin. Two days later he developed a rash over his face and trunk accompanied by chills, fatigue, and headache. He was afebrile with a diffuse maculopapular rash over his face, chest, and abdomen. Although a drug reaction was suspected, HIV serologies were sent. His HIV antibody was indeterminate but his viral load was over 500,000. DISCUSSION: Acute HIV infection is often underdiagnosed, but should be routinely considered in clinical care. More than 80% of people with acute infection seek medical care for their symptoms, yet the diagnosis is considered less then 25% of the time. Nonspecific symptoms such as fever, myalgias, rash, and lymphadenopathy are typical. Initial testing should include HIV antibody as well as HIV RNA viral load or HIV DNA. Early treatment decreases viral burden as well as improves, and perhaps restores, host immune responses. HIV is highly contagious during the acute infectious stage. Early diagnosis is critical in decreasing HIV transmission and has significant impact on public health, treatment, and possibly prognosis. Because the majority of patients present to primary care physicians, they have an important role in recognition of early infection. Further education regarding symptoms and serologic testing, as well as screening strategies, may be needed to increase awareness of acute infection in primary care settings.\nAN UNUSUAL CASE OF HYPERTENSION. S. Kripalani 1 ; Emory University, Atlanta, GA LEARNING OBJECTIVES: 1) Explore causes of secondary hypertension in a young man. 2) Recognize the appearance and pathophysiology of Page kidney. CASE INFORMATION: A 27 year-old narcotics agent presented for evaluation of previously untreated high blood pressure. Manual readings over the past year ranged from 140 ± 151/96 ± 110. He complained of daily bifrontal headaches, as well as occasional palpitations and diaphoresis, but only when working in high-stress situations. Review of systems was otherwise negative, as were past medical history and medication use, including OTC products. He denied smoking and illicit drug use, but he used chewing tobacco and had 2 ± 3 alcoholic drinks per week. He used to play high school football and had been in a car accident years ago, with no apparent injury. His father received a heart transplant for idiopathic cardiomyopathy. Family history was otherwise negative. Blood pressure was 162/108 in the left arm, 152/104 in the right arm, and 154/106 in the right leg. Heart rate was 64, height 71 inches, and weight 196 pounds. He appeared comfortable and well-developed. Physical examination was unremarkable. EKG, chest x-ray, blood count, basic chemistry panel, TSH, urinalysis, and drug screen were normal. The patient was started on amlodipine, to avoid interfering with anticipated testing, and his blood pressure responded appropriately. Initial 24-hour urine catecholamines were elevated, prompting a CT scan to help localize a potential pheochromocytoma. The CT showed normal adrenals, but unexpectedly, the left kidney had a 5x5x11cm subcapsular fluid collection, known as Page kidney. An MRI/MRA confirmed the CT findings and demonstrated normal vasculature. Renin levels were slightly elevated Ð supine 1.7 ng/mL/hr (0.2 ± 1.6) and upright 4.9 ng/mL/hr (0.5 ± 3.3) . Cortisol, aldosterone, and upright aldosterone/renin ratio were within normal limits. Head CT and repeat urine studies were normal. Given the imaging results, the initial catecholamine elevation was deemed a false positive. DISCUSSION: Potential causes of secondary hypertension included renal parenchymal and renovascular diseases, coarctation of the aorta, mineralocorticoid excess (either through hyperaldosteronism or use of chewing tobacco or licorice), renin-producing tumors, pheochromocytoma, Cushing's syndrome, congenital adrenal hyperplasia, substance abuse, exogenous hormones or sympathomimetics, sleep apnea, hypo-and hyperthyroidism, hyperparathyroidism, acromegaly, carcinoid, and increased intracranial pressure. Page kidney is rare cause of hypertension resulting from compression of the renal parenchyma. High blood pressure is mediated by elevated renin levels, which are thought to be secondary to local ischemia or parenchymal scarring. In the model which he described in 1939, Page used cellophane wrapping to constrict the kidneys. However, most cases in clinical practice are due to a subcapsular hematoma resulting from blunt trauma, usually associated with contact sports or an accident. If blood pressure cannot be controlled medically, percutaneous drainage or nephrectomy can be curative.\nNOT``JUST ANOTHER GI BLEED''! A. Krishnamurthy 1 , R. Ruffner 1 , D. Weber 1 ; 1 University of Pittsburgh Medical Center Shadyside, Pittsburgh, PA LEARNING OBJECTIVES: 1) Consider small bowel tumors in the evaluation of GI bleeding with the upper endoscopy and colonoscopy are negative. 2) Small bowel series, enteroclysis and enteroscopy have a role in their diagnosis. CASE INFORMATION: 84-year-old Asian male presented with his second episode of melena. He also complained of``fullness of the abdomen after meals''. He had no nausea, vomiting, abdominal pain, constipation or diarrhea. The patient had been admitted two weeks before with melena and anemia requiring blood transfusions. He had both upper DI endoscopy (showed mild esophagitis) and colonoscopy (showed two polyps). PMH of hypertension. His medications included iron, famotidine and Metoprolol. He did not smoke or abuse alcohol. ROS: Negative. Examination: His temperature was 36.8, pulse 72 bpm, BP 120/60 and was not orthostatic. His conjuntiva was pale. Abdomen was soft with normal bowel sounds, no tenderness, no masses, liver/spleen not palpable. Rectal exam reveled heme positive stool. Examination of all other systems was normal. Labs: WBC 5.4, Hemoglobin 8.9, BUN 42 & Cretinine 1.2, Iron 28, TIBC 258 Ferritin 25. Small bowel entersocopy was normal upto the duodenum but the scope could not be advanced beyond the fourth part of duodenum because of narrowing. Small bowel follow through shoed an apple-core defect at the duodeno-jejunal junction. Surgical resection of lesion confirmed a 4-cm adenocarcinoma of jejunum without lympnode involvement. DISCUSSION: Small bowel neoplasms are relatively uncommon. Small bowel accounts for less than 3% of GI malignancies despite contributing to more than 90% of the surface area of GI tract. The possible reasons are:\n1. in November 1999 complaining of weight loss, bulky stools, fatigue, abdominal distention, and anorexia. Review of those hospital records revealed an extensive evaluation that yielded a diagnosis of``protein losing enteropathy\". She improved with total parenteral nutrition (TPN). Small bowel biopsy and serologies were not reported, nor was there mention of treatment with a gluten-free diet. In May 2000, the patient presented to our institution with similar complaints and worsening lower extremity edema and weight loss. Physical examination revealed an extremely cachectic woman with flat affect, hypotension, tachycardia, inspiratory crackles, hepatomegaly, and 2+ pitting lower extremity edema. Laboratory exam showed severe hypoalbuminemia, hyponatremia, and hypocalcemia, mildly elevated transaminases and alkaline phosphatase, and iron deficicency anemia. Antigliadin and antiendomysial antibodies were elevated. Histologic examination of biopsies obtained from the proximal small bowel was consistent with collagenous sprue. Due to her severe presentation, short-term corticosteroids and TPN were initiated. She improved and was discharged with instructions to continue a gluten-free diet. DISCUSSION: Adult patients with celiac sprue may present with a variety of gastrointestinal as well as extraintestinal symptoms, many of which were present in our patient. The diagnosis is usually made by histological evidence of villous atrophy in the small intestine along with symptomatic improvement on a gluten free diet. Serological tests (antiendomysial and antigliadin antibodies) further aid in diagnosis and screening of celiac sprue. Collagenous sprue was first described (in 1970) as continued malabsorption, failure of a gluten-free diet, and a subepithelial collagen layer on small bowel biopsies in a patient previously diagnosed with celiac sprue. Most published case reports of collagenous sprue have not detected these serological markers typical in celiac sprue. Recently, a relationship was postulated when serological markers were present in a patient with collagenous sprue. Our patient exhibited biopsy proven collagenous sprue, but also demonstrated positive serology for adult celiac sprue, supporting the notion that collagenous and adult sprue may be related. Whether this simply reflects an improvement of antibody assays remains to be established as more cases are evaluated. American female presented with abdominal pain, nausea and emesis for two days, and 10 lb. weight loss over a month. Physical examination revealed a cachectic female in moderate distress, hypotension, tachycardia, and diffuse abdominal pain with rebound tenderness. Laboratory exam showed an anion gap metabolic acidosis with a mild lactic acidosis, acute renal failure due to acute tubular necrosis, hyponatremia, leukocytosis with bandemia, and elevated Troponin associated with anteriorlateral ischemic EKG changes. Swan-Ganz readings indicated cardiogenic shock. Emergent cardiac catherization revealed diffuse``beading'' of small and medium coronary vessels consistent with vasculitis. Renal angiography revealed a similar picture. A primary vasculitis workup was negative. Hepatitis studies were consistent with acute HBV: + Hb SAg, À Hb SAb, + Hb Core IgM. Quantitative HBV-DNA was 6290 pg/ml. Hepatitis A, C, and D studies were negative. Initially, high-dose parenteral steroids were started for suspected PAN, but when the HBV serologies became known steroids were discontinued and Lamivudine initiated. Hemodialysis was required. Clinically, the patient improved and was discharged home. Three months later the patient' condition deteriorated acutely, and an acute cardiorespiratory arrest lead to her death. Histological examination revealed a multisystem, segmental, necrotizing vasculitis of medium and small-size arteries (most notably the coronary and renal vasculature) in various phases of progression consistent with classic PAN. DISCUSSION: HBV-related PAN is most commonly described with chronic HBV infections. In our case, laboratory evidence indicated acute HBV infection. Cardiopulmonary involvement of PAN is fairly common, but rarely associated with cardiogenic shock. Traditional treatment of PAN cannot be utilized when PAN is associated with an acute HBV infection. Good therapeutic outcomes have been reported with immunosuppressive agents along with plasma exchanges, or treatment with two weeks of high-dose steroids followed by immnuosuppresives and plasma exchanges. \nA 45 year old woman with a history of hypertension, s/p total hysterectomy was admitted for unstable angina. She had a positive stress test and underwent a left heart catheterization (LHC) during her hospitalization that was normal. Her catheterization site was sealed with a vascular sealing device and was without external hematoma. Her precatheterization Hgb was 13.3 mg/dL. One day post-LHC, she complained of dysuria. A urinalysis was normal and she was discharged for outpatient evaluation of her chest pain. The patient developed worsening dysuria, frequency, and urgency and was prescribed antibiotics over the telephone for the treatment of a presumed UTI. Her symptoms improved but did not resolve and she was seen in the office 2 weeks later now also complaining of new constipation. Examination revealed normal vitals, a shuffling gait and rebound tenderness on abdominal exam. Pelvic exam revealed a tender, non-pulsating mass in the right lower quadrant. The catheterization site was identified as above the inguinal ligament and did not show any external signs of hematoma. An ultrasound and CT of the pelvis and abdomen revealed a 12Â7Â8cm mass overlying the iliac artery consistent with a hematoma and blood tracking up the right paracolic gutter. The bladder and colon were compressed by the mass. Doppler studies did not reveal active arterial flow from the iliac arteries. Her hemoglobin was 9.6 mg/dL. The patient was admitted for supportive care and her symptoms improved. DISCUSSION: Cardiac catherization is a common procedure with serious potential complications including a 1% risk of vascular injury including intraperitoneal bleeding. Risk factors for complications include female gender, congestive heart failure, percutaneous transluminal coronary angioplasty, valvuloplasty and peripheral vascular disease. The recent increased use of vascular sealing devices may decrease the risk of hemorrhage from anterior arterial perforation, but will not provide hemostasis from a posterior wall puncture. Thoughtful surveillance for post-LHC complications is essential. Management of peritoneal hematomas should include serial hemoglobins, diagnostic imaging and surgical consultation when necessary. In this patient, her post-LHC bleeding was misdiagnosed as a urinary tract infection. The Internist must recognize that post-LHC complications may masquerade as more common pathology leading to delayed diagnosis and potentially poor outcomes. presented to an outpatient clinic seeking primary care. Her complaints were depression and high blood pressure. Her primary language was Korean, but she spoke moderately fluent English. On exam she was oriented to person and place, but was hard to interview as she shifted from topic to topic. Depressive symptoms were not present, nor were symptoms of psychosis. Over the course of a few months she came often but erratically to clinic with vague complaints. She often discontinued her medications without any clear reasons. The nurses noted that although they understood her English, they could never tell what she wanted. They wondered if she was schizophrenic. After struggling to understand her clearly a Korean interpreter was obtained. This interpreter said that in Korean she was difficult to understand as well. After psychiatric evaluation which confirmed lack of mental illness, neuropsychometric testing was done with an interpreter. This revealed mental retardation with developmental disability especially in areas of language. DISCUSSION: Mental retardation is often defined by a composite IQ of less than 70. Developmental disabilities are very prevelent and encompass overt mental retardation as well as less pervasive cognitive deficits. As in this case, these patients can have difficulty with reading and communicating clearly, which can be a barrier to effective healthcare. Most people in the US are screened for this in school or childhood clinic visits. Many adult immigrants likely were not screened. When this diagnosis is known accomodation can be made to improve compliance and effective communication. Also, a diagnosis of mental retardation may qualify a patient for government benefits and outpatient social services. This case highlights the need to diagnose developmental disabiltiy and differentiate it from mental illness especially in adult immigrants. To recognize the preliminary management for IPA. CASE INFORMATION: A 28 year old female presented requesting prenatal vitamins and offered no complaints. PMH was significant for exploratory laparoscopies for abdominal pain and endometriosis. The patient was in graduate school and had been married for 1.5 years. When asked``do you always feel safe at home?'', she was noted to hesitate, whereas on previous questions, she answered rapidly. This question was immediately followed by``does your husband ever hit you, yell at you, or make you feel afraid?'' She replied that while she knows her husband loves her, on occasions he does get``upset''. They have frequent arguments, during which he breaks furniture and hits her. She stated``he never hits me hard'', he``always apologizes immediately'', and his hitting has``never gotten out of hand''. She denied having a plan of action should that happen. There were no weapons in the house. Patient was immediately referred to a psychologist for counseling and who helped her to formulate a safety plan. DISCUSSION: Intimate partner abuse (IPA) is common, with an estimated prevalence of 5 ± 20% women per year. It is defined as a pattern of intentionally coercive and violent behavior toward an individual with whom there is or has been an intimate relationship. These behaviors can be used to establish control over an individual and include physical and sexual abuse, psychological abuse with verbal intimidation, progressive social isolation or deprivation, and economic control. IPV transcends social classes and so all women should be asked about safety in their current setting as part of routine primary care and when presenting for emergency care with traumatic injuries. A screening question can be``have you been hit, kicked, punched, or otherwise hurt by someone within the past year? If so by whom?'' The physician must then make an assessment of the patient's safety, ascertaining the presence of weapons in the house, violence that is increasing in severity or intensity, threats to kill the woman or her children, and the woman having told her partner she is``planning to leave.'' It is important to give the patient validation and support and help her to develop a safety plan. Physicians should be aware of resources within their institution, which include social workers or domestic violence advocates. If these are unavailable the physician can provide counseling and give referral to a local domestic violence hotline. A 32 year old woman presented with bright red blood per rectum associated with tenesmus and urgency. She then developed left lower quadrant pain. There was no fever, rash, eye or joint pain. There was no foreign travel, unusual food intake, or history of antibiotic, NSAID or oral contraceptive use. Her past medical history was unremarkable. She had no known drug allergies, used alcohol on occasion, and did not use illicit drugs. Examination revealed postural hypotension, scant bowel sounds, and tenderness in the left lower quadrant. Rectal exam showed gross blood. Investigations revealed hemoglobin 141, platelet count 270, and wbc of 14.7. Electrolytes, INR, PTT, liver profile, amylase, and beta-HCG was negative. Abdominal films were normal. Micrologic examination of the stool was negative. Colonoscopy showed erythematous, friable mucosa from 75 cm (mid descending colon) to 35 cm distal to the anus. Pathology revealed patchy disease with inflammatory exudates on the surface, consistent with ischemic colitis. No crypt abscesses or granulomas were identified. A vasculitic workup was performed, with negative ANA, pANCA, cANCA, and normal complement levels. Hepatitis B and C serology were negative. Antithrombin III, Protein C, Protein S, homocysteine, Factor V Leiden genotype, and anticardiolipin antibody were all normal. Her diarrhea improved within 3 days of admission with conservative treatment, and she was discharged on ASA. She has had no recurrence. DISCUSSION: Internists frequently consider ischemic colitis in the differential diagnosis of the elderly patient who presents with bloody diarrhea. However, ischemic colitis in young patients is more common than realized. Case series in younger patients have described an increased risk in women, and a lack of vascular risk factors, when compared to the typical older patient with ischemic colitis, who has multiple vascular risk factors. Etiologic agents that have been implicated include estrogen, collagen vascular disease, diabetes, hypercoagulable states, cocaine use and even long distance running. It is also common to find no identifiable etiologic cause. In an analysis of 68 consecutive cases diagnosed by early colonoscopy, 23 (34%) of the patients were younger than 50 and nineteen of those were women. Habu found that the associated factors for ischemic colitis were an increased incidence of chronic constipation and a prior history of abdominal surgery. Our case illustrates the importance of consideration of ischemic colitis in the differential diagnosis of bloody diarrhea in the younger patient.\nBATS AND RABIES: IT DOESNT HAVE TO BE A BITE. D.S. Lindes 1 ; 1 University of California, San Francisco, San Francisco, CA LEARNING OBJECTIVES: 1) Recognize the risk of rabies from non-bite exposure to bats. 2) Provide post-exposure prophylaxis against rabies infection. CASE INFORMATION: A 40 year old woman living in the San Francisco Bay Area presented to her primary care physician with concern about the risk of rabies from contact with a bat. Three weeks prior to presentation, she had found a bat flying around in her living room. Her two cats had tried to catch it, without success. She used a window screen, held in front of herself and above her head, to gradually guide the bat towards an open window. The bat collided with this screen several times before it flew out the window, and the patient spent approximately 45 minutes in the room with the bat. She was not bitten, nor did she recognize any direct contact with the bat's saliva. At the time of presentation, she had no signs or symptoms suggestive of rabiesvirus infection. DISCUSSION: Rabies is a rare but nearly always fatal viral infection. Its clinical course is characterized by a nonspecific prodrome, with associated paresthesias or fasciculations at the inoculation site, followed by an encephalitis with agitation, confusion, hyperesthesia, autonomic dysregulation, paralysis, and progressive brainstem dysfunction. Death generally results from respiratory center depression. Wild animals, including racoons, skunks, and bats, are the principal vectors for human rabies. Since 1980, 21 (58%) of the 36 human rabies cases diagnosed in the United States have been associated with bats. Of these cases, there was definite history of a bite in only one or two. In another 10 ± 12, contact but no bite was reported; the other 7 ± 10 cases had no clear contact, but an undetected bat bite was the most plausible route of exposure. Rabid bats have been documented in all 49 Continental U.S. States. Current Centers for Disease Control and Prevention guidelines recommend consideration of post-exposure prophylaxis (PEP) against rabies whenever direct contact between human and bat has occurred, unless the exposed person can be certain that there was no bite, scratch, or mucous membrane exposure. Rabies PEP consists of the following measures: 1) thorough cleansing of all wounds with soap and water, as well as povidone-iodine irrigation if possible; 2) administration of rabies immune globulin, 20 IU/kg, with as much as possible infiltrated around the wound(s) and the remainder given intramuscularly at a site distant from vaccine administration (only needed for individuals not previously vaccinated); 3) rabies vaccine (human diploid cell vaccine [HDCV] , rabies vaccine adsorbed [RVA], or purified chick embryo cell vaccine [PCEC]), 1.0 ml administered intramuscularly on days 0, 3, 7, 14, and 28 . Because incubation periods of over one year have been reported, PEP is indicated regardless of the amount of time after exposure. If at all possible, the involved animal should be collected for rabiesvirus testing. In the above case, because of prolonged contact and possible mucous membrane exposure, the woman underwent PEP, as did her previously unvaccinated cats. Recognition of the significance of non-bite exposure to bats is important for primary care physicians because they are likely to encounter patients with such exposures, many of whom may be candidates for post-exposure prophylaxis.\nPERIPARTUM CARDIOMYOPATHY: DIAGNOSIS AND IMPLICATIONS. C.L. Long 1 , R. Deversa 1 ; 1 University of Tennesee College of Medicine-Chattanooga unit, Chattanooga, TN LEARNING OBJECTIVES: 1-Identify theories regarding etiology of peripartum cardiomyopathy. 2-Recognize physical findings of peripartum cardiomyopathy. 3-Recognize the morbidity and mortality associated with peripartum cardiomyopathy. CASE INFORMATION: A 23 year old caucasian female, G 1 P1, who is status post caesarean section two weeks prior presented with increasing shortness of breath. She had elevated JVP, bibasilar crackles, and a S3 gallop. ECG showed no acute changes. CXR revealed cardiomegaly with pulmonary venous congestion. An echocardiogram revealed a dilated left ventricle and an ejection fraction of 15%. Despite appropriate treatment during admission, patient became hypotensive, and went into asystole. Aggressive resuscitation was unsuccessful. DISCUSSION: Peripartum cardiomyopathy is defined as the development of heart failure in the last month of pregnancy or within five months after delivery without any determinable cause and without previous heart disease before the last month of pregnancy. The etiology of peripartum cardiomyopathy is unknown. Some actually question whether this is a distinct disorder or another form of idiopathic cardiomyopathy. Several ideas have been proposed including an immunologic response to the fetus, increased hemodynamic load of pregnancy, nutrition, and hormone response. Several studies have actually shown that these patients have a high incidence of myocarditis, possibly implicating a viral trigger like enterovirus. Signs and symptoms are typical for congestive heart failure. However, these can sometimes be confused with normal pregnancy. Therefore, peripartum cardiomyopathy may not be diagnosed until postpartum. Evaluation would include ECG, chest xray, and an echocardiogram. Cardiac catheterization should be considered in those at risk for atherosclerosis. Endomyocardial biopsy remains controversial. Serum can be tested for bacterial and viral cultures and Coxsackie B virus titers. Therapy consists of digoxin, diuretics, sodium restriction, and afterload-reducing agents. Thromboembolic phenomena are common and anticoagulation should be strongly considered. Cardiac transplantation may be considered as well. The mortality rate ranges from 25 ± 50%. Nearly half of these deaths occur within the first 3 months postpartum. Death is often caused by chronic progressive congestive heart failure, an arrhythmia, or a thromboemolic event. If the patient's heart is to recover, it willusually do so within the first 6 months. If ventricular function does not improve, it is often recommended that the patient not become pregnant again due to the high risk of death.\nWHEN ANTIBIOTICS DONT WORK, TIME TO THINK BEYOND BACTERIUM. S. Malhi1 1 , K. Gopal 1 ; 1 Fairview Hospital, Fairview Park, OH LEARNING OBJECTIVES: 1) Work up of non resolving pulmonary infiltrate. 2) Recognize the clinical manifestations of Disseminated Histoplasmosis. 3) Suspecting HIV disease in the presence of opportunistic infection CASE INFORMATION: A 54 yrs. old white male presented to the ER with complaint of fever, shaking chills, myalgias, fatigue, and episodes of profound sweating. He had been sick for the past 4 weeks. His primary care physician ordered a chest radiograph that revealed an infiltrate & he was treated with Levofloxacin for 10 days. Patient continued to do poorly with fevers in the range of 100 ± 104 F and was then treated with another 7 ± 10 days of antibiotics. He lost 20 lb. of weight and hence presented to the ER. His other medical problems included hypertension and remote history of alcohol abuse. He denied smoking, current alcohol or drug use. He was divorced, had 3 children and worked as a social worker. Physical exam was remarkable for a fever of 102 F and mild tachycardia. A CXR on admission showed worsened left lung infiltrate. Initial Lab studies revealed WBC 3.0, HGB 11.0, HCT 31.8, MCV 81, PLT 192 DISCUSSION: A lung biopsy may be the last recourse to obtain a diagnosis and ascertain the correct treatment for a non resolving pulmonary infiltrate. Disseminated Histoplasmosis occurs in elderly and about 5% of AIDS patients in endemic areas and is characterized by fever, malaise, pulmonary infiltrates, hepatosplenomegaly, lymphadenopathy and weight loss. Where as asymptomatic infection is most common; CXR of patients with acute infection show focal mid lung infiltrates (30%), hilar or mediastinal lymphadenopathy (30%) or both (30%).\nA COUGH THAT LINGERED: DIAGNOSING LEGIONELLA PNEUMONIA. R. Mann 1 , D.W. Brady 1 ; 1 Emory University, Decatur, GA LEARNING OBJECTIVES: 1. Diagnose legionella pneumonia from its clinical manifestations. 2. Recognize the limited utility of the legionella urinary antigen in excluding the diagnosis of legionella in a patient with a pneumonia that has been refractory to treatment. CASE INFORMATION: A 40 year old black male presented complaining of a 3-week history of cough (productive of dark yellow sputum), fevers, chills, nightsweats, and malaise. He initially was treated at an out of town facility with a 5 day course of azithromycin, with some improvement in symptoms; however, within 2 ± 3 days after completing the medication, the symptoms returned. He presented to a second emergency room, where he received a chest xray and was discharged home with a prescription for guafenesin. One week later, he presented again. A second chest xray revealed interval worsening of a right middle and right upper lobe pneumonia with a patchy airspace opacity in the left upper lobe consistent with pneumonia. On exam his blood pressure was 156/75, pulse 119, respiratory rate 30, temperature 36.9C, with bilateral rales throughout. Pulse ox-70% on room air ( ABG on 100% FiO2-pH 7.44; pC02 30; pO2 100); WBC count 16.5 (77%N,6%Mono,16%L), ; HIV test was negative. He was admitted and treated with IV ceftriaxone and erythromycin for the first 72 hours with worsening hypoxemia. Erythromycin was replaced with IV Azithromycin for the next 48 hours with no improvement. Levofloxacin 750mg IV qd was added to replace azithromycin to cover Legionella. A Legionella urine antigen was negative. After 48 hours of high dose Levofloxacin, the patient showed little clinical improvement. Legionella, mycoplasma, and influenza serologies were sent, as preparations were being made for bronchoscopy. On the 3rd day of high dose Levofloxacin the patient began to require lower FiO2's and subsequently the Legionella Pneumophilia IgM serology was found to be positive (1:64) . Within the next week, the patient was discharged home to complete a 21 day course of high dose Levofloxacin for treatment of Legionella pneumonia. DISCUSSION: This patient's final diagnosis was made after a protracted outpatient and subsequent inpatient course of therapy. His pneumonia ultimately was found to have been caused by Legionella pnuemophila; however, the legionella urinary antigen was negative. Clinicians should be aware that the legionella urinary antigen only tests for serogroup I of L. pnuemophila, and a negative urinary antigen does not, in fact, rule out infection caused by L. pneumophila (the most common Legionella species causing community-acquired pneumonia in the U.S.). Often, serum antibodies are needed to confirm or exclude the diagnosis.\nTHROMBOTIC THROMBOCYTOPENIC PURPURA FOLLOWING THE FLU VACCINE. A. Markarian 1 , R.D. Hobbs 1 ; 1 Oakwood Healthcare System, Dearborn, MI LEARNING OBJECTIVE: To recognize thrombotic thrombocytopenic purpura (TTP) as a potential complication of the influenza vaccine. CASE INFORMATION: A 58 year-old man with COPD, diabetes mellitus, and hypertension presented with mental status changes and oliguria. He had felt fatigued for several days and had noted a rash on his shins. His chronic medications were Metformin, Glipizide, Nitrobid, Metoprolol, Omeprazole, and Quinine. He received the flu vaccination two weeks prior to admission. He was afebrile but tachycardic. Physical exam showed flaccid paralysis, diminished deep tendon reflexes and bilateral Babinski signs. The heart, lungs and abdomen were unremarkable. A petechial skin rash was noted on both shins. Laboratory studies revealed mild anemia (Hgb 11.7), schistocytes on the peripheral smear, LDH (1927) and a low haptoglobin. The platelet count was 13,000. PT, PTT, INR, fibrinogen and fibrin degradation products were normal. The BUN was 84 and the creatinine 3.4. ANA was positive with a titer of 1:160; anti ds DNA and Lupus anticoagulant were negative. Total compliment levels were normal. SSA/RO and SSB/LA were negative. Antiglo-merular basement membrane Ab, ANCA, ACLA-IgG and IgM, and C1 Esterase inhibitor were normal. HIV was non reactive. Cultures were negative for enteric pathogens. Von Willebrand' factor activity was normal. Quinine dependant antibodies against platelet glycoprotein were negative but non drug dependant IgM antibody was positive. The patient was treated with plasmapheresis and hemodialysis and recovered. DISCUSSION: The classic pentad of TTP includes microangiopathic hemolytic anemia, thrombocytopenia, neurologic symptoms, renal disease and fever. The etiology is poorly understood although underlying immune complex formation is frequently found. Plateletassociated antibodies and serum reactivity against endothelial cells are positive in some cases. Most cases are idiopathic. Associated conditions include infections (bacterial, viral, and fungal), autoimmune diseases, AIDS, cancer, vaccinations and drugs such as quinidine, quinine and ticlopidine. Cases of TTP following influenza vaccine have been reported (Brown et al, Br Med J 1973; 2(861):303; Symmers, Br Med J 1973; 2(866) :614). In the present case, it is likely that the influenza vaccine triggered the antibody formation and immune complex deposition and led to the development of TTP. CASE INFORMATION: A 55-year old male post myocardial infarction was transferred for cardiac catheterization from a small community hospital. His past medical history per family included closed head trauma as a child, with residual cognitive impairment, hypertension, gout, and some hearing loss over the past 2 months. He has worked as a janitor for a local school for the past 13 years. He was described by his family as``slow'' and had a 6th grade education. On hospital day 6, after stabilization, his disposition became a concern. That morning, he sat in bed watching cartoons, laughing, with the sound off. When examined, he became confused and cried. His answers to questions were intermittently unintelligible. He frequently answered questions not asked. An uncle confirmed that this was his baseline, and that he had exhibited odd behavior since a child; his family had attributed it to falling off a truck and hitting his head. His primary team asked psychiatry if evaluating the level of his cognitive functioning would help, and his supervisor at work was called. She said that he was an excellent and very dependable employee as long as he was shown exactly what to do. When asked if he would follow verbal orders, she again stated that he needed to be shown what to do. Suspecting severe hearing impairment, a stethoscope was placed in his ears. With the communicator shouting into the diaphragm, he could hear clearly and responded appropriately. Audiometry on day #10 revealed complete sensory hearing loss on the right, severe loss on the left, with 10% speech discrimination on the left, which increased to 20% when allowed to lip-read. Further questioning revealed that he remembered being able to hear to some degree as a very young child. He was not aware that his family did not know how profoundly deaf he was. DISCUSSION: Chartlore, the process by which diagnoses are assigned via undocumented notes in the chart, is usually a product of loosely applied terms that, once used, tend to``stick.'' This particular example of chartlore is interesting in that the original misdiagnosis/assumption appears to have been made, not by the medical staff, but by the patient's own family, and tragically for him, perpetuated over decades, preventing its correction. Physicians must recognize the often deceptive nature of chartlore and the need to confirm history gleaned from medical records and family members. This fact is especially true when the``facts'' obtained conflict. 1) Recognize that McArdle disease is the number one glycogen storage disease diagnosed in adults, 2) Recognize the typical history of a patient with McArdle disease, and 3) Increase awareness of the disease for possible earlier detection. CASE INFORMATION: CASE: CC is a 31-year-old male who presented with a one-day history of dark urine. CC had to swim strenuously when a barge he was on sank. He then developed diffuse body aches and dark urine. He had a long history of exercise intolerance, easy fatigability, and on several occasions had dark colored urine after exertion. CC's younger brother had a similar history but his symptoms were not as severe. Meds: none, otherwise negative PMH. Pertinents on PE were BP 131/78, P 101, RR 24, WNWD male in moderate distress from diffuse whole body pain. CV: RRR with mild tachycardia, abd was normal with no HSM. The patient had diffuse muscular tenderness over the thighs, arms, forearms, anterior chest, and back. Labs BUN of 14, Cr of 1.2, AST of 461, ALT of 138, ALK PHOS of 97, LDH of 7,090, and a CK of 87,000. UA showed specific gravity of 1.015, pH 5.0, 4+ prot, 3+ blood, + LE, + NIT, 0 ± 3 RBC's, and +3 myoglobin. The patient had a biceps muscle biopsy which showed the absence of myophosphorylase. DISCUSSION: McArdle Disease or muscle phosphorylase deficiency results in limited ATP production from glycogenolysis and results in glycogen accumulation. It is the most common glycogen storage disease in adults. Inheritance is autosomal recessive, and the abnormal allele is on chromosome 11. Patients usually are diagnosed in the second or third decade but most have had symptoms since childhood. Exercise intolerance with muscle cramps, myoglobinuria, and a second wind phenomenon are common. Most patients have baseline elevated CK's. Diagnosis can be made by after ischemic exercise testing by measuring lactate levels and ammonia. Definitive diagnosis can be made by muscle biopsy and demonstrating myophosphorylase deficiency. DNA based diagnosis and carrier detection are available. Prognosis is generally good since longevity is not affected. Patients should avoid strenuous exercise and know the symptoms for seeking medical care during an exacerbation. High protein diets and oral glucose or fructose may increase exercise endurance.\nOH NO, NOT``NPO''. UNCOVERING OCCULT DIABETES INSIPIDUS. S. Mcelhattan 1 , R. Granieri 1 ; 1 University of Pittsburgh, Pittsburgh, PA LEARNING OBJECTIVES: 1. To recognize that occult diabetes insipidus can be unmasked when a patient is no longer able to maintain adequate free water intake. 2. To recognize diagnosis and discuss pathophysiology of lithium-induced diabetes insipidus. CASE INFORMATION: An 81 year-old male was admitted with aspiration pneumonia and dehydration two weeks after head and neck surgery. Postoperatively, he was maintained on nasogastric tube feedings but otherwise kept NPO. His past medical history was notable for bipolar disorder requiring lithium for 20 years. On physical, he was lethargic and orthostatic with dry mucous membranes. His sodium was 157 and creatinine was 2.6. The free-water deficit was calculated, and appropriate IVF therapy was initiated. However, despite increasing IVF administration and free-water boluses, hypernatremia and polyuria persisted. Urine osmolality was 171 and serum osmolality was 324. A water restriction test was not performed as the clinical occurrence of nephrogenic diabetes insipidus was suspected. The patient was given larger and more frequent free-water boluses with improvement. DISCUSSION: Nephrogenic diabetes insipidus (DI) is resistance to endogenous ADH at the level of the kidney. Up to 20 percent of patients on chronic lithium therapy develop nephrogenic DI. Patients generally adjust their lifestyle and maintain normal fluid balance to keep up with the polyuria. Although the etiology is not totally understood, it is thought that lithium enters the renal collecting tubule cells via a sodium channel and inhibits adenylate cyclase. Lithium may also reduce the density of ADH receptors or downregulate the expression of aquaporin-2 molecules. The diagnosis is substantiated by a water restriction test. As water is restricted, urine and plasma osmolality are measured hourly. If a steady urine osmolality is measured for three successive hours or if the plasma osmolality reaches 300 and the urine osmolality remains below normal, exogenous ADH is given. If urine osmolality increases, central DI is diagnosed but if the urine osmolality does not change, a diagnosis of nephrogenic DI is made. In our patient, it may have remained clinically silent for many years if he had not been NPO. It is essential that all patients on lithium who are made NPO be monitored for the development of hypernatremia and fluid imbalance. Abdominal pain is one of the most common ambulatory patient complaints. Its etiologies can be numerous, leaving the meticulous physician to pursue leads by the history, physical examination and laboratory or imaging tests. We present an unusual cause of abdominal pain that presented in the outpatient setting. CASE: A previously healthy 58 yearold male presented to the office with a two-day history of``heartburn.'' His abdominal pain worsened post-prandially and he noted early satiety, but denied nausea, vomiting, melena, or hematochezia. Alka-Seltzer and aspirin provided no relief. He denied usage of tobacco or illicit drugs, and reported occasional alcohol use, though none over the preceding days. His exam was significant only for mid-epigastric tenderness, without rebound or guarding. His rectal exam revealed brown colored stool, negative for occult blood. Laboratory data was significant only for a mildly elevated ALT. He was treated empirically with cimetidine for possible dyspepsia, gastritis, or peptic ulcer disease. Two days later, the patient was emergently seen for worsening abdominal pain. His exam was unchanged and laboratory data continued to be unremarkable. An urgent abdominal ultrasound surprisingly revealed acute portal, superior mesenteric, and splenic vein thromboses and an enlarged prostate. He was admitted for treatment and diagnostic work-up, which failed to reveal an underlying etiology for the thromboses. DISCUSSION: Portal vein thrombosis (PVT) is a rare condition associated with inherited hypercoagulable states, underlying myeloproliferative disorders, neoplasms, infections, and other inflammatory processes. It remains idiopathic in 8% to 15% of cases. Abdominal pain is commonly reported when thrombosis also involves the superior mesenteric vessels and produces bowel ischemia, though not present in our patient. The treatment has traditionally focused on complications, most commonly acute gastrointestinal bleeding. The role of anticoagulation therapy remains controversial, but appears to be indicated in patients with acute thrombotic events without intestinal ischemia, as was the case with our patient. While rare, PVT remains an important cause of abdominal pain. A high index of clinical suspicion is necessary for successful diagnosis.\nJ. Messler 1 , V. Nunez 1 , T. Jacobson 1 ; 1 Emory University, Atlanta, GA LEARNING OBJECTIVES: 1. Learn the differential diagnosis of pulmonary cavities in HIV patients. 2. Recognize the importance of bronchoscopy for diagnosis. 3. Recognize the various pulmonary manifestations of cryptococcus. CASE INFORMATION: A 41yo homeless man presents to the walk-in clinic with a chief complaint of cough. He complains of blood-tinged sputum for two months, worsening over the past three weeks. He denies fever or chills but reports night sweats, dizziness, decreased appetite, and weight loss. Except for an episode of``bronchitis'' treated 2 months earlier, his past medical history is unremarkable. A former intravenous drug user, he has lived in and out of shelters for three years. A recent PPD was negative. He admits to multiple, unprotected bisexual contacts in the past year. He has never been tested for HIV. On admission, his temperature is 39.1C, pulse 98, respirations 16, and blood pressure 100/60, sitting and lying down. Significant findings on his exam include bitemporal wasting, pale conjunctiva and oral thrush. Chest x-ray on admission reveals a 3cm cavity in the right posterior upper lobe. His PPD is negative and sputum cultures for AFB are negative. He tests positive for HIV with a CD-4 count of 6mm3. Bronchoscopic washings grow budding yeast. Serum cryptococcal antigen returns positive at a titer of greater than 1:512. The opening pressure from the lumbar puncture is normal. Cerebrospinal fluid (CSF) analysis contains 4 WBC, glucose 40, and protein 50. CSF yields a positive india ink stain and CSF cryptococcal antigen titer is positive. Subsequently, he begins therapy for cryptococcal pneumonia and cryptococcal meningitis. DISCUSSION: In HIV patients, the differential diagnosis for pulmonary cavities is extensive. The most common etiology is tuberculosis. However, as the CD-4 count falls below 200mm3, tuberculous cavities decrease in prevalence. Various other etiologies include atypical pneumocystis presentation, toxoplasmosis, invasive fungal infections, bacterial infections, and neoplasms including Kaposi's sarcoma. Sputum samples are a start to diagnosis, but bronchoalveolar lavage or biopsy would likely be required to obtain a definitive diagnosis for a cavitary process. A cavitary lesion involving cryptococcosis is a rare occurrence. Usual radiographic presentations for pulmonary cryptococcosis include interstitial infiltrates or pleural effusions. Additionally, there is no typical clinical picture. Interestingly, although the majority of cryptococcal patients are initially diagnosed with disseminated CNS disease, the pulmonary complaints of cryptococcosis often precede the diagnosis of meningitis by months. The variety of clinical and radiographic presentation underscore the need for definitive diagnosis, especially in the patient with clinically silent cryptococcal meningitis. \nA 17 year old incarcerated male presented to our emergency department complaining of headache, nausea, vomiting, diarrhea, subjective fever and weight loss for one week. There were no sick contacts or recent travel. The patient had no prior medical history, and had tested negative for HIV six months before presentation. He was taking no medication and denied any family history. Sexual history revealed prior homosexual intercourse. On presentation, the patient's blood pressure was 99/44 mm/Hg with a temperature of 39.4 8C. Physical exam revealed seborrhea over the face and cervical, axillary and inguinal lymphadenopathy. Laboratory studies revealed a WBC count of 2,000, hematocrit of 48% and platelet count of 48,000. Urinalysis showed 2+ protein. Cerebrospinal fluid analysis, stool studies, and blood cultures were negative. Chest X-ray was normal. The patient underwent bone marrow aspiration, which revealed a hypoplastic marrow with decreased erythroid precursors, consistent with viral infiltration. The patient clinically improved and remained afebrile with resolution of all of his symptoms. His platelet count returned to normal. Parvovirus serologies were equivocal, and Epstein Barr serologies did not reveal acute infection. The patient underwent HIV testing which revealed a positive ELISA with an indeterminate Western blot. The CD4 count was 420. After consultation with the infectious disease team and the pathology department, we were permitted to perform viral load testing in the hopes of confirming the diagnosis of acute HIV infection. The viral load was greater than 750,000 copies per mL. DISCUSSION: Acute HIV infection presents with a variety of non-specific symptoms, including fever, lethargy, rash, myalgias and headache. Symptoms are present in 50 ± 90% of patients with acute infection, and the complex is known as acute antiretroviral syndrome. Because of the non-specific symptoms, acute HIV infection in adolescents often resembles, and may be mistaken for, more common viral illnesses such as infectious mononucleosis. The diagnosis should be suspected in any patient at risk for HIV infection who presents with this constellation of symptoms or fever of unknown cause. Early diagnosis of HIV, particularly during acute infection, is crucial so that antiretroviral therapy may be initiated without delay. The diagnosis is based on a HIV-1 RNA level of greater than 50,000 copies per mL or positive p24 antigen in the absence of a positive ELISA and confirmatory Western blot test for HIV. If a patient is HIV enzyme immunoassay-negative and HIV RNA-positive, follow-up antibody testing should be performed 2 ± 4 weeks after the resolution of symptoms to document seroconversion.\nCOMMON COUGH,UNCOMMON CAUSE. F. Millhouse 1 , L.J. Schultz 1 ; 1 Emory University, Atlanta, GA LEARNING OBJECTIVES: 1. Learn when to evaluate further young patients with``recurrent pneumonias.'' 2. Recognize the clinical presentation of pulmonary sequestration. 3. Learn the pathology and pathophysiology surrounding the formation of sequestration cysts. CASE INFORMATION: A 26-year-old male presented with the complaint of a 2 ± 3 week history of cough productive of approximately one cup of yellow green sputum per day. On the day prior to admission, he noted 1 ± 2 teaspoons of blood with each cough. Five years earlier with a similar presentation, he was noted to have a cavitary lesion on chest x-ray. He was treated with antibiotics for a presumed lung abscess and was asymptomatic until this admission. Vital signs on admission revealed a blood pressure of 137/76, heart rate of 137, respirations of 22, temperature of 38.4 degrees Celsius, and a pulse ox of 88%. On physical exam he was a young well-developed male in no apparent distress with dullness to percussion and tubular breath sounds at the right base. Chest x-ray showed an interval increase of the air-fluid level within a large right middle and lower lobe cystic lesion and a left mid lung infiltrate. The patient was admitted and started on antibiotics for pneumonia and presumed lung abscess. The patient was unable to tolerate a CT scan or bronchoscopy secondary to uncontrollable coughing and hemoptysis. With a high suspicion for pulmonary sequestration, an aorto-bronchial arteriogram was obtained that confirmed the diagnosis. It showed an aberrant vessel arising off the infradiaphragmatic aorta just above the level of the celiac artery supplying a right lower lobe sequestration. DISCUSSION: Pulmonary sequestration is defined as an area of nonfunctioning lung tissue that lacks normal communication with the tracheobronchial tree and derives its blood supply from systemic vessels. Pulmonary sequestration is divided into two types: intralobar and extralobar sequestration. The former usually presents in early adulthood. The clinical presentation may range from asymptomatic to chest pain, chronic cough or hemoptysis. Radiographic findings can include either consolidation with irregular margins, multicystic lesions, or cavitation. The diagnosis should be considered in young patients with recurrent or persistent pneumonias. The treatment is surgical removal of the sequestration via segmentectomy or lobectomy. SEVERE POSTURAL HYPOTENSION AS A DELAYED SEQUELA OF LIGHTNING INJURY. V. Mukerji1 1 , R. Nonneman 1 ; 1 Southern Illinois University, Springfield, Illinois LEARNING OBJECTIVES: 1) Review the acute and delayed injuries that may result from lightning strike. 2) Recognize postural hypotension as a possible delayed sequela of lightning injury. CASE INFORMATION: A 27-year-old woman was referred to us for increasing dizziness and palpitations. About 8 years ago while sitting indoors with her foot on the fireplace grill she was struck by lightning. She suffered a brief syncopal episode followed by severe lower extremity weakness and inability to walk. With physical therapy her weakness had almost cleared but she continued to experience increasing dizziness and palpitations. Her physical examination was unremarkable except for a resting heart rate of 110 beats per minute. The EKG and echocardiogram were normal. On tilt table testing the patient became dizzy and lightheaded as the blood pressure fell from 116 to 76/42 mm Hg and her heart rate rose from 94 to 150 beats per minute. Her symptoms cleared and the heart rate and blood pressure returned to normal as soon as the table was brought back to the horizontal position. The tilt table test was then repeated with both lower extremities wrapped with Ace bandage. This time there were only minor changes in her blood pressure and heart rate and the patient remained asymptomatic. Compression stockings were recommended for the patient with significant improvement in her symptoms.\nDISCUSSION: Lightning is the second most common cause of environmental death in the United States. It is estimated that the number of human injuries may be as high as 5000 per year with 300 deaths. Permanent sequela may occur in 75% of survivors. The serious injuries from lightning strike are usually cardiovascular or neurologic. Most cases develop burns but they are rarely serious. Eye and ear injuries each occur in 50% of cases. Cardiovascular problems include asystole, dysrhythmias, cardiac contusion, pericardial effusion, EKG changes and cardiac enzyme elevations. Neurologic problems include syncope, confusion, paresthesias, and neuropsychiatric disorders. Respiratory center paralysis may occur. Less commonly seizures, cerebral edema, cranial nerve dysfunction and cerebellar ataxia have been reported. This patient developed autonomic dysfunction with severe disabling postural hypotension. The onset was gradual over several years after the actual lightning strike. A 61 year old man was admitted with atrial fibrillation and congestive heart failure. Pharmacologic treatment to limit the ventricular rate was complicated by episodes of bradycardia. Heart rates as low as 30 beats per minute in the presence of continued tachycardia necessitated the implantation of a pacemaker. A pacemaker was implanted, and anticoagulants were restarted twenty-four hours after the surgery. A hematoma was noted at the site. Warmth and erythema developed, yet the patient was afebrile. Empiric antibiotics were started and an aspirate culture was drawn. The culture grew Corynebacterium Urealyticum. The patient was treated with vancomycin, and the pacemaker was explanted. Cultures of the pacemaker also grew Corynebacterium Urealyticum. DISCUSSION: The incidence of pacemaker infections post-implantation ranges from one to six percent, with the majority of cases being caused by staphylococcus species. This case represents an unreported etiologic organism of pacemaker pocket infections. Corynebacterium Urealyticum is a slow growing, multi-drug resistant, gram positive aerobe that is often found in skin flora in hospitalized patients. 2. Generalized erythroderma is rare in Psoriasis and any such a change should prompt a biopsy and further evaluation. CASE INFORMATION: Peripheral T-cell lymphoma is classified among aggressive group of non-Hodgkin lymphomas by R.E.A.L. classification and accounts for < 15% of lymphomas in US. It presents as generalized disease with pruritis. Lymph nodes, skin, liver spleen and other viscera maybe involved. Presentation with generalized erythroderma is rare with peripheral Tcell lymphomas and is usually seen with primary cutaneous lymphomas. Case History: 78 year old Hispanic female with past medical history of hypertension, psoriasis and eczema for the last ten years presented to dermatology service with two month history of gradually worsening generalized erythematous macular rash with mild itching. Patient denied HIV infection or risk factors. Home medications included plendil, monopril, aspirin and lidex oint. There was no change in her medications recently. Physical exam. revealed generalized erythmatous non scaly macular rash with violacaeous discoloration at some areas and cervical lymphadno -pathy . CBC revealed leukocytosis with eosinophillia. LDH was 441. CXR was unremarkable. Skin biopsy was performed which revealed atypical lymphocytic infiltrate with abundant eosinophills. These cells were positive for CD3, CD4, CD5 (T cell markers) and negative for CD20 & CD30 (B cell markers). Lymph node and bone marrow biopsies were performed to further characterize the disease. Both specimens showed neoplastic infiltrates of lymphoid cells, which were positive for CD3, CD4, and CD5. Review at NIH confirmed the above findings and a diagnosis of peripheral T cell lynphoma-unspecified was made. Patient, who was visiting US from Santo Domingo, declined chemo-herapy and elected to return to her country. DISCUSSION: Peripheral T cell lymphomas-unspecified is a rare kind of lymphoma. Whereas erythro-derma is not an uncommon presentation of primary cutaneous T cell lymphomas especially Sezary syndrome, it is very rare in perpheral T cell lymphoma-unspecified. In a patient with psoriasis, such as ours, generalized erythroderma is rare and such an evolution of the disease should prompt skin biopsy and further workup. Treatment of peripheral T cell lymphomas-unspecified is not clear due to rarity of this disease. These lymphomas are usually very aggressive and have only 20% cure rate.\nS. Narreddy 1 , M. Snyder 1 , P. Khan 1 , N. Lekas 1 , R.D. Hobbs 1 ; 1 Oakwood Healthcare System, Dearborn, MI LEARNING OBJECTIVE: To recognize the dilemma posed by multiple enhancing ring lesions in the brain and emphasize the need for brain biopsy in equivocal cases. CASE INFORMATION: A 67 year-old Arabic male presented with a one-month history of progressive weakness and sensory changes of the left arm and leg. He had lived in the US for thirty years before returning to Yemen two years ago to work on a farm. On examination, he had right homonymous hemianopia, left hemipareisis and left hemianesthesia but nothing that would suggest either an infectious process or a malignancy. Basic work-up including HIV test was negative. An MRI revealed multiple enhancing ring lesions in the left and right occipital lobes, the right parietal lobe, and left cerebellar hemisphere. The findings were interpreted as metastatic lesions from an unknown primary. When no primary focus was found a brain biopsy was performed. Gross pus was aspirated. Histology was consistant with abscess. Smears revealed filamentous gram-positive organisms and cultures grew Nocardia asteroides. DISCUSSION: Multiple ring enhancing lesions in the brain typically represent either metastatic lesions (3 ± 15% of brain tumors; bronchogenic, breast, melanoma, renal, lymphoma are most common primaries) or abscesses. Differentiating between the two by clinical or radiographic means has been difficult, even with the development of CT and MRI scanning. Although advanced radiographic technologies (i.e., diffusion-weighted echo planar imaging, technetium-99m labeled leukocyte scintigraphy) accurately image brain appearance, to clarify treatment it is important to recognize that a brain biopsy is warranted when the diagnosis is in question and a primary focus cannot be identified. A 44 year old Hispanic male presented to clinic with multiple complaints including: red itchy eyes, productive cough with occasional blood, rhinorrhea, nasal congestion, myalgias, abdominal pain, and subjective fevers and chills. Except for a gastric ulcer diagnosed 1 month earlier, he was otherwise healthy. He was diagnosed with a viral syndrome, reassured, and given symptomatic treatment. The patient returned for a second visit 2 weeks later without improvement in his symptoms. A CXR, PPD, and HIV test were all negative. An ophthalmology exam confirmed the diagnosis of viral conjunctivitis. The patient, concerned that his symptoms were not improving, returned a week later, appearing very ill, and was admitted for further work up. On physical exam, notable findings included a T = 38.8 C, scleral injection, nasal and oropharyngeal erythema, decreased breath sounds at both lung bases, and mild abdominal tenderness in the right upper quadrant. Labs were significant for proteinuria, hematuria, and elevated liver function tests. To work up his abdominal symptoms, a CT of the abdomen was obtained which showed gastric thickening and a right lung nodule. Subsequent testing included an EGD showing diffuse erythematous mucosa; a repeat CXR and CT of the chest revealing multiple opacities in both lungs with hilar lymphadenopathy; bronchoscopy showing erythematous friable mucosa; and a repeat slit lamp examination revealing bilateral episcleritis. C-ANCA titers were 1:160. A nasal biopsy was performed showing granulomatous disease with giant cells and chronic vasculitis confirming the diagnosis of Wegeners Granulomatosis. The patient was treated with Cytoxan and Prednisone with resolution of symptoms. DISCUSSION: Wegeners Granulomatosis is a form of small vessel vasculitis with prevalence of 3:100,000. The mean age of onset is 40 years, with men affected slightly more than women. It classically involves upper and lower respiratory tracts and kidneys, but can involve virtually any organ system. This case illustrates how Wegeners can mimic colds and sinusitis, obscuring the diagnosis and delaying treatment. Early diagnosis is crucial because untreated Wegeners has a poor prognosis with median survival of 5 months and up to 90% mortality. Furthermore, renal failure can progress rapidly even in the absence of symptoms. Timely initiation of therapy with Cytoxan and Prednisone can be life saving and may markedly decrease morbidity and mortality. On the morning of admission, he was unable to arise from bed. The weakness involved both upper and lower extremities and was associated with mild abdominal pain. After 5 hours, he alerted a family member who assisted him to his feet. Upon standing the patient lost consciousness. He denied vomiting or recent illness but noted decreased food intake for the 2 days prior to admission. His only past medical history was a penetrating knife wound to the forehead 20 years prior to admission. He was taking no medications and did not use intravenous drugs. The blood pressure was 90/54 mmHg, the heart rate 110 beats/min and the temperature 36C. He had a normal thyroid exam and there were no carotid bruits. Examination of the heart, lung and abdomen were normal. Reflexes were decreased throughout; proximal and distal muscle strength was diminished. There were no focal deficits; sensation and cerebellar functions were normal. His sodium, potassium, calcium, magnesium, and creatinine were normal. The blood glucose was 54 mg/dL, and despite multiple ampules of dextrose he remained hypoglycemic. Persistent hypotension and refractory weakness prompted an evaluation of his adrenal function. The random cortisol was 1.6 mg/dl; the ACTH was low. DISCUSSION: The diagnosis of adrenal insufficiency should be considered when refractory hypoglycemia, orthostatic hypotension, and weakness simultaneously present. The causes of acute adrenal insufficiency include iatrogenic withdrawal of steroids, adrenal hemorrhage, tuberculosis and autoimmune disease. An important and under-recognized cause of adrenal insufficiency is inhibition of the hypothalamic-pituitary-adrenal axis by heavy alcohol ingestion. A repeat history revealed that the patient had a 6-day period of heavy alcohol and cocaine use. The normal electrolytes were clues that the adrenal insufficiency was not due to Addison's disease. The functional aldosterone axis and lack of hyperpigmentation implied a pituitary etiology which was confirmed by the low ACTH and robust rise in cortisol following cortrosyn stimulation. The weakness and hypoglycemia corrected with administration of hydrocortisone. Clinical reasoning identified the specific etiology of this patient's Addisonian syndrome and prevented unnecessary therapy.\nPROGRESSIVE PNEUMONIA IN AN IMMUNOCOMPETENT HOST. L. Orlando 1 ; 1 Tulane Medical Center, New Orleans, LA LEARNING OBJECTIVES: Suspect fungii in antibiotic refractory pneumonias and treat the presence of hyphae as invasive aspergillosis in these patients. Recognize that chronic diseases can increase the risk for invasive aspergillosis.\nA 71 year-old man with severe COPD (not on steroids) presented with one week of a non-productive cough, fever and dyspnea. His exam revealed crackles and egophony over the right upper lobe (RUL). The chest X-ray confirmed a community acquired pneumonia (CAP), and he was started on Azithromycin. His symptoms did not improve. A chest CT showed extension of the RUL consolidation, but no cavities or endobronchial lesions. Blood and sputum gram stain, AFB, KOH, and cultures were negative, except one sputum with branching hypae, which was dismissed as a contaminant. Fluconozole and Vancomycin were added, but his condition worsened. Acute renal failure occurred on day seven. Bronchoscopy was performed on day eight, and revealed black mucus and white nodules within the bronchioles. Lavage and biopsies were negative for organisms or cancer. The patient died from an acute lung hemorrhage on day ten. Autopsy revealed invasive Aspergillus Niger with an acute lung hemorrhage. DISCUSSION: A progressive CAP despite antibiotic therapy implies one of the following: the wrong antibiotic selection, a post-obstructive pneumonia (POP), a fungal pneumonia, immunosuppression, alveolar proteinosis or BOOP. In our patient the acute presentation, imaging studies without fleeting infiltrates, and bronchoscopy without obstruction were clues against BOOP, alveolar proteinosis, and POP. The patient's brisk immune response, severe COPD and negative bacterial cultures were important hints towards a fungal infection. Although Aspergillus pneumonia usually occurs in the immunocompromised, it can occur in immunocompetent patients so the presence of branching hyphae on cultures should not be disregarded as colonization or contamination when a clinical pneumonia is present, especially in those with chronic diseases. Empiric Amphotericin B and lung biopsy should be considered when clinical evidence suggests Aspergillus since cultures and even bronchoscopy are frequently negative. This patient had Aspergillus Niger which produces oxalic acid creating a pathognomonic black acidic sputum and an immune mediated glomerulonephritis. Sputum and BAL cultures are positive in less than 10% and dissemination occurs in 25% of patients.\nA. Pai 1 , A.L. Riba 1 , R.D. Hobbs 1 ; 1 Oakwood Healthcare System, Dearborn, MI LEARNING OBJECTIVE: To recognize the unique radiologic and therapeutic features associated with impending paradoxical emboli. CASE INFORMATION: A 61 year old female presented with an acute onset of intractable headaches, vertigo, dyspnea and melena. A CT scan confirmed an embolic CVA involving the right occipital lobe. The presence of an asymptomatic, swollen right leg, and an acute axis change on EKG lead to the suspicion of a DVT with PE. Large thrombi were seen in both pulmonary arteries on spiral CT. Additionally, a TEE demonstrated a large embolus straddling a patent foramen ovale. Therapy was problematic due to the patient's history of complications following a CABG in the past and active GI bleeding. A conservative treatment approach involved careful anticoagulation with heparin and Greenfield filter placement. The patient success-fully recovered without further embolic episodes or complications. DISCUSSION: Although 25% of patients at autopsy have a patent foramen ovale that can be detected by probing, the actual occurrence of paradoxical emboli (as with most pulmonary emboli) can safely be assumed to be under-diagnosed. Although newer technology (spiral CT, TEE) has increased our ability to diagnose such conditions, impending paradoxical embolus is still rarely reported in the literature. The optimal therapy is unknown although embolectomy with closure of the patent foramen ovale, thrombolysis and anticoagulation are the commonly available options. Treatment should be individualized depending on the patients age, existing co-morbidities and the availability of cardiothoracic surgical services.\nPOLYARTERITIS NODOSA PRESENTING AS AN ACUTE CEREBROVASCULAR ACCIDENT IN A YOUNG MALE. P.J. Bennett 1 , J.J. Yium 1 , M. Panda 1 ; 1 University of Tennesee -Chattanooga, Chattanooga, TN LEARNING OBJECTIVES: To understand the differential diagnosis of CVA in young patients. To recognize clinical features and proper diagnosis of polyarteritis nodosa (PAN) CASE INFORMATION: A 24-year-old black male with a questionable history of multiple sclerosis presented to the ER with acute onset of right-sided weakness, slurred speech and dysphagia. The patient denied any visual symptoms, headache, fevers or recent illnesses. On exam the patient was afebrile, pulse 60 beats per minute, and BP 180/128mm/Hg, neck was supple without bruits, and a 2/6 systolic murmur was noted at the apex. No skin lesions were noted. On neurologic exam the patient had dysarthria, right facial droop and right hemiplegia. Routine lab work was unremarkable with a normal CBC, metabolic panel and sedimentation rate. Urinalysis revealed 2+ proteinuria with benign sediment. LP revealed no oligocional bands. An MRI of the brain revealed a recent deep left-sided infarct involving the internal capsule as well as a small aneurysm of a temporal branch of the right middle cerebral artery. Hypercoaguable work up was negative, p-ANCA was negative but myeloperoxidase antibody was positive. Echocardiogram revealed mild left ventricular hypertrophy with normal LV function. Cerebral and renal angiography revealed innumerable aneurysms involving almost all branches of medium-sized arteries. A biopsy of the sural nerve with accompanying vasculature confirmed the diagnosis of PAN. The patient was treated with steroids, monthly pulse dose Cytoxan, and aggressive physical therapy. Nine months later he shows marked improvement and no further neurologic insults. DISCUSSION: PAN is a systemic vasculitis involving small and medium sized arteries and is more common in middle-aged men. Any organ can be affected but skin, peripheral nerves, joints, intestinal tract and kidneys are most commonly involved. The onset of disease can be abrupt or gradual and usually involves symptoms such as fever, malaise, palpable purpura, infarctive ulcers, joint pain and livedo reticularis, although multiple mononeuropathies are the most typical neurologic manifestation. The sedimentation rate is usually elevated but, as in our case, can be completely normal. Antibody to hepatitis B surface antigen can be positive. Tissue biopsy confirms the diagnosis. Untreated the 5 year mortality rate is greater than 85%. High dose steroid therapy has improved the five-year mortality to 30 ± 45% and the addition of immunosuppressive has reduced relapses. Plasmapheresis offers no additional advantages. Treatment of any associated viral illness (hepatitis, HIV) should be part of the initial therapy.\nRAPIDLY PROGRESSIVE CARCINOID SYNDROME IN AN IMMUNOSUPPRESSED MALE. J.R. Pederson 1 , J.J. Yium 1 , M. Panda 1 ; 1 University of Tennessee±Chattanooga, Chattanooga, TN LEARNING OBJECTIVES: 1. Recognize the increased risk for malignancy in an immunosuppressed patient 2. Recognize the signs and symptoms of carcinoid syndrome. CASE INFORMATION: A 61 year old male, status post renal transplantation in 1995 for chronic renal insufficiency, was admitted in 9/1999 with complaints of weight loss, diarrhea, anorexia and early satiety. The patient noted a gradual onset of symptoms since 1996, with worsening over a six month period prior to presentation. He was on immunosuppressive therapy since 1995, Physical exam revealed a cachetic white male with redness and telangectasias of the face and neck. His physical exam was otherwise unremarkable. CBC, electrolytes and liver function tests were all within normal limits. Ultrasound of the abdomen, CT pelvis and colonoscopy in 1996 and panendoscopy in 1999 were normal Work up was negative for malabsorption, H. pylori, Giardia and C.difficile. He was readmitted a month later for right hip fracture. He still had diarrhea and weight loss but also noted prominent bilateral facial, neck, and arm flushing. Punch biopsy from the skin of the neck was essentially unremarkable. Post-op CT of the abdomen revealed innumerable hepatic lesions in all lobes of the liver. Ultrasound guided liver biopsy revealed metastatic carcinoid tumors. Subsequent 24 hour urine collection revealed elevated 5-HIAA levels. The patient was treated with sandostatin and antihistamines. He rapidly deteriorated and died within several months of diagnosis. DISCUSSION: Carcinoid accounts for 30 ± 40% of all small intestinal tumors. Over 95% of all GI carcinoids occur in one of 3 sites: the rectum, appendix or small intestine (usually the ileum). Tumors less than 2 cm are less likely to metastasize. These tumors secrete vasoactive materials responsible for the clinical manifestations of carcinoid syndrome which is seen only in patients with hepatic metastasis. With advanced disease, treatment is symptomatic and true cure is seldom achieved. Rapid progression of indolent carcinoid has been reported inpatients who are immunosuppressed. As our patients preliminary extensive GI work-up did not reveal an obvious primary, we postulate that our patient may have primary hepatic carcinoid which rapidly progressed due to his immunosuppressed state. There has been only one case reported in the literature of primary hepatic carcinoid in a renal transplant patient. With the world wide increase in the incidence of tumors among immunosuppressed patients, physicians should have a low threshold for early thorough workup of suspected tumors. A 34 year old black male presented with a 2 week history of low grade fever, night sweats, generalized fatigue, weakness and myalgias. He had a waxing and wanning rash on his face and arms. Past medical history was significant for hypertension since age 26 and a similar rash and fever about 12 years ago. On examination he was febrile (temperature of 103F) and tachycardic. Physical examination was unremarkable except for a 2Â2cm right submandibular lymph node, a few lymph nodes in the right posterior cervical triangle and a hyperpigmented maculopapular skin rash involving the upper extremities and the face. The laboratory data showed WBC: 2th/mm3, Hbg./Hct.À8.4gm/dl 25.8%. The blood cultures, urine cultures, chest x-ray, imaging studies of the head, neck, thorax, and abdomen were all negative. ESR: 98, CRP 8.4mg/dl. Rheumatologic workup, viral and rickettsial serology, RPR and HIV were negative. Bone marrow biopsy and culture was also negative. The right submandibular lymph node biopsy showed histiocytic necrotizing lymphadenitis. He was started on Vioxx for the fever and myalgia. The patient's fever subsided after a total of 5 weeks. He returned with vision problems and was found to have retinal hemorrhages bilaterally with normal disc margins. Steroids were initiated for ocular symptoms with some improvement. DISCUSSION: Kikuchi's disease was first described in Japan in 1972 by Kikuchi and Fujimoto. It is a self limiting disease involving different ages, races and geographic regions. The etiology of this disease is uncertain, although its association with certain viruses like CMV, EBV, HSV, Parvo B-19, HIV, Yersinia and Toxoplasmosis has been described. These viruses or other antigens can induce immunological abnormalities in a susceptible individual with progression to autoimmune diseases like SLE and Still's disease. The most common clinical features of Kikuchi's disease are fever, weakness, fatigue, myalgia, arthralgia and lymphadenopathy (especially cervical). Skin rash is non-characteristic particularly involving the upper regions of the body. Hepatosplenomegly and neurological manifestations like aseptic meningitis and ataxia have also been described. However this is the first case described with retinal changes. Kikuchi's syndrome needs to be differentiated from other diseases like malignant lymphoma, SLE, tuberculosis, sarcoidosis, and cat scratch disease. As the course of Kikuchi's disease is generally self limited, and histopathology is diagnostic it is important to include it in the differential diagnosis of FUO.\nA CHALLENGING CASE OF DYSPNEA IN A PATIENT WITH BREAST CARCINOMA. P.J. Bennett 1 , M. Panda 1 ; 1 University of Tennesee, Chattanooga, TN LEARNING OBJECTIVES: Use of appropriate diagnostic testing to differentiate the pulmonary tumor emboli syndrome from a thromboembolic event. Understand the diagnosis and management of pulmonary tumor emboli CASE INFORMATION: A 75 year old white female with a past history of poorly differentiated ductal adenocarcinoma of the right breast diagnosed in 7/95 presented with progressive shortness of breath for 4 ± 5 days. On exam, she was afebrile, tachypneic and tachycardic. She had decreased breath sounds bilaterally with crackles posteriorly in the right lower hemithorax. ABG revealed: pH 7.46, PCO2 26mmHg, PaO2 43mmHg, HC03 19meq, 82% saturation on room air. Chest films and CT were unchanged from previous of 8/98. There was no evidence of lymphangitic carcinomatosis. Echocardiogram revealed an EF of 72%, evidence of new moderate pulmonary hypertension and new tricuspid regurgitation, without tamponade. VQ scan revealed small unmatched peripheral deficits in the left upper lobe consistent with intermediate probability of pulmonary embolus. Subsequent pulmonary angiogram revealed no evidence of pulmonary embolism. Due to worsening hypoxia, bronchoscopy with transbronchial biopsy was performed. Biopsy revealed metastatic poorly differentiated carcinoma with prominent intravascular growth consistent with a breast primary. DISCUSSION: Pulmonary tumor embolism is more often a postmortem diagnosis in patients with known solid tumors such as breast, liver, prostate, and kidney. Potential mechanisms of tumor emboli to the lung include direct extension into the IVC, extension via the thoracic duct to pulmonary lymphatics or via the SVC & right side of the heart to the pulmonary arterioles. The tumor cells occlude the smaller arterioles without parenchymal involvement. Resulting intimal proliferation leads to increased pulmonary pressures and right ventricular strain. Progressive dyspnea is the most common symptom. Chest X-ray is often unremarkable. VQ scans may be normal, indeterminate, or reveal focal defects. Pulmonary angiography may only reveal slow blood flow. Occasionally, CT/MRI scans may reveal a tumor mass occluding the pulmonary artery. Diagnosis is proven by tissue biopsy. Treatment of patients with pulmonary tumor emboli is difficult. Tumor embolectomy may be an option in large proximal emboli. Chemotherapy and radiotherapy usually produce a poor response. Together with fat and amniotic fluid embolism, tumor embolism cause hypoxia with a normal pulmonary angiogram. As in our patient unrelenting hypoxia and new development of hypertension may be important diagnostic clues.`I A 40 year old white female with no significant medical history presented with a 5 month history of a slowly expanding rash on her lower trunk and proximal extremities associated with generalized fatigue. The rash was painless, nonpruritic and not associated with arthralgias, fever or chills. The patient admitted to taking several herbal medications which she had recently stopped. Her physical exam was unremarkable except for a pink, blanchable, nontender, confluent, macular rash involving the lower abdomen and proximal portions of the arms and legs. There were no bullae or pustules. Laboratory evaluation revealed a negative ANA, rheumatoid factor, SS-A and SS-B antibodies. The patient had normal levels of aldolase, CPK and sedimentation rate. Her CBC was unremarkable except for the presence of 26% eosinophils. A deep fascial biopsy of one of the left thigh lesions revealed an inflammatory infiltrate of the fascia consistent with eosinophilic fasciitis. Upon review of the patient's herbal medication list and available literature, the probable causative agents were glucosamine sulfate and the fever few leaf. The patient was started on steroids with slow improvement. DISCUSSION: Eosinophilia-myalgia syndrome was first described in 1989 when it was observed in 3 group of patients who developed scleroderma-like skin changes, myalgias, eosinophilia, and histologic findings similar to eosinophilic fasciitis after ingestion of Ltryptophan manufactured by a single company. However, as this case illustrates, other agents, including herbal medications can precipitate a similar syndrome. Other clinical manifestations include low grade fever, fatigue, dyspnea, cough, arthralgias/arthritis, erythematous rashes and myalgias. Myocarditis and pulmonary hypertension can occur. Usually significant peripheral eosinophilia is noted and striking tissue eosinophilia may be noted as well. Diagnosis is dependent on histologic findings from a deep biopsy including fascia and muscle. Response to treatment is variable but patients may respond to steroids, plaquenil, methotrexate, Dpenicillamine and cimetidine. The course of the disease is poorly defined but many patients spontaneously regress or remain unchanged for years. Use of herbal medications is becoming very common as these products are widely advertised and readily available. However clinical trails and adequate information on efficacy and adverse reactions are limited. This case stresses the importance of incorporating questions on use of herbal medications routinely on medication history and the need to have a hightened awareness of their possible implication in disease. A 74 year old previously healthy male presents complaining of a 2 month history of progressively worsening scrotal swelling. The patient denied urinary hesitancy, frequency, hematuria, dysuria, or incontinence, but admits to mild decrease in urine output with occasional urgency. Past medical history was negative, and the patient denied using any medications including NSAIDS. Physical exam was significant for marked scrotal, suprapubic, penile shaft, and foreskin edema with normal testicular size. No masses or hernias were detected. Routine labwork showed BUN 67, Creatinine 9.6. When seen for the same complaint 5 weeks earlier, the patient had normal electrolytes and creatinine 1.2. Foley catheter was successfully placed with approximately 50cc of residual volume obtained. Renal ultrasound showed mild to moderate right hydronephrosis, mild prominence of the left collecting system without hydronephrosis. Percutaneous nephrostomy of the right kidney failed to improve renal function, and hemodialysis was initiated. Left renal biopsy revealed normal glomerular structure. CT scan of pelvis showed calcification of the prostate, with thickened mucosa of the sigmoid colon and rectum and bilateral hydroceles. Prostate biopsy was negative for malignancy. Rectal biopsy revealed poorly differentiated carcinoma of probable uroepithelial etiology. After 7 days, urine output began to improve, and dialysis was discontinued. Hepatitis serologies, urine and serum protein electrophoresis, ANCA, and anti-GBM antibodies were negative. Cystoscopy showed bladder thickening with locally invasive disease and bilateral ureteral obstruction. Results of bladder biopsy showed high grade papillary transitional cell carcinoma with vascular invasion and extension to the pelvic wall and rectum. DISCUSSION: Approximately 4% of cases of renal failure due to obstructive uropathy present without dilation of the urinary tract. Of these cases, 60% are due to intrapelvic malignancy (prostate, bladder, colorectal). Proposed mechanisms for lack of dilation include ureteral encasement by tumor, ureteral edema, reabsorption, and interrupted peristalsis. Although renal ultrasound is often used to rule out urinary tract obstruction, the absence of hydronephrosis does not exclude obstructive uropathy as the cause of acute renal failure. In the presence of high clinical suspicion, additional studies such as cystoscopy, intravenous pyelogram, or retrograde pyelography must be pursued to evaluate patency of the upper and lower urinary tracts.\nACQUIRED HEMOPHILIA IN SLE. J. Pedersen 1 , J. Paty 1 , M. Panda 1 ; 1 University of Tennessee College of Medicine -Chattanooga Unit, Chattanooga, TN LEARNING OBJECTIVES: 1. Recognize the entity of acquired hemophilia secondary to antifactor VIII antibodies and its association with autoimmune diseases and occult malignancy. 2. Understand the indications for treatment of acquired hemophilia. CASE INFORMATION: A 76 year old white female with a past history of SLE and polymyalgia rheumatica, in remission on daily prednisone and Plaquanil, presented to the office with a one week history of spontaneous, extensive bruising of the right hip and thigh after arising from a chair. She denied trauma, a personal or family history of easy bruising or prolonged bleeding. Physical examination was unremarkable except for an extensive area of ecehymosis along the right inner and outer thigh. MRI revealed iliospoas hemorrhage. Initial lab revealed a normochromic, normocytic anemia with a hemoglobin of 6 (one week prior to admission her hemoglobin was 10). She had a normal platelet count, prothrombin, thrombin and bleeding times, but an elevated partial thromboplastin time (57.1 seconds). The PTT partially corrected with the addition of normal plasma, so Factor VIII assay was done which revealed decreased activity at 8% with positive titers for factor VIII inhibitor (72 Bethesda, n1 0.1 ± 0.8). Lupus anticoagulant and antiphospholipid antibodies were negative. Subsequently the patient had a spontaneous right retroperitoneal and left upper extremity hemorrhage and required factor VIII transfusions, high dose corticosteroids and oral cytoxan. Six months later she remains symptom free on 5mg of prednisone and Plaquenil 400mg daily. Her hemoglobin is 11.8; PTT-28.6 and there is no detectable Factor VllI inhibitor. DISCUSSION: Hemophilia A, as a result of Factor VIII deficiency, can be inherited or acquired. Acquired inhibitors to Factor VIII are endogenous IgG/IgM immunoglobulins which interfere with normal blood coagulation. Acquired Factor VIII inhibitors can occur in patients with inherited hemophilia A, postpartum women, patients with collagen vascular diseases such as SLE, or idiopathicafly, especially in older patients. They are also associated with occult malignancy such as lymphoma and solid organ tumors. The syndrome usually presents with significant bleeding and soft tissue ecehymoses with minimal or no trauma. Treatment of active episodes of bleeding may involve administration of exogenous porcine or human Factor VIII or treatment with activated prothrombin complex concentrates as in our patient. Prophylactic treatment with steroids, with or without immunosuppressive agents, is indicated in patients with significant bleeding or with high titers of inhibitor. year-old African-American woman with no significant past medical history presented to clinic with complaints of bilateral lower extremity swelling, easy fatiguability and generalized weakness of 2 weeks duration. She also reported increased sensitivity to cold, dryness of skin and menorrhagia. On exam, she was moderately obese, did not appear acutely ill. Vitals signs were normal. Physical exam remarkable for mild conjunctival pallor, soft heart sounds, slow relaxation phase of deep tendon reflexes and bilateral lower extremity non pitting edema. EKG showed low voltage complexes in all leads. Echocardiogram demonstrated large pericardial effusion with no tamponade. Labs revealed normocytic, normochromic anemia. Thyroid profile with TSH 2.07 (0.4 ± 6) and free thyroxine 0.30 (0.6 ± 1.7) pointed towards the diagnosis of central hypothyroidism. Other hormonal assays were done which also revealed coexisting adrenal insufficiency. MRI of brain showed flattened pituitary along the floor of sella and majority of the sella was filled with CSF.These findings were consistent with empty sella . No focal lesion in the pituitary gland were identified. Pt was diagnosed to have hypopituitarism due to primary empty sella syndrome . She was treated with thyroxine and steroids to which she responded appropriately. The pericardial effusion was presumed to be secondary to the hypothyroidism and pericardiocentesis was not performed as she was hemodynamically stable. DISCUSSION: Primary empty sella syndrome has been classically described in obese hypertensive women. It is characterized by the presence of an arachnoid herniation filled with fluid that compresses the pituitary against the sellar wall.It is often asymptomatic but may be associated with endocrine disorders. Less than 30 percent of these patients presents with symptoms suggestive of hormonal deficiency. Recent literature reports only about 10 percent of these patients present with hypopituitarism as in our patient. The most common causes of central hypothyroidism are pituitary adenomas,pituitary apoplexy, infiltrative lesions. Pericardial effusion is one of the common cardiovascular manifestation in hypothyroid patients but most of these cases are described in patients with primary hypothyroidism. There are only few case reports describing pericardial effusion as presenting manifestation in patients with central hypothyroidism. These pericardial effisions are usually large but rarely progress to tamponade. They usually respond well to thyroxine supplementation. for unexplained anemia (Hb 6 g/dl). The patient had a history of chronic diarrhea with weight loss secondary to a diagnosis of CVID in 1994. Multiple colonoscopies revealed only nonspecific colitis. CT scan showed extensive mesenteric lymphadenopathy and thickening of the small bowel wall. He underwent EGD which showed healed duodenal erosions with copious bile in the esopaghus and stomach. A small bowel follow-through revealed proximal bowel dilatation and a partially obstructing process in the mid-jejunum (see Figure) . Exploratory laparotomy with partial small bowel resection revealed malignant lymphoma (large B-cell type). Bone marrow biopsy was normal. Tests for HIV antibody were repeatedly negative. The patient was discharged for outpatient oncological treatment. DISCUSSION: GI lymphomas have been reported rarely in patients with CVID, but are not usually considered among the initial differential diagnoses. We present this case to call attention to non-specific symptomatic presentation of lymphomas of the GI tract (Lai Ping So and Mayer, Semin Gastrointest Dis 1997; 8:22; Gottesman et al., Leuk Lymphoma 1999; 32: 589) . Figure Legend . Barium failed to progress rapidly to the distal jejunum (left panel), but was present after 15 minutes (right panel), indicating a partially obstructing process with considerable proximal bowel dilatation. An 84 year old woman with end-stage renal failure, congestive heart failure, insulin-dependent diabetes, depression, and osteoarthritis begins to express a desire to discontinue hemodialysis after many years. She feels sad, exhausted, and a burden to her family. The patient has been widowed for five years. Her medications include Celexa and Vicodin. She has four attentive children who all live in the same city. She is cared for primarily by her youngest daughter, who lives with the patient, administers medications and transports the patient to dialysis. Intent on caring well for her mother, and worried that pain was interfering with the patient's appetite, the patient's daughter has been administering increasing doses of Vicodin. While the patient has made a number of comments about discontinuing dialysis to each of her children, none of the children have talked with each other, hoping to avoid such discussions, as well as differences of opinion between them. The patient's thoughts have not been assessed during the last year by either her nephrologist or primary care physician. An endof-life consultation team met with the patient and her daughter. Clarification of the appropriate use of pain medicine allowed the patient to control her pain with only occasional use of Vicodin, leading to increased energy, independence, and enthusiasm. Assessment of the patient's CESD depression score showed an increase from 14 (six months earlier) to 22. The patient's Celexa dose was adjusted. The patient's daughter was invited to attend support classes for family taking care of patients with end-stage illness and grew more comfortable talking about her fears and sense of responsibility. With renewed energy and improved mood, the patient withdrew her desire to discontinue dialysis for now. In a family meeting, all four children were able to recognize their own feelings and to better coordinate care. DISCUSSION: Even among closely-knit families and well-supported patients, miscommunication is possible, especially around the highly-charged issues of advance care planning for patients with end-stage illness. Assessing for medication over-and underprescribing, and ruling out depression as an etiology for withdrawal of support requests, are good first steps during discussions. Understanding the motivations and influences of relevant family members is necessary as well. The key to good advance care planning is to begin the discussions well in advance of the decisions as there is often much to learn and minor clinical interventions can have major effects on planning variables.\nHYPERTROPHIC GASTROPATHY -ANEMIA. S. Ramalakshmi 1 , J. Lloyd 1 , R. Gregorio 1 , H. Dubner 1 ; 1 UPMC Shadyside Hospital, Pittsburgh, PA LEARNING OBJECTIVES: Hypertrophic gastropathy is a diverse disease characterized by giant mucosal hypertrophy of gastric rugae. The disease is usually confined to gastric body and fundus and can manifest with hypoalbuminemia. We present a case of an extensive hypertrophic gastropathy involving the antrum and body of the stomach associated with iron deficiency anemia. CASE INFORMATION: A 41year-old male patient during a routine physical, with symptoms of fatigue was diagnosed with iron deficiency anemia. Physical exam was unremarkable with guiac negative stools. Hemoglobin 6.0, Ferritin 1, Protein 4 and Albumin .9. Work-up of anemia included endoscopy, which showed diffuse nodularity of the body and a nodular mass in the antrum, which obscured pylorus and extended into second portion of the duodenum. Biopsies of stomach and duodenum were inconclusive. Upper GI series confirmed endoscopic findings. Due to the extensive involvement of the lesion as well as uncertainty of the diagnosis, gastrotomy was done. Large polypoid lesions were seen in the gastric antrum prolapsing into the duodenum and were excised. The mucosal surface of the duodenum was normal. Pathology was consistent with hypertrophic gastropathy. Stains and serum antibodies were negative for H. pylori. Patient was treated with acid suppressive therapy and iron supplementation. On follow-up patient was free of symptoms with Albumin 3.7 and Hemoglobin 12.2. DISCUSSION: This case is a relatively new variant of the already known hypertrophic gastropathy, in regards to its site and clinical presentation.\nHIV RELATED HEMOLYTIC UREMIC SYNDROME. Physical exam in the ER showed a distressed lady with tachycardia and tachypnea. She had petechiae over the legs, 3+ bilateral pedal edema and a right pleural effusion. Labs revealed severe anemia (hematocrit 21); normal platelet count (227k/ml), increased BUN and creatinine (81/5.3); increased LDH (579) and near normal PT and PTT (14.8/28.6) . Peripheral smear showed schistocytes. Factor V and VIII were normal. Hospital stay was initially represented by persistent anemia, worsening thrombocytopenia (down to 29k/ml), bleeding from multiple sites, worsening renal and pulmonary function. Neurological exam remained normal. LDH remained high whereas PT and PTT remained mostly unchanged. CD4 count and viral load sent at this time came back as 89 (Â106/L) and 42 (k copies/ml) respectively. A diagnosis of HUS was made on clinical and laboratory parameters. No known risk factors were identified. Patient underwent plasmapheresis and hemodialysis. Her condition however continued to deteriorate and she died seven weeks post admission. Autopsy confirmed HUS and ARDS. DISCUSSION: HUS has been seen mostly in terminal AIDS patients and thrombotic microangiopathy in AIDS patients is increasingly being considered a consequence of direct pathogenic effect of the virus over glomerular capillaries and arterioles. We were unable to identify any other risk factor for HUS in our patient as well. Prior to this admission our patient did not have any AIDS defining illness. We concur with others that HUS may be considered as an AIDS defining illness. Plasmaphersis was not associated with survival in our patient. The role of this mode of therapy in HIV patients needs to be studied further. MRI showed mild periventricular white matter disease and audiometric testing showed severe bilateral vestibular defects. In late 1997 she presented with recurrent pulmonary infections and was diagnosed with common variable immunodeficiency (CVID). In October 1998 she began having 10 ± 15 minute episodes of ascending right or left hand tingling and weakness followed by slurred speech and a reported mild facial droop that resolved with verapamil in December. In April 2000 she presented with declining memory, concentration problems and diarrhea. Workup revealed secondary adrenal insufficiency, hyperprolactinemia and decreased insulin-like growth factor 1 (IGF-). Extensive testing over six years included three head MRIs, two lumbar punctures, audiology testing, psychosocial evaluation, bronchoscopy, muscle biopsy, abdominal CT and numerous blood tests. Positive studies included high CSF protein of 64, oligoclonal bands, cortisol base 3.2 \"g/dL (6 ± 25) after stimulation 14.6 \"g/dL (!18 at 60 minutes after injection) and ACTH 5, prolactin 109ng/mL (1 ± 24) , and IGF1 50 ng/mL (71 ± 240). The lack of linear plaques and atrophy of the corpus callosum on the MRI scans were inconsistent with MS and neurology and otolaryngology consultants concluded that her progression of symptoms, imaging studies, and lab testing were most consistent with a diagnosis of Susac Syndrome. DISCUSSION: There have been less than 50 reported cases of Susac syndrome in the literature. This is the first reported case of immune deficiency and endocrine abnormalities accompanying Susac's syndrome which is described as a clinical course of hearing loss, encephalopathy, and visual changes consistent with a microangiopathy of the cochlea, brain and retina. High suspicion and extensive testing is required to rule out other etiologies of this triad of symptoms.\nA REFRACTORY NEUTROPENIC PNEUMONIA. S. Regenbogen 1 , A. Rosen 2 ; 1 University of California, San Francisco, CA LEARNING OBJECTIVE: 1) Diagnose and treat Bronchiolitis Obliterans Organizing Pneumonia (BOOP) in patients with hematologic malignancies who present with pneumonia. CASE INFORMATION: A 59-year-old man with cryptogenic pancytopenia was admitted with 3 weeks of cough, fatigue and fever. Bone marrow was markedly hypocellular, but nondiagnostic. He had no hematuria, rheumatic symptoms, or toxin or radiation exposure, except for 6 months on a Naval nuclear submarine. He attributed his condition to myelosuppression from chronic ibuprofen use. Admission vitals were temp 39C, BP 133/71, pulse 78, 02 sat 97% on room air. He had no bleeding or bruising, and no lymphadenopathy. Lung exam showed crackles and egophony at the right mid-chest. Spleen and liver were not palpable. CBC showed WBC 0.4, Hct 34, Plt 102, and ANC 0.2. CXR showed dense right-sided infiltrates. Blood and induced sputum cultures were repeatedly negative and broncho-alveolar lavage was nondiagnostic. Despite treatment with broad-spectrum antibiotics and antifungals, his lung exam and CXR continued to worsen. His neutropenia persisted despite 3 weeks of stem cell growth factors. On day 15, immunoperoxidase staining confirmed the diagnosis of hairy cell leukemia. He underwent open splenectomy, which increasd his WBC to 3.13, and ANC to 2.35. Open lung biopsy (OLB) on day 22 was consistent with BOOP. He was treated with high dose steroids and discharged on a prolonged prednisone taper. DISCUSSION: BOOP is an inflammatory response to lung injury, with edematous plugs of granulation tissue and interstitial fibrosis. It is associated with infection, autoimmunity, hematologic cancers, and other conditions. Patients present with a subacute, febrile illness with dry cough and dyspnea that is unresponsive to antibiotics. CXR shows patchy alveolar infiltrates with airspace consolidation, and PFTs reveal a mixed restrictive/obstructive pattern. Transbronchial biopsy is often nondiagnostic, therefore requiring OLB for definitive diagnosis. BOOP is usually responsive to steroids, but relapse is common. In one series (Am J Resp Crit Care Med 161:723, 2000), among patients with hematologic cancers undergoing OLB for an unknown pulmonary process, BOOP was the most common finding±20% of cases where a diagnosis was made. BOOP accounted for 30% of the diagnoses missed by bronchoscopy but found on OLB. Identification of a specific diagnosis on OLB significantly increased survival. In the setting of refractory pneumonia in a patient with a hematologic malignancy, the diagnosis of BOOP should be considered. OLB is advised if other procedures fail to identify a cause. LEARNING OBJECTIVES: 1. Participants will learn to recognize skeletal muscles as a rare metastatic site for the cancers. 2. Participants will learn diagnosis and treatment of esophageal cancer. 3. Participants will learn the incidence, pathophysiology, diagnostic strategy and treatment of skeletal muscle metastasis. CASE INFORMATION: Introduction: Although comprising 50% of body mass with a very rich blood supply, skeletal muscle is rarely the site of metastatic disease with only 242 cases having previously been reported (1) . Although primary cancers of the lung, colon, genitourinary tract and blood are most frequently involved (1, 35) , gastroesophageal adenocarcinoma has never been reported. We report a case of thigh muscle metastasis from primary adenocarcinoma of the gastroesophageal junction. CASE REPORT A 71-year-old African American male presented with severe right thigh pain, causing him to be unable to walk. His medical diagnoses included Stage IV Adenocarcinoma of the Gastroesophageal junction, hypertension, COPD (chronic obstructive pulmonary disease), and GERD (gastroesophageal reflux disease). He was an active smoker and Ex-alcoholic. Physical examination was remarkable only for his cachectic state and an extremely tender right upper thigh. Despite NSAID (non-steroidal anti-inflammatory drug) and full dose narcotic analgesia, the patient continued to complain of severe pain in his thigh. Thigh X-rays showed no lytic or blastic lesions. Bone scan did not show any metastasis. A MRI was done and showed a deep 2 x 4-cm soft tissue muscle mass in the right thigh without bony involvement, just anterior and medial to the femur. Diagnostic considerations included a soft tissue metastasis or a sarcoma. A CT guided needle biopsy of the right thigh mass was positive for metastatic adenocarcinoma consistent with the primary esophageal cancer. Radiation therapy was started to the patient's right thigh with a good response. At the time of discharge, the patient was walking without pain receiving narcotic analgesia. Upon returning to the clinic after 2 weeks for follow-up, he continued to walk without difficulty receiving outpatient radiation therapy. DISCUSSION: The incidence of adenocarcinoma of the esophagus and esophagogastric junction has been increasing over the past 25 years (2, 3, 4, 5) , with cancer of the esophagus (used to be predominantly squamous cell carcinoma) ranking among the ten most frequent cancers in the world. Although direct muscle invasion by carcinoma is well recognized, distant metastasis to skeletal muscle is uncommon (6). The present case is unique in that the localized thigh pain was produced by metastatic involvement to the thigh musculature without any osseous or perineural lymphatic extension. Even though autopsy series report a 0.8% to 20% (8,9,10,11,12) incidence of microscopic intramuscular metastasis, only a few cases of visible metastasis to the muscle have been described in the literature (7, 13, 14, 15, 16) . Accordingly, muscle metastasis often remains asymptomatic often undetected by both physical examination as well as diagnostic imaging procedures. The reported incidence, therefore, might be infrequent because of either a lack of recognition, underreporting, or infrequent autopsy evaluation for muscle metastasis. (7). Furthermore, it may be that only a fraction of patients with metastases to muscle survive long enough to allow clinical detection (17). Just why metastases to skeletal muscle is so rare is still unknown (17, 18) . Multiple factors, such as blood flow, intramuscular blood pressure, blood flow per unit of weight (millilitres/minute per gram); local changes in pH, as well as local temperature distribution may be involved. (6, 9, 11, 19, 20, 21, 22) . Organs with a high incidence of metastatic carcinoma such as lung, liver and bone have a constant blood flow. Although equally rich in its vasculature, it has been suggested that the blood flow in skeletal muscle is variable, is under the influence of Betaadrenergic receptor control, and is subject to varying tissue pressure that may affect tumor implantation (20,23). Although some have suggested that protease inhibitors in the muscle's extracellular matrix may resist tumor cell invasion (24), it may be the production of lactic acid and other metabolites by skeletal muscle that inhibits tumor cell growth. (20, 18) Whereas two thirds of all cancers metastatic to muscle are carcinomas, about one third are from leukemias and lymphomas with rare cases originating from melanoma. (25). Accordingly, although factors in the recipient site may be responsible for the relatively low rates of metastasis to muscle, properties of the primary tumor may also be involved. Furthermore, the differentiation between a primary soft tissue sarcoma and metastatic carcinoma to muscle is important as the treatment and prognosis is markedly different. Although the presence of a soft tissue mass caused by metastatic carcinoma may be misdiagnosed easily as a soft tissue sarcoma on physical examination and imaging studies (10), the current literature does not provide any clinical or radiographic characteristics that helps distinguish the two (17). Whereas 50% of carcinomas and sarcomas metastatic to muscle occur in the lower extremity, a greater percentage of upper extremity (26%) carcinomatous metastases occurs then has been reported for soft tissue sarcomas (10%). In addition, most of the cases reported are located in one muscle group or in one part of the body (26, 27, 28) . Although various imaging studies were used to identify metastases to muscle, none were specific for differentiating among carcinoma, sarcoma, or other muscle disorders. Plain radiographs, radionuclide scanning, and angiography have not been beneficial in differentiating carcinoma from sarcoma (17) although it has been thought that MRI imaging is superior to CT scanning (29, 30) . CT guided fine needle aspiration provide a rapid, minimally invasive means of diagnosis (17). Treatment options include radiation therapy, surgery or a combination of the two. Reports suggest surgical resection followed by adjuvant radiation or chemotherapy provides excellent palliative results (31,32,33). Although solitary metastases less then 4 cm in diameter can be treated by excisional biopsy (34), other reports suggest a less invasive approach may be better due to a low incidence of functional disability and because of the poor survival of these patients. Surgical resection is often reserved for those lesions that fail to be controlled locally with radiation or when the tumor growth results in neurologic deficit. SUMMARY: Although metastasis to skeletal muscle is extremely uncommon, physicians must remain aware of its occurrence, as its detection often requires specific evaluation. Differentiation between a primary soft tissue sarcoma and metastatic carcinoma to muscle is important, as their treatment and prognosis are so markedly different. The current literature does not provide any clinical or radiographic characteristic (plane x-ray or bone scans) that distinguishes metastatic carcinoma to muscle from soft tissue sarcoma (17). MRI, however, appears promising, and it should be considered earlier in the diagnostic work up. Treatment continues to remain palliative as metastatic carcinoma to muscle continues to remain a late event, with an overall poor prognosis.\nSHOULD WE SCREEN FOR HEPATOCELLULAR CARCINOMA? G. Roehrig 1 ; 1 UCSF Primary Care Internal Medicine Program, San Francisco, CA LEARNING OBJECTIVES: 1)Recognize screening methods for Hepatocellular carcinoma (HCC) and the evidence for or against them. 2)Identify who should be screened for HCC. CASE INFORMATION: A 46 year old woman with a history of injection (IV) drug use and alcoholism presents for establishment of primary care, without specific medical complaints. Her last IV drug use was over 10 years ago, is recently HIV negative, and doesn't recall hepatitis testing. She reports 15 yrs of intermittent heavy drinking. Her past medical history is also significant for Diabetes mellitus Type 2 and obesity. Her medications include methadone and metformin. Reveiw of systems: negative history of jaundice, ascities, bruising, or gastroentestinal bleeding. Exam: Obese woman with normal vital signs and no scleral icterus. She had normal cardiac and pulmonary exams and her abdomen was obese few spider angiomata. Initial lab values: Hepatitis C antibody positive, B negative, Hct: 35.8, Platelets: 109, INR 1.1, Albumin 3.0, AST: 94 (Normal < 40), ALT: 87 (Normal < 40), Total Bili 1.1, Alk Phos 174 (Normal < 130). Given the lab results and spider angiomata, an abdominal ultrasound (U/S) was ordered which showed signs of cirrhosis as well as a 1.4 cm liver``hemangioma''. An alpha-fetoprotein (AFP) subsequently obtained was 42.0 (normal < 20). The patient was referred to Hepatology, but then lost to follow-up. Repeat AFP 6 monts later was 316. An abdominal CT was obtained to better characterize the lesion showed growth to 2.7cm. A biopsy of the lesion was not done due to its location but a routine liver biopsy confirmed cirrhosis. The patient underwent transarterial chemoembolization for presumed HCC, her AFP at this time was 551. DISCUSSION: The major risk factor for development of HCC is cirrhosis, at a rate of 1 ± 7% per year. The most common etiologies of cirrhosis are viral Hepatitis (B or C) and alcohol. Data for time to development of HCC range from 15 ± 25 years. Survival rates are 50% at 3 years for resectable lesions and 20% at one year for non-resectable HCC. Screening tools: AFP > 20 sensitivity of 39 ± 70%, U/S = 58 ± 85%, CT = 46 ± 84%, AFP and U/S combined = 79 ± 90%. Unofficial screening guidelines, followed by many surveyed by Chalasani et al, aren't necessarily supported through evidence-based medicine (EBM). The 6 month screening periods initially arose from data on tumor doubling time. Currently, most hepatologits do every 6 month AFP or U/S in patients with documented cirrhosis, with CT imaging alternatively being used. A recent cost-effective analysis by Sarasin et al concluded that screening offered minimal survivial benefits for the majority. The most significant benefits were found in well-compensated Child's A cirrhosis, especially with resectable tumors. There is a logical argument for screening liver transplant candidates. In summary, there is currently no EBM to support screening beyond the select group of patients mentioned above. There is a need for more research to better establish the mortality benefit and cost-effectiveness of HCC screening. A 52 year old man with a history of low back pain and alcoholism presented to his PMD with severe low back pain. He was given Vicodin, but returned to clinic 5 days later with worsening pain, constipation, difficulty walking, and a fever. Pt was sent to a local hospital where he was found to be confused and febrile with bilateral chest infiltrates; last drink had been 2 days prior. He was treated for pneumonia and presumed alcohol withdrawal. 4/4 blood cultures grew out S. aureus, but TTE showed no valvular vegetations. 4 days later, pt was transferred to our hospital where he was febrile to 103, agitated, disoriented, unable to follow commands, but spontaneously moving all extremities. To the extent testable, neurologic exam was nonfocal except for marked low back pain on neck flexion and possible nucchal rigidity. He had no spinal tenderness. He was pan-cultured and a head CT showed no acute event. MRI and TEE were planned for the following morning to rule out SEA and endocarditis, respectively. Lumbar puncture was initially deferred because of concerns for possible SEA and because pt was already on meningitis-dose Nafcillin. The following morning (5 hrs after admission) the patient no longer spontaneously moved his lower extremities and his toes were upgoing bilaterally. Emergent MRI showed a large T9 epidural abscess with posterior and anterior extension. Patient underwent emergent neurosurgical evacuation and had slow but progressive improvement in lower extremity function over the next 2 weeks. One week after transfer to rehabilitation, he expired in the middle of the night. Autopsy showed reaccumulation of the epidural abscess with infarction of nearly half of his spinal cord. IMPLICATIONS/DISCUSSION: Spinal epidural abscess is a rare but serious infection with potentially disasterous consequences, as highlighted in this case. Entertaining the diagnosis early is the key to prompt discovery and treatment. Severe back pain in a febrile patient, particularly with other risk factors for SEA (IVDU, past back surgeries, bacteremia), warrants rapid workup. Spinal MRI should be done as soon as the diagnosis is entertained, ideally before any neurologic deficits develop. In most cases, spinal cord damage occurs suddenly secondary to cord infarction from thrombosis or interruption of the local venous or arterial supply rather than via cord compression. Management requires neurosurgical evacuation and antibiotics (etiology is S. aureus in > 60% of cases). Early surgical intervention ( < 24hrs) is associated with markedly better prognosis.\nWHEN THE DATA ARE NOT ENOUGH: SHARED MEDICAL DECISION MAKING. A. Rosen 1 ; 1 University of California, San Francisco, CA LEARNING OBJECTIVES: 1) Increase awareness of decision analysis as a tool for shared medical decision making (SMDM), 2) Recognize the importance of patient preferences in making medical decisions. CASE INFORMATION: A 77yo male with paroxysmal atrial fibrillation and recent diagnosis of colon cancer with hepatic metastases presented to my clinic. His quality of life (QOL) and functional status were excellent and he desired no adjuvant chemotherapy unless his QOL significantly worsened. He presented questioning his need for coumadin. We constructed à`b ack-of-the-envelope'' decision tree to address this question.\nIn conjunction with a review of the literature and discourse with the cardiology and oncology services, his risk of significant bleeding on coumadin with known liver metastases was thought equal to his risk of stroke off coumadin. In discussing his preferences for health states, his fear of mental incapacity and other potential sequelae of stroke far outweighed his disutility for a bleeding complication of the coumadin. These preferences were so strong that coumadin remained the preferred treatment modality even when aspirin was presented as an option. While the patient initially desired cessation of coumadin, this decision changed following the presentation of a simple decision tree and open discussion of his preferences. DISCUSSION: Decision analysis allows for the implicit decisions made everyday in clinical medicine to be made more explicit. The process of SMDM is often very illuminating, as it encourages better communication between patient and physician as they consider the consequences of a particular medical decision together. These consequences include the probabilities of health outcomes and the values, or utilities, that the patient places on these outcomes. Explicit knowledge of patient utilities may have a major impact on the medical decision particularly in a setting such as this one in which quality of life may be valued over life expectancy. SMDM is an excellent way to engage patients in the important dialogue needed to best inform medical decision making. Heparin infusion was discontinued and warfarin was continued. On the same day he had a burning pain over his right shoulder and forearm. Day 6, well demarcated edema and erythema of his right shoulder and forearm and later purpura and skin necrosis occurred. Warfarin was discontinued and he was offered lepirudin. Biopsy of the skin confirmed anticoagulant related skin necrosis. Day 11, platelet count increased to 150. Platelet factor 4 antibody was positive confirming heparin induced thrombocytopenia (HIT). The liver biopsy showed metastatic adenocarcinoma. DISCUSSION: Warfarin skin necrosis is a rare complication but could lead to extensive skin loss with its associated problems such as is seen in major burns. The patient usually complains of pain of the affected area and this should prompt the physician to consider it in any patient on anticoagulation who has unexplained cutaneous pain. It may be minimized by discontinuing the warfarin and substituting with heparin, heparinoids or lepirudin if anticoagulation is needed. If INR is high, reverse anticoagulation. Avoid initial``loading'' dose of warfarin because this will lead to rapid depletion of protein C leading to a relatively hypercoagulable state. This risk appears to be particularly high in inherited hypercoagulable states and in patients with HIT. Heparin skin necrosis may occur with all types of heparin and could be at or away from the site of injection. Antibodies to heparin-platelet-factor 4 is suggestive. Give warfarin or lepirudin if needed. It is not possible to tell if this patient had warfarin or heparin skin necrosis. A method of distinguishing these two conditions is needed as treatment is different. week history of increasing confusion and 3 day history of right-sided frontal headache. His medications included thyroxine, oxybutynin, and theophylline. One week following recent knee surgery, he stated that his energy level was decreased and he was having persistent memory difficulties. Physical examination was unremarkable except for delirium and a resting tremor. The patient was alert, but oriented only to self and location. He had difficulty cooperating with the examination and was easily distracted from tasks. He was admitted to the hospital, where outpatient medications were restarted. Chemistry panel, CBC, LFT's, thyroid function tests, blood cultures, and cardiac enzymes were all normal. Theophylline level was high normal (19.9 mg/L). An EKG was read as sinus arrhythmia. A head CT showed ventriculomegaly with no intracranial bleed or mass. Results of lumbar puncture, EEG, MRI of the brain, and overnight pulse oximetry were nondiagnostic. On day 4 of hospitalization, confusion persisted. The patient experienced a 9 beat run of ventricular tachycardia and a cardiology consult was obtained. Review of EKG's and a rhythm strip revealed multifocal atrial tachcardia. A theophylline level was obtained and reported at 39 mg/L. Theophylline was discontinued. Over the next 7 days, the patient's mental status gradually cleared and he was discharged. DISCUSSION: Theophylline has a narrow therapeutic index (10 to 20 mg/L). Hypotension, cardiac arrhythmias, and seizures have been reported in association with toxicity. Levels often do not correspond to symptoms in chronic toxicity. Treatment is mainly supportive, although dialysis is often used in management of severe toxicity. The case illustrates: 1) the broad differential diagnosis and resource intensive evaluation of delirium in the elderly and 2) the insidious nature of chronic poisoning from a prescribed medication. A bladder scan demonstrated a residual urine of greater than 800 cc; bladder catheterization produced one liter of clear, yellow urine. Urinalysis was within normal limits. A review of the patient's medication list revealed that he had most recently been prescribed phenylpropanolamine and guaifenesin and had also been taking oxybutinin for``incontinence''. DISCUSSION: Benign prostatic hypertrophy (BPH) is a common problem, ranging from 40% ± 50% in males 51-60 years old, to over 80% in males above 80 years of age. Males with BPH have symptoms suggestive of overflow incontinence. Pharmacologic therapy includes antiadrenergic agents to assist with detrusor contraction and relaxation of the urinary sphincter. In addition, current literature provides some evidence for 5-alpha reductase inhibitors (finasteride) to decrease testosterone-dependent prostate gland enlargement. Phenylephrine, a popular over the counter and prescription medication, is commonly used as decongestant. It has been associated with acute urinary retention. It would follow that phenylpropanolamine (PPA), also a sympathomimetic, has similar capabilities. In this scenario, the patient's BPH in combination with PPA and the anticholinergic effect of the oxybutinin precipitated acute bladder outlet obstruction and anuria. Although PPA has been taken off the market recently, other sympathomimetic drugs can be inferred as equally hazardous in these patients. One of the many ways to prevent adverse outcomes from polypharmacy is to take a medication history every time one sees a patient and to be coginzant of potential adverse medication interactions. By reviewing all current medications, we may reduce iatrogenesis. DVT but an MRI demonstrated extensive soft tissue bleeding. Anticoagulation was withheld and her coagulopathy resolved as the hyperthyroid state was treated. DISCUSSION: Atrial fibrillation (AF) is a common arrhythmia, found in 1% of patients over age of 60. It is the most common cardiac condition associated with cerebral embolism. Underlying hyperthyroidism occurs in up to 12% of patients with this arrhythmia and therefore, a TSH is recommended in patients presenting with new onset AF. Our patient had an embolic right middle cerebral artery stroke due to AF and her AF was undoubtedly due to unrecognized hyperthyroidism. In addition, an atypical manifestation of hyperthyroidism was present. As has been reported in the literature, the hypermetabolic state of hyperthyroidism, exacerbated by a recent iodine load from her CT scan, led to consumption of clotting factors and a clinically relevant coagulatopathy which resolved with treatment of the hyperthyroid state. Finally, in this patient, the extrinsic compression of the venous system by the bleed was initially incorrectly interpreted as a DVT. Only by careful physical exam and attention to pathophysiologic and laboratory details of the case was potentially harmful treatment with heparin avoided.\nPHEOCHROMOCYTOMA RELATED CARDIOMYOPATHY. Papules that progress to hemorrhagic pustules develop on the trunk and extensor surfaces of the distal extremities. Asymmetric joint involvement of the knees, elbows, wrists, MCPs and ankles occurs. The cutaneous and articular findings are due to an immune reaction to circulating gonococci and to immune-complex deposition. Synovial fluid cultures are consistently negative and blood cultures are positive in less than 45% of patients. If cultures are negative, alleviation of symptoms in 12 to 24 hours after antibiotic therapy supports a clinical diagnosis. Initial treatment is IV ceftriaxone. A similar arthritis-dermatitis syndrome may occur with N. meningitides. If cultures are negative, this must be considered in the differential. However, gonococcemia and meningococcemia respond to similar antibiotic treatment.\nCASE INFORMATION: C.D. is a 44-year-old female with Grave's disease treated with propylthiouracil for two years. She presented with a 2 week history of productive cough, fevers, pleuritic chest pain and fatigue. She was diagnosed with right lower lobe pneumonia and admitted for IV antibiotics. Despite appropriate therapy, the patient's condition deteriorated and she developed hemoptysis and progressive hypoxemia that required intubation. CXR showed bilateral alveolar infiltrates. Bronchoscopy was consistent with diffuse alveolar hemorrhage. Admission CBC revealed pancytopenia. Her PTU was discontined. Additional labs included serum perinuclear antineutrophil cytoplasmic antibody (p-ANCA) > 160, proteinase 3 antibody 10 ( > 2 positive), myeloperoxidase antibody 8 ( > 6 positive), decreased C3 and C4, microscopic hematuria and negative ANA, anti-Smith, anti-RNP and HIV. The patient was diagnosed with p-ANCA vasculitis secondary to PTU and was treated with pulse dose steroids with stabilization of alveolar hemorrhage. She was extubated several days later. DISCUSSION: PTU is the most common antithyroid drug prescribed for Grave's disease.\nThere have been 5 reported cases in the English literature of PTU-associated ANCA-positive syndrome with the development of diffuse alveolar hemorrhage. All patients had a preceding influenza-like prodrome, and all improved with discontinuation of PTU. Two were treated with corticosteroids and cyclophosphamide, and one with steroids alone. The mechanism of PTUinduced ANCA-positive vasculitis is unknown. It has been proposed that activated neutrophils in the presence of hydrogen peroxide release myeloperoxidase (MPO) which converts PTU into cytotoxic products. Alternatively, in the presence of MPO, PTU is converted to PTU-sulfonate which is immunogenic for T cells. CASE INFORMATION: CASE: A 23-year-old G8P1 female with diet controlled gestational diabetes presented at 33 weeks gestation with dyspnea and productive cough. She was febrile, tachypneic and tachycardic. Her chest film revealed bilateral perihilar infiltrates and left lower lobe airspace disease. She was empirically treated for a community-acquired pneumonia. Initial laboratory results showed a HCO3 of 9, an anion gap of 20, and a glucose of 94. Urinalysis showed greater than 80 ketones and no glucose. Arterial blood gas was 7.32/18/68. Subsequent investigation revealed a lactic acid level of 0.6 and large serum ketones. Shortly after admission, the patient exhibited worsening respiratory distress and polydipsia. Repeat labs showed a HCO3 of 5, an anion gap of 24, a glucose of 113, and an ABG of 7.16/17/68. The patient was started on D5 1/2 NS and a continuous insulin infusion. Continued therapy led to resolution of the anion gap acidosis and clearance of serum ketones. DISCUSSION: Pregnancy is a state of relative insulin resistance marked by enhanced lipolysis and ketogenesis. Under certain circumstances, this hormonal milieu may lead to DKA. DKA can present differently in the pregnant patient, making the diagnosis difficult. The mechanism responsible for insulin resistance in pregnancy involves human placental lactogen, progesterone, cortisol, and prolactin, all of which impair glucose uptake by insulin sensitive cells. The cause of DKA with euglycemia is not well understood, but may result from constant fetal removal of glucose, the expanded blood volume of pregnancy, or the rapid clearance of glucose due to increased glomerular filtration. As seen in our patient, DKA in pregnancy may not present classically. As such, high clinical suspicion of DKA is required in ill gestational diabetics to prevent adverse maternal and fetal outcomes. LEARNING OBJECTIVES: 1). Review the newly described association of osteoporosis and HIV disease 2). Identify the secondary causes of osteoporosis in AIDS patients. CASE INFORMATION: A 39 year old man with AIDS presented to the emergency department with the acute onset of back pain over his thoracic spine. He denied trauma or radicular symptoms. His past medical history included Kaposi's Sarcoma of the lower legs and esophagus, CMV esophagitis, and adrenal insufficiency. He had received multiple antiretroviral regimens over the past 10 years. His current combination therapy included abacavir, lamivudine, and nelfinavir. His exam revealed marked tenderness over the thoracic spine from T4 to T10. His WBC was 3.1, hematocrit was 31%, electrolytes, renal and liver function tests were normal. A recent CD4 count was 10 cells/ul with a viral load of 85,000 RNA copies/ml. Spine radiographs revealed multiple new vertebral fractures at T5, T6 and T9 confirmed by MRI. Bone biopsy at T6, however, revealed normal cellularity without infiltrate, granuloma or obvious infection. A subsequent lumbar DEXA bone mineral density demonstrated a T score of -2.9, consistent with severe osteoporosis. Laboratory evaluation of secondary causes of osteoporosis demonstrated hypogonadism with a free testosterone of 0.8pg/ml (NL 47-244) and a total testosterone of 7ng/dl (NL 400-1080). The patient was managed with opioid analgesics, calcitonin, and calcium with vitamin D supplimentation. DISCUSSION: Metabolic bone disease may represent an important complication of HIV antiretroviral therapy. A few published reports have noted a prevalence of osteopenia or osteoporosis as high as 50% in some groups of HIV patients. This case, however, illustrates the multifactorial nature of osteoporosis and the importance of identifying all of the potential secondary causes. Since AIDS patients have a higher rate of GI malabsorptive syndromes, poor PO intake, immobility, and hypogonadism they are already at a substantial risk for osteoporosis regardless of the potential risk from antiretroviral therapies. As AIDS patients continue to live longer, the cumulative consequences of these risks may lead to a substantial rise in complications from osteoporosis. It is important for clinicians caring for HIV patients to be cognizant of this risk. Recognize accessory spleen as a rare cause of a pancreatic mass 3. Identify strategies to differentiate accessory spleen from a pancreatic hypervascular tumor CASE INFORMATION: A 41 year old female with a past medical history of uric acid nephrolithiasis presented to the Emergency Department complaining of right flank pain and blood in her urine. Her only medication was allopurinol 300 mg daily. Physical exam revealed right flank tenderness to palpation. A spiral CT of the abdomen revealed two renal calculi in the right and one in the left kidney. In addition, a mass was seen in the tail of the pancreas. A CT scan of the abdomen with contrast confirmed a 4.1  4.8 cm hyperenhancing, hypervascular, well defined mass within the tail of the pancreas consistent with an islet cell tumor. Upon further questioning, the patient denied symptoms of peptic ulcer disease (gastrinoma), hypoglycemia (insulinoma), diarrhea and renal failure (VIPoma), hyperglycemia (glucagonoma), or diabetes, steatorrhea and cholelithiasis (somatostatinoma). A presumptive diagnosis of a nonfunctioning islet cell tumor was considered and the patient was referred to a surgeon. A distal pancreatectomy was performed and pathology revealed an intrapancreatic accessory spleen. DISCUSSION: Accessory spleen has been recognized as a rare cause of an intrapancreatic mass. It is seldomly detected clinically due to its lack of symptoms. Due to the rise in abdominal imaging procedures performed, though, the detection of accessory splenic tissue in the pancreas can be expected to increase. Its importance lies in the fact that it can mimic a pancreatic hypervascular tumor. Strategies to differentiate an accessory spleen from a pancreatic hypervascular tumor include angiographic demonstration of splenic blood supply, similar enhancement of the accessory spleen and the spleen on CT or MRI, radionuclide testing or biopsy. In this case, the possibility of an accessory spleen should have been considered and potentially could have avoided unnecessary surgery. (1) to recognize that thyrotoxicosis can potentiate warfarin's anticoagulant effects; (2) to manage warfarin therapy in thyrotoxic patients. CASE INFORMATION: A 59 yo woman with hypertension noted new-onset palpitations and \"shakiness\" at a routine clinic visit. She had no prior history of cardiac or thyroid disease and denied dizziness, loss of consciousness, chest pain, use of new medications, dietary supplements, drugs or alcohol. Current medications included benazepril and aspirin. Physical examination revealed a thin, anxious woman with a HR of 128 and BP of 100/60. She had a prominent, smooth, non-tender thyroid gland. Cardiopulmonary exam was notable for an irregularly irregular tachycardia, a normal S1 and S2 without gallops, and a II/VI short, systolic ejection murmur at the apex. An ECG showed rapid atrial fibrillation, prompting transfer to the emergency department, where work-up revealed a normal PT/INR, creatinine, and hepatic function. After cardiopulmonary stabilization, she was discharged on metoprolol and warfarin 2.5 mg/d. Subsequent TSH < 0.03 mIU/L, free T4 > 71 pmol/L, total T3 of 667 ng/dL, elevated anti-TSH receptors, and abnormal thyroid uptake scan confirmed Graves' disease. The patient's palpitations resolved, but anti-coagulation proved challenging, with INR' s above 4 on average warfarin doses of < 1.0 mg/d (Table I) . She exhibited exquisite warfarin sensitivity until her free T4 levels normalized three weeks after starting methimazole. DISCUSSION: Warfarin exhibits many drug-drug interactions, including an increased effect with l-thyroxin administion. Warfarin potentiation by endogenous thyroid hormone is a less appreciated phenomenon documented in case reports dating to 1972. As this patient's initial INR illustrates, thyrotoxicosis alone does not prolong bleeding times. Rather, thyroxin enhances the metabolic clearance of vitamin K-dependent clotting factors II, VII, IX, and X, resulting in relative hypoprothrombinemia and warfarin potentiation. As in this case, potentiation closely parallels free T4 levels and can occur after one dose. Thus, in thyrotoxic patients, appropriate initial warfarin doses may range from 1.0 mg QOD and require upward titration as hyperthyroidism improves. Awareness of such patients' potential enhanced warfarin sensitivity and judicious anti-coagulation may minimize hospital stays and prevent bleeding complications. A 17-year-old male presented to the emergency room with a history of myalgias for two weeks. Two weeks prior to admission he underwent a dental extraction that required general anesthesia with succinylcholine. He developed symptoms of myalgias four hours after procedure that persisted until readmission. While in the emergency room, he described severe muscle pain in the upper and lower extremities. Enzyme analysis revealed a creatine kinase of 22, 000, and he was admitted for rhabdomyolysis. The patient was treated with IV fluids and rest. His urinalysis was negative for myoglobinuria after hydration. He subsequently underwent a muscle biopsy that revealed a generalized myopathy. After hydration, pain control and initial rest, he gradually improved over the ensuing weeks. DISCUSSION: Growth hormone excess in childhood results in an uncommon condition of giantism. Gigantism causes inappropriate growth of both bones and muscles that results in a large individual. With the continued muscle growth, gigantism can cause an underlying myopathy. Succinylcholine provides muscle relaxation during anesthesia; however, it can cause rhabdomyolysis. This normally occurs in patients with an underlying muscle disorder. Succinylcholine may damage muscle in patients with muscular dystrophy or other primary muscle disorders. This is the first case of succinylcholine-induced rhabdomyolysis in a patient with primary myopathy from gigantism. While unusual, rhabdomyolysis from succinylcholine must be considered in all patients with potential myopathy. Treatment involves IV fluids and supportive care. Sugar water test was negative. Occult mediastianl lymphadenopathy was not found with contrasted chest CT. Bone marrow biopsy showed normocellularity with blasts of less than 5%, myeloid with left shift and one lymphoid aggregate, with adequate numbers of megakaryocytes. The iron was absent. CD59 and CD55 cell markers were normal. Comprehensive studies for lymphoma/leukemia were normal. Autoimmune pancytopenia was confirmed. He was treated with platelet pheresis transfusion initially, which hemolyzed shortly. He responded to IVIG and solumedrol with normalized WBC and platelet count. He was discharged home on prednisone taper. One month follow up showed normal WBC and platelet, but with persistent iron deficient anemia. Pt remained asymptomatic. DISCUSSION: There are three general causes of pancytopenia: splenic sequestion, insufficient production, and excessive destruction. In our case, drug-induced autoimmune processes were suspected after thorough work-up. Treatment for autoimmune hemolytic processes includes steroid, IVIG. If not effective, splenectomy may be an option. Immunosuppressive drugs such as azothioprine and cyclophosphamide, have been used with satisfactory results. Recently, we have had two patients present with autoimmune hematologic disorders within one to two weeks after using Levofloxacin. This association has not been reported previously in the literature. Submission of these cases may assist primary care providers recognize this adverse effect of Levofloxacin.\nPROLONGED MOOD DISORDER AFTER 3,4-METHYLENEDIOXYMETH-AMPHETAMINE (MDMA)USE: A CASE REPORT. S.D. Thakur 1 , L. Coberly 1 ; 1 University of Cincinnati, Cincinnati, OH LEARNING OBJECTIVES: 1) Recognize the potential for prolonged neuropsychiatric illness after methylenedioxymeth-amphetamine use. CASE INFORMATION: A 23-year-old male with no prior psychiatric history except polysubstance abuse, presented with sudden onset of anhedonia, mutism, avolition, feelings of guilt and worthlessness, and hypersomnia after an MDMA binge. The patient had severe paranoid delusions about eating and ultimately quit eating altogether. Hospitalization and a feeding tube were required for his malnutrition. The patient was diagnosed with major depressive disorder with psychotic features, and was started on venlafaxine, olanzapine, haloperidol, and benztropine. His symptoms have continued to persist for several months. He was discharged once but decompensated and attempted suicide within the week. Upon rehospitalization lithium augmentation therapy was implemented. It is postulated that his prolonged psychiatric illness was precipitated by his MDMA use. MDMA ingestion has been linked to acute psychosis, mood disorder, memory and cognitive disorders. Recent reports have implicated its involvement in prolonged mental disturbances as well.\nMDMA is thought to cause potent and possibly permanent neurotoxicity in the serotonin (5HT) pathways. These pathways are the same ones that underlie chronic neuropsychiatric illnesses such as major depressive disorder, psychosis, and panic disorder, supporting the view that MDMA can lead to prolonged neuropsychiatric problems. As in our patient, recovery from the neuropsychiatric complications of MDMA tends to be slow and tedious. Improvement may be explained by compensatory metabolic changes within remaining 5HT and non 5HT neurons, or regeneration of damaged neurons. These mechanisms serve to reverse the 5HT deficit and lead to clinical improvement. MDMA has gained popularity recently as a recreational drug in the high school and college populations. It is commonly referred to as``ecstasy'',``X'',``E'', or``Adam''. It has hallucinogenic and stimulant properties. Due to its potential for severe and chronic complications, it must be considered in the differential diagnosis of new onset psychiatric illness, especially in adolescents and young adults. DISCUSSION: He was discharged home after alcohol counseling. DISCUSSION: Hyponatremia is the most common electrolyte abnormality in hospitalized patients. Symptoms are related to CNS dysfunction and include headache, nausea, vomiting, anorexia, disorientation and depressed reflexes. Symptom severity is correlated with how rapidly the hyponatremia developed. Coma and seizures can occur with acute drops to less than 120 meq/L. The initial approach to diagnosis is measurement of serum and urine osmolality and urine sodium. The most common finding is hypotonic hyponatremia, which is further subdivided by volume status. Our patient was hypovolemic, and low urine sodium implied appropriate renal sodium retention. The diagnosis of beer potomania was also considered, although this is classically a euvolemic state with a low urine osmolality. Treatment should be initiated in all symptomatic patients and those with levels less than 120mEq/L. For hypovolemic hyponatremia, the treatment of choice is normal saline. Overly rapid correction is avoided to prevent central pontine myelinolysis. year-old male with a longstanding history of steroid-dependent asthma and recurrent sinusitis presented with bilateral upper extremity weakness, beginning during a steroid taper. Symptoms then progressed to generalized weakness, malaise, anorexia, and a peripheral eosinophilia. He was given stress dose steroids with prompt resolution of both symptoms and eosinophilia and kept on prednisone 10 mg a day. He then began to develop wasting of his forearms, difficulty in performing tasks using his hands, and difficulty arising from a chair. Medications included salmeterol, fluticasone 220 mcg MDI, and prednisone. On physical examination, he was cushingoid. Lungs were clear. Pertinent neurologic findings included moderate proxminal muscles weakness and motor weakness with atrophy in distribution of the left and right median and ulnar nerve, left radial nerve and bilateral peroneal nerves. Deep tendon reflexes were absent in the right biceps, brachioradialis and left achilles. White count was 22,000 with 30% eosinophils. ACTH stim test was consistent with adrenal insufficiency. Chest X-ray was clear. EMG was consistent with a mixed motor and sensory loss in the ulnar and median nerve distribution. Peroneal muscle biopsy showed eosinophilic infiltration of a blood vessel consistent with a vasculitis. DISCUSSION: Our patient presented with adult-onset asthma, peripheral eosinophilia, and mononeuropathy multiplex. These features are consistent with Churg-Strauss Syndrome (CSS). The muscle biopsy showing eosinophilic infiltration of a blood vessel ensured the diagnosis. The American College of Rheumatology has developed classification criteria by comparing 20 patients who had CSS to 787 control patients with other forms of vasculitis. The six criteria more typical of CSS include: asthma, eosinophilia of greater than 10% , mononeuropathy or polyneuropathy, nonfixed pulmonary infiltrates on CXR, paranasal sinus abnormalities, and biopsy containing a blood vessel with extravascular eosinophils. The presence of any four or more yielded a sensitivity of 85% and a specificity of 99.7%. The natural history of the syndrome follows three clinical phases: (1) prodromal allergic phase (2) allergic rhinitis, nasal polyposis, and asthma and (3) peripheral and tissue eosinophilia. The differential diagnosis includes Wegener's granulomatosis, hypereosinophilic syndromes, polyarteritis nodosa, and microscopic polyangiitis. LEARNING OBJECTIVES: 1) Recognize that an asymptomatic mass on an extremity can be a soft tissue sarcoma. 2) Consider an incisional biopsy in an extremity lesion, particularly if it is larger than 5 cm in diameter. CASE INFORMATION: A 32-year-old African American woman with no significant past medical history presented with a 4 week history of right thigh pain, swelling and hemoptysis. She noted that her right thigh had been swollen without pain for several weeks prior to her presentation. On examination she had a swollen, tender, firm right thigh with right inguinal adenopathy. She had no skin lesions, clear lungs, and a normal pelvic exam. A pap smear was normal. Laboratory data were notable for a hematocrit of 27.7% and a serum iron of 24 ug/dl. Doppler ultrasound studies of the legs were negative for deep venous thrombosis. Chest x-ray showed multiple pulmonary nodules of variable sizes in both lungs, confirmed by a CT scan of the chest which also showed no mediastinal or hilar lymphadenopathy. MRI studies revealed a 6.5Â4.5Â4.5 cm soft tissue mass in the right thigh with lymphadenopathy in the right inguinal, external iliac and common iliac chains, and retrocaval region at the level of the renal veins. A biopsy of the right thigh mass revealed sheets of large monomorphic epithelioid cells which were positive by immunohistochemical studies for cytokeratin and vimentin. These findings were consistent with a diagnosis of epithelioid sarcoma metastatic to the lungs. DISCUSSION: Soft tissue sarcomas account for fewer than one percent of all malignancies and often present as an asymptomatic mass on the extremities of young adults, most often in the third decade of life. Fewer than 20% present as metastatic disease. Because of their slow-growth (over months) and initial presentation, they may be mistaken for other entities (eg. lipomas). Compression of a nerve or blood vessel can cause symptoms. Sarcomas usually spread hematogenously to the lungs, although epithelioid sarcomas can also metastasize through the lymphatic system. Patients with soft tissue sarcomas < 5 cm in diameter have a metastasis-free 5year survival rate of 81%, regardless of tumor grade, depth, and location. In contrast, there is only a 20% 5-year survival rate for patients with lung metastasis. Diagnostic workup should include radiographs, MRI, referral for an incisional biopsy, and chest x-ray or chest CT to search for possible metastatic disease. Because sarcomas are rare and histological interpretation can be difficult, a primary care physician should refer patients to surgeons or centers experienced in the diagnosis of soft tissue sarcomas. The early consideration of this diagnosis can greatly improve patient outcome.\nHYPOGLYCEMIA PRESENTING AS BIZARRE BEHAVIOR. On the day of admission, he was found in his home unresponsive. Emergency medical services were called, and they found his glucose to be 34. Glucose was administered en route to the hospital with an increase in his glucose to 154 and return of consciousness. He had no history of diabetes and denied use of exogenous insulin. Physical exam at the time of admission was normal. Routine labs were all normal except for a glucose of 52. The patient was admitted for a supervised 72-hour fast. Several hours into the fast, the patient's accucheck was < 50, and he again began to exhibit bizarre, child-like behavior. Stat chemistries, serum insulin and C-peptide level were drawn revealing a glucose of 36, insulin 97.1 mU/ml and C-peptide 7.6ng/ml, values consistent with excess endogenous insulin production. A sulfonylurea level was ordered, and a CT scan was scheduled. The CT revealed a 5Â2.5 cm mass in the body of the pancreas and numerous masses throughout the liver. DISCUSSION: This case illustrates the importance of recognizing the signs and symptoms of hypoglycemia in the context of a low glucose value. Low glucose without neuroglycopenia or sympathoadrenal activation may not require further evaluation. However, the presence of these symptoms in an otherwise healthy-appearing, non-diabetic patient with low glucose necessitates a work-up for a hypoglycemic disorder. Careful measurement of insulin, C-peptide, glucose, and sulfonylurea levels can quickly narrow the differential. Whipples Triad (symptoms of hypoglycemia, a glucose of < 50, and relief of symptoms with glucose) is present in the initial presentation of most hypoglycemic disorders.\nCEREBRAL HERNIATION AND FATAL HYPERAMMONEMIA WITH NORMAL HEPATIC FUNCTION. R. Tripathi 1 , E. Warm 1 ; 1 University of Cincinnati, Cincinnati, OH LEARNING OBJECTIVES: 1) Determine differential diagnosis and treatment of severe hyperammonemia and microvesicular steatosis, 2) Describe the mechanism of action of a previously unreported enzyme deficiency (Hepatic Glutamine Synthetase Deficiency) that has fatal consequences, and 3) Discuss appropriate screening for this deficiency. CASE INFORMATION: A 60 year-old white male with a past medical history significant only for chronic obstructive lung disease was admitted to the hospital with shortness of breath and pneumonia. He was treated with piperacillin/tazobactam, steroids and inhalers. On day 2 of hospitalization, the patient developed mental status changes and had an ammonia level of 41. By day 3 he developed seizures, and on day 4 he was comatose with an ammonia level of 2808. At that point, family withdrew all support. Throughout his course, the patient had essentially normal liver enzymes. Autopsy showed pulmonary embolism with infarction, ischemic colitis, cerebellar tonsil herniation, and multifocal areas of microvesicular steatosis in less than 50% of liver. DISCUSSION: Microvesicular steatosis has a limited differential including Reye's syndrome and it is usually not associated with normal liver function enzymes and tremendous increases in ammonia. Similar histopathological and clinical findings as in our patient have been described in patients with heart-lung transplantation. A case series of these orthotopic lung transplant patients with fatal hyperammonemia have shown them to have a hepatic glutamine synthetase deficiency with normal urea cycle and liver function enzymes. The increased ammonia is shunted to the cerebrospinal fluid. Initial increase in ammonia is thought to be secondary to an increased protein load (i.e. gastrointestinal bleeds, TPN) and/or increased catabolic processes (i.e. major surgery). Patients with intact glutamine synthetase enzymes are able to handle this increased ammonia production and there is a pulmonary process in patients with the deficiency for clearing the excess ammonia. A combination of hepatic enzyme deficiency, increased protein load and acute pulmonary disease could be postulated to be the cause of rapid hyperammonemia, cerebral herniation and death in our patient. This is a recently reported entity that may have been underdiagnosed in the critical care setting in the past. In the future, screening for this disorder may be important before patients are candidates for heart-lung transplantation or other pulmonary surgery. It may be important currently to screen patients with mental status changes and no known liver disease with ammonia levels because the only proven treatment modality for severe hyperammonemia is emergent hemodialysis. LEARNING OBJECTIVES: Identify the causes of acute paralysis in the patient who uses drugs.Illustrate the importance of using the history and physical exam to quickly discern the correct diagnosis while definitive studies are pending CASE INFORMATION: A 20 year-old man presented with acute onset of bilateral lower extremity weakness and anesthesia shortly after arriving in New Orleans. He reported passing out for 12 hours after injecting heroin and cocaine. When he regained consciousness, he could not ambulate and had to pull himself to the telephone to call for help. On physical exam, he was afebrile with normal vital signs. He had no heart murmurs and there were no skin ulcerations. He had bilateral loss of sensation in the L4-S2 distribution. He was unable to dorsiflex, plantarflex, invert or evert his ankles. Patellar reflexes were normal; ankle reflexes were absent bilaterally. Flexion of the hips and knees, and sphincter tone were normal. The remaining neurologic exam was normal. The urinalysis showed large blood, but few red blood cells. His serum creatinine was 4.1 mg/dL and the creatinine kinase was 43,900 U/dL. All other laboratory values were normal. The patient was diagnosed with rhabdomyolysis and a compression neuropathy of the peroneal nerves. He received aggressive intravenous hydration; his creatinine decreased to 1.3mg/dL at discharge. He regained sensation and progressive motor function in his lower extremities. DISCUSSION: Acute paralysis is a medical emergency, which requires prompt diagnosis and treatment. The differential diagnosis in the intravenous drug user includes wound botulism, paradoxical embolism with stroke, HTLV-1 with spastic paraparesis, spinal cord compression from epidural abscess, nerve compression and rhabdomyolysis. The history and physical exam can quickly discern the correct diagnosis while definitive studies are pending. The abscence of skin wounds and cranial nerve deficits excluded botulism; the distribution of nerve deficits were inconsistent with a cerebral stroke and there was no murmur on cardiac exam. The time course for the paralysis was too rapid for HTLV-1 infection, and the normal sphincter tone and hip function argued against spinal cord compression. Deductive reasoning led to the diagnosis of nerve compression and rhabdomyolysis, which was confirmed by laboratory testing. Upon further questioning, the patient reported finding himself with his buttocks on the floor, his knees draped over the metal railing of the shower doors and his torso slumped over his lower extremities. This resulted in compression of the peroneal and tibial nerves proximal to the popliteal fossa with subsequent paralysis.\nSHOULD WE SCREEN YOUNG HYPERTENSIVE PATIENTS WITH ACUTE ISCHEMIC STROKES FOR HYPERHOMOCYSTEINEMIA? L. Vaidyanathan 1 , K. Barnard 1 ; 1 University of Pittsburgh Medical Center Shadyside, Pittsburgh, PA LEARNING OBJECTIVES: There is data suggesting that increased plasma homocysteine confers an independent risk for vascular disease (cardiac, cerebral and peripheral). It also (powerfully) increases the risk associated with smoking and hypertension. Therefore, consider obtaining homocysteine levels in young patients with strokes despite the presence of other risk factors. CASE INFORMATION: A 40 year old hypertensive African American male was admitted to the hospital with sudden onset right sided weakness and slurred speech of 8 hours duration. Past medical history includes hypertension (diagnosed at 24 years). Patient is a nonsmoker. Initial evaluation revealed a blood pressure of 200/110 mmHg, right upper and lower extremity weakness with an upgoing right plantar reflex and dysarthria. MRI of the brain showed small vessel ischemia in the lenticulostriate distribution in the posterior limb of the left internal capsule extending into the anterior aspect of the thalamus and corona radiata. EKG showed normal sinus rhythm and there were no arrhythmais on the monitor. Transthoracic echocardiogram only showed left ventricular ejection fraction of 50%. Carotid and transcranial Dopplers were normal. Fasting homocysteine level was increased at 14.4 Umoles/liter (reference < 9Umoles/liter). DISCUSSION: More data is required to determine if hypertensives and smokers under 60 years with acute ischemic stokes, should be screened for hyperhomocysteinemia and if treating these patients with folate and vitamin B6 will decrease their risk of a recurrent cerebrovascular event. We treated our patient with asprin, folate and Vitamin B6 for secondary stroke prevention. LEARNING OBJECTIVES: 1. Recognize the increasing prevalence of Tuberculosis (TB) in the U.S. due to the immigration of high-tech workers. 2. Highlight the diagnostic dilemma a physician can face when the traditional work-up for TB is negative. 3. Highlight the Adenosine Deaminase Test (ADA) as a quick and highly sensitive and specific test to aid in the diagnosis of TB. CASE INFORMATION: A 26-year-old high-tech worker from Madras, India presented with new, severe headache. He denied cough or constitutional symptoms. The patient had a history of``Intestinal TB'' two years prior, with 16 months of medical therapy, the details of which were not known at the time of admission. His physical exam was completely normal, his chest X-ray showed a diffuse miliary pattern, and a head CT showed multiple ring-enhancing lesions. An extensive work-up for TB was undertaken, all of which was negative. Additionally, tests for fungi, HIV, bacteria, viruses and malignancy were negative. The patient's headache continued to worsen, so anti-tubercular therapy was started along with prednisone to cover for possible neurosarcoidosis. The patient did quite well and was discharged home soon thereafter. One week later, the patient returned with altered mental status. A new CT scan showed massive hydrocephalus. A ventriculostomy was placed and the CSF was analyzed for TB; the AFB smears and PCR were once again negative. An Adenosine Deaminase Test of the CSF was positive. Treatment was initiated to cover for multi-drug resistent TB. An open lung biopsy was performed to harvest tissue for culture and a few AFB were identified by smear. The original sputum culture finally turned positive for TB eight weeks after the patient's initial presentation. The patient improved slowly over the next several weeks and was eventually discharged in stable but neurologically impaired condition. DISCUSSION: CNS TB is uncommon, but is in the differential diagnosis of ring-enhancing lesions. It occurs in only 10% of immunocompetent patients and is highly lethal with mortality exceeding 25%. Even if a patient has widespread TB, the diagnosis of TB can be very difficult and require an extensive, invasive investigation. The ADA test is highly sensitive (100%) and specific (99%) for TB when compared to culture-proven disease in the CNS and lungs. It is an excellent test to aid in the diagnosis of TB and should be considered in the routine work-up whenever CSF or pleural fluid are analyzed.\nWHEN NEW ONSET POLYURIA AND POLYDIPSIA DOES NOT EQUAL DIABETES. E.W. Vogel 1 ; 1 MCP Hahnemann University, Philadelphia, PA LEARNING OBJECTIVES: 1. Recognize severe hypercalcemia as a cause of polyuria and polydipsia, and check for hypercalcemia in patients with polyuria when diabetes has been ruled out. 2. Recognize parathyroid carcinoma as a cause of severe hypercalcemia. CASE INFORMATION: A 37 year old African American female with no significant past medical history presented with a two to three week history of increasing thirst, leading to intake of approximately three liters of water per day, associated with urination every 30 minutes. She also noted some polyphagia, dizziness, and mild blurred vision. She had a strong family history of type 2 diabetes, with her mother, brother, and uncle all having diabetes. On a brief initial physical examination, no significant abnormalities were detected. Somewhat surprisingly, her office finger stick glucose measurement was only 106 mg/dl. A basic sevenitem chemistry panel, urinalysis, and hemoglobin A1C were sent to the lab from the office, and the patient was released to home. Although not specifically ordered initially, a calcium level included with the chemistry panel came back at 14.5 mg/dl; her glucose and hemoglobin A1C were normal. The patient was admitted to the hospital for treatment, and on reexamination was found to have a hard left thyroid mass. Her parathyroid hormone level was markedly elevated at 485 pg/ml, with normal thyroid function tests. After resolution of her hypercalcemia with intravenous normal saline and pamidronate, she underwent surgical neck exploration, and was found to have a tumor extending from her thyroid to local skeletal muscle and the esophagus. Pathological examination revealed parathyroid carcinoma. The patient later was found to also have metastatic disease in her lungs. Despite primary tumor resection, local neck irradiation, chemotherapy, and multiple outpatient doses of pamidronate, the patient has required several admissions for management of recurrent, severe hypercalcemia. DISCUSSION: Severe hypercalcemia can have a number of clinical manifestations, including polyuria and polydipsia. Interestingly, this patient manifested none of the more common findings of hypercalcemia at the time of presentation, such as bone pain, renal colic from nephrolithiasis, or gastrointestinal or neurological symptoms. Clinicians should therefore consider hypercalcemia in the differential diagnosis of polyuria, especially when diabetes has been ruled out. In this case, the serum calcium was not ordered initially, but luckily this laboratory routinely includes a calcium level with the chemistry seven panel, which established the diagnosis. Parathyroid carcinoma is a rare cause of hypercalcemia; an underlying parathyroid cancer causes only 1 to 2 percent of cases of primary hyperparathyroidism. This patient had two of the warning signs for parathyroid carcinoma as the cause for primary hyperparathyroidism: severe hypercalcemia at the time of presentation ( > 14 mg/dl), and a palpable neck mass. This patient also typifies the clinical course of parathyroid carcinoma that is not cured by primary surgery, in that she has developed recurrent episodes of severe hypercalcemia. This persistent hypercalcemia eventually results in renal and cardiac complications, accounting for most of the morbidity and mortality associated with this disease.\nNOT ALL VOMITING IN PREGNANCY IS HYPEREMESIS. S. Vora 1 , R.O. Powrie 1 ; 1 Brown University School of Medicine, Providence, RI LEARNING OBJECTIVES: 1. Recognize the differential diagnosis of hyperemesis gravidarum and illustrate the role of a general internist in diagnosis and management of medical illness in pregnancy. 2. Demonstrate the clinical presentation of mesenteric thrombosis. 3. Emphasize the fact the pregnancy is a hypercoaguable state and briefly outline the management of thrombosis in pregnancy. CASE INFORMATION: A 37 year-old primigravid Nigerian woman presented in her 19th week of an IVF pregnancy to the emergency room with complaints of two days of fever and constipation and three weeks of malaise, anorexia, and vomiting. She had been treated in the ER on three prior occasions with hydration and antiemetics for hyperemesis gravidarum. On this last presentation, she produced her photograph from one month prior to demonstrate the extent to which her appearance had deteriorated. Her past medical history was significant for only childhood malaria and sickle cell trait. She had not suffered from protracted nausea and vomiting of pregnancy during the first trimester. She was taking only prochlorperazine suppositories at the time of presentation. She had no personal history of smoking, alcohol or recreational drug use. Her examination was significant for a temperature of 101.6 F, a heart rate of 114 per minute, respiratory rate of 12 per minute, and BP 103/60. She was mildly jaundiced with sunken orbits. Her cardiopulmonary examination was normal. Her abdomen was markedly tender in the right upper quadrant with voluntary guarding but no rebound. No fluid wave was present and her rectal examination was normal. Her uterus was gravid and nontender to palpation. Her fetal heart tracing was reassuring. Neurologic examination was unremarkable. Her laboratory examination was remarkable for a leukocytosis of 36.6 with a left shift. Her chemistries revealed a hypochloremic metabolic alkalosis with a serum bicarbonate of 34 mEq/ L. She had a mild elevation of her serum AST to 40 U/L. The remainder of the laboratory data was unrevealing. A malarial blood smear was negative. An abdominal ultrasound revealed absent portal venous flow, but was otherwise normal. A subsequent CT scan of the abdomen demonstrated thrombosis of the portal vein and superior and inferior mesenteric venous branches. The patient was admitted, treated with intravenous unfractionated heparin, gentamycin and piperacillin and achieved significant improvement in pain. She defervesced by the second hospital day. Her liver enzymes normalized. Her hypercoaguable work-up revealed only a relative protein S deficiency. Once therapeutic on intravenous heparin, she was changed to low molecular weight heparin, which she continued until 36 weeks gestation. At that time her therapy was changed to adjusted dose subcutaneous unfractionated heparin until her uncomplicated cesarean delivery of a healthy 2475 g girl. DISCUSSION: Nausea and vomiting occurs frequently during pregnancy, especially during the first 20 weeks, afflicting 70-90% of all pregnant women. Causes can be physiologic, due to progesterone effects on the lower esophageal and pyloric sphincters, or due to mechanical compression of the diaphragm on the enlarging uterus. However pregnant women are also subject to all other causes of nausea and vomiting, such as severe gastroenteritis, cholecystitis, pyelonephritis, hyperthyroidism, primary hyperparathyroidism, liver dysfunction, and rarely, small bowel obstruction, mesenteric thrombosis or mesenteric ischemia. Internists can be instrumental in assisting in these less common medical diagnoses during pregnancy. Portal and mesenteric venous thromboses are rare conditions, even in nonpregnant patients. Most patients present with at least two weeks of abdominal pain, anorexia, vomiting, and change in bowel habits. Many patients are also febrile. In 20% ndash;40% of cases of mesenteric thrombosis no underlying cause can be found. Other investigated cases of mesenteric thrombosis during pregnancy have revealed inherited thrombophilias such as activated protein C resistance, antithrombin III deficiency, and protein C and S deficiency. The latter is a difficult diagnosis in the face of both active thrombosis and in pregnancy, since protein S normally decreases in both of these settings. The treatment of acute thrombosis involves heparin (either low molecular weight or unfractionated), as warfarin is contraindicated in pregnancy. Special care is required in the management of anticoagulation in the peripartum period. Recognize the importance of ductography and fiberoptic ductoscopy, the later being a new technology for nipple discharge. CASE INFORMATION: A 45-year-old woman presented with a 1 day history of a spontaneous amber right nipple discharge. Breast examination revealed fibrocystic changes. A small amount of clear, non bloody fluid could be expressed from one of the right breast ducts. There was no palpable lymphadenopathy. A mammogram revealed fibroglandular densities. Ultrasound (US) of the right breast showed slightly dilated ducts. Ductography was performed as follows: by applying focal pressure a single draining orifice was identified, cannulated and 0.2 cc of Conray 60 were injected. Post-mammographic images showed the presence of a cystically dilated duct that contained a small 3 ± 4 mm intraluminal filling defect, which now could also be visualized by repeat US examination. Multiple core biopsies were obtained under US guidance using a vacuum assisted biopsy (bx) needle. A small clip was deployed in the bx cavity following the procedure. Histology revealed high grade ductal carcinoma in situ (DCIS). The patient was seen in surgical consultation and underwent an attempted wide local excision, which revealed extensive DCIS, 5 cm in greatest extent. There were multiple microscopic foci of invasive cancer. The surgical resection margins were positive for DCIS after multiple re-excisions. After thorough discussion the patient elected to undergo bilateral mastectomies. Two sentinel lymph nodes (blue dye and lymphoscintigram) contained micrometastatic adenocarcinoma. The patient is presently undergoing adjuvant chemotherapy. DISCUSSION: This case exemplifies the importance of an extensive evaluation of a pathologic nipple discharge. While a bilateral, inducible nipple discharge involving multiple ducts is a physiologic finding in many women, patients presenting with a spontaneous, unilateral discharge (bloody or watery) confined to one duct need careful evaluation for an underlying neoplasm (papilloma, carcinoma). A small malignancy, which in our patient had already involved axillary nodes, can be easily missed on physical examination and mammogram. In this setting an US examination in the \"trigger zone\" may identify an occult lesion. Cytologic evaluation of the discharge or ductal lavage fluid may be helpful. An important diagnostic tool is ductography, as performed in our patient. With the recent introduction of fiberoptic ductoscopy direct, realtime intraductal images, allowing detection of lesions as small as 0.2 mm can be obtained offering a safe alternative to ductography in guiding subsequent breast surgery in the treatment of nipple discharge. (Shen K-W. Breast Cancer Research and Treatment, Vol. 64, No 1, Nov. 2000, p. 30) ACUTE METHEMOGLOBINEMIA SECONDARY TO TOPICAL BENZOCAINE SPRAY. J. Walker 1 , H. Houston 1 , S. Miller 1 , G. Rouan 1 ; 1 University of Cincinnati, Cincinnati, OH LEARNING OBJECTIVES: 1) Define the pathophysiology associated with methemoglobinemia, and 2) Recognize the clinical settings likely to be explained by methemoglobinemia. CASE INFORMATION: JT is an 82-year-old white female with a history of HTN, paroxysmal atrial tachycardia, and hypothyroidism who was admitted for elective laparoscopic left adrenalectomy, which proved to be a benign adenoma. Post-operative course was uncomplicated until POD #3 when the patient developed nausea and vomiting with a distended abdomen and decreased bowel sounds. A diagnosis of paralytic ileus was made and a nasogastric tube was placed, preceded by a 3 second dose of Hurricane spray (20% Benzocaine, approximately 600 ± 885 mg total dose) to the oropharynx. Within several minutes, the patient developed lethargy and confusion. The patient then became dyspneic, syncopal, and developed a cyanotic appearance. She did not respond to 100% O 2 by non-rebreather mask despite an arterial blood gas (ABG) with a PaO2of 310 and an O2saturation of 99.7%. A simultaneous pulse oximetry revealed an O2sat of 86%. The arterial blood was extremely dark in appearance. An ECG revealed supraventricular tachycardia with a rate of 170 ± 180 and ST depression, which converted after 6mg adenosine to sinus rhythm, with a rate of 120 and resolution of the ischemic changes. A diagnosis of methemoglobinemia was confirmed by co-oximetry, which revealed a methemoglobin level of 30.9% (normal = 0%). A 50 mg dose of methylene blue was administered, and over the course of 1 hour, the patient developed dramatic improvement in mental status and cyanosis. Methemoglobin levels decreased to 6.4%, 3.7% and 2.4% over the next 1, 3, and 6 hours, respectively. DISCUSSION: Methemoglobinemia is a condition in which iron in hemoglobin becomes oxidized (Fe 2+ to Fe 3+ ) at an overwhelming rate, resulting in a drastic decline in the oxygen carrying capacity of the RBC. Pharmacological agents, such as benzocaine, are capable of inducing methemoglobinemia by direct or indirect oxidation of the hemoglobin molecule. The ABG is not an accurate indicator of oxygenation status in a patient suffering from acute methemoglobinemia. The treatment of choice is methylene blue (1mg/kg, IVP). This case of benzocaine-induced methemoglobinemia is similar to others that have been reported in the past and indicates the need for a high index of suspicion for this syndrome and a need for close supervision of patients receiving benzocaine spray for clinical signs and symptoms of cyanosis. 1) Recognize the importance of the life narrative in the comprehensive understanding of the adolescent who presents with somatic distress and chronic fatigue. 2) Distinguish between diagnoses that denote a disease process (eg anemia) and those that are strongly influenced by cultural``memes'' (eg chronic fatigue syndrome). CASE INFORMATION: CASE 1: A 17 y/o white teenager developed increasing fatigue beginning at age 15. Other symptoms included daily fever spikes, visual changes, myalgias, and headaches. Her grades suffered greatly as a result of repeated absences from school, & she also had to quit her part-time job. At the age of 5, she witnessed her 3 y/o brother drown in their pool while her mother was in the house. Her parents subsequently divorced when she was 12, & she has a strained relationship with her alcoholic father. The patient received counseling after the drowning and at the time of the divorce. CASE 2: A 18 y/o high school senior was diagnosed with chronic fatigue syndrome (CFS) at the age of 13. She was unable to attend school fulltime because of sleep disturbance, diffuse pain, & disabling fatigue. Both her parents were alcoholic, and her dad was verbally & physically abusive. She served as a caretaker for her wheelchair-bound mother. At the age of 12 she was raped by her half-sister's 29 y/o boyfriend. At the age of 17 while being treated with an antidepressant for depression, her psychiatrist would not allow concurrent oral contraceptives & she became pregnant and had an abortion. DISCUSSION: Many hypotheses have been put forth to explain CFS, an illness in search of an etiology. Characterized by a variety of somatic symptoms, CFS often has an abrupt onset. We postulate that it may be a form of \"post-traumatic stress disorder,\" particularly in young patients. Children with CFS have been reported to exhibit more psychological morbidity, such as anxiety and depression, than those with chronic disease, but these psychological categories are not necessarily helpful in patient management. We have found that the life narrative which will often include antecedent traumatic events serves to illuminate the unique personal issues of the adolescent with CFS. This approach can create a doctor-patient relationship that will engender trust and perhaps lead to better clinical outcomes. LEARNING OBJECTIVES: 1) Recognize Salmonella Hepatitis as a cause of acute hepatitis in a patient with typhoid fever. 2) Mortality rate is as high as 20% particularly with delayed treatment. The prognosis is good if treated early with specific antibiotic therapy. CASE INFORMATION: A 22 year old female ,immigrant from Bangladesh with no past medical history was admitted with complaints of fever, nausea ,vomiting and headache of one week duration. The patient had visited Bangladesh three weeks back. Prior to this presentation she had two visits to the ER in the same week and was diagnosed as viral syndrome. On admission now the patient was delirious and agitated .Her BP was 112/62; Pulse-70; Temperature-105 deg. Farenheit. Physical Examination -Right hypochondriac tenderness, no hepatosplenomegaly, no neck rigidity, no icterus, no skin rash. Lumbar puncture and CT Scan of head were normal. USG Abdomen was normal and HIDA Scan showed no uptake. There was a moderate elevation in the Transaminase level with AST/ALT of 294/205 respectively, LDH 579,Total bilirubin of 1.3 and direct-0.5.Albumin PT/APTT and Reticulocyte counts were normal. WBC count was 8300 with 10% band cells. Blood cultures later came positive for Salmonella Typhi sensitive to ceftriaxone. Hepatitis serology was negative for A,B and C.The patient was started on ceftriaxone .LFT's improved and patient was discharged home after 7 days in stable condition. DISCUSSION: Salmonella hepatitis is a rare condition and 20% of the patients with salmonella hepatitis may not be bacteremic. A diagnosis of salmonella hepatitis was made on the basis of 1)ALT/LDH < 4 (0.35 in this case).This happens to be the best discriminator between Salmonella hepatitis and viral hepatitis as is appreciated in this case.2) Positive blood culture for Salmonella.3) Relative bradycardia. 4) High-grade fever.5) Left shift of WBC and 6) Positive travel history to an endemic area. The prognosis is good if treated early with specific antibiotic therapy, however the clinical course can be severe with a mortality rate as high as 20% particularly with delayed treatment. the malar eminences, neck and trunk with bilateral axillary and submental lymphadenopathy. Neurological exam was normal and no joint deformity was noted. There was generalised abdominal tenderness with no guarding or organomegaly. Investigations revealed Hb-10.6 gm/dl and total WBC-3.4K/ul, BUN-23mg/dl, creatinine-0.9 mg/dl. Liver function tests showed albumin-2.3 g/dl, AST-54, ALT-26, GGT-53U/L, LDH-485 U/L. Serum amylase was 401 and serum lipase 358. Serum calcium was 8.9 with triglycerides of 187mg/dl. Urine analysis showed specific gravity of 1015 with 3+ proteinuria. CT scan of abdomen showed fullness of pancreatic head and ultrasound showed a normal sized CBD with no evidence of cholelithiasis. Biopsy of skin rash showed intravascular thrombi, vasculitis consistent with early lupus with atypical lymphocytic infiltrate. Diagnosis of SLE with acute pancreatitis was made on the the basis of above and ANA of 1:1280 with homogenous pattern and Anti ds DNA positivity. Patient was started on intravenous steroids. Patient's clinical status continued to worsen and required mechanical ventilation. Repeat CTscan confirmed pancreatitis with bilateral pleural effusions and no evidence of necrosis or abscess formation. She improved over the next few days and was successfully extubated. However, two weeks later she developed diffuse and severe abdominal pain accompanied with fever. Repeat CTscan now showed Grade E pancreatitis(multiple fluid collections +/À gas in or adjacent to pancreas)with necrosis. CT guided aspirations of necrotic material was negative for gram stain but culture grew pseudomonas, MRSA and candida. The patient had an exploratory laprotomy with necrotectomy. Despite aggressive antibiotic and antifungal treatment with multiple abdominal explorations the patient's condition deteriorated. She died of multiple organ failure. DISCUSSION: Acute pancreatitis is an uncommon manifestation of SLE. It is even rarer for such patients to present with pancreatitis as the sole major organ affected. The role of corticosteroids in such setting is controversial. Review of literature suggests that corticosteroids do not cause pancreatitis in patients with SLE and they should be used during episodes of pancreatitis if required. He also noted pruritic, non-tender skin lesions on his face and arms. He denied the use of any medications. Review of systems revealed a 10 lb. weight loss, blurry vision, nonproductive cough, hoarseness and mild dyspnea on exertion. He denied any HIV risk factors. Physical examination revealed widespread, small, hyperpigmented, nodular and papular lesions. A 3Â2 cm subcutaneous nodule was noted on the left side of the neck. Breath sounds were decreased bilaterally, with dullness to percussion on the left. The testicles were each twice normal size and tender with enlarged epididymi. Vital signs and the remaining exam were unremarkable. Scrotal ultrasound confirmed the physical exam and showed no discrete masses. Chest x-ray and CT revealed a moderate left pleural effusion with mediastinal and hilar adenopathy but no infiltrates. PFTs were consistent with a mild restrictive pattern. Serum ACE level was elevated at 87. Skin biopsies revealed non-caseating granulomas, negative for AFB or fungi. Follow-up scrotal ultrasound after corticosteroid therapy showed regression of testicular abnormalities. DISCUSSION: Sarcoidosis is a disease of unknown etiology, which can present with a wide variety of symptoms and involve virtually any organ system. In this unusual case, the patient presented for care due to testicular involvement but also had dermatologic and pulmonary manifestations, which he had ignored. While many cases of sarcoidosis spontaneously remit within 5 years and do not need treatment, therapy is indicated in patients such as this one, with symptomatic disease or systemic involvement. The goals of treatment include a reduction in symptoms and the avoidance of systemic complications. Despite attempts with steroid-sparing agents, the mainstay of treatment remains systemic corticosteroids. Monitoring with chest x-ray and PFT's is often warranted and patient education remains essential. SARCOIDOSIS AND STONES. K. White 1 , U. Mason 1 ; 1 Denver Health Medical Center, Denver, CO LEARNING OBJECTIVES: 1) Diagnose renal manifestations of sarcoidosis, and 2) Recognize the need for monitoring calcium levels and renal function in sarcoidosis. CASE INFORMATION: A 43-year-old man with a long history of cystic sarcoidosis presented with acute renal failure. He had chronic renal insufficiency with a baseline serum creatinine of 1.4 mg/dL. He had been treated with prednisone for several years and his dose had been tapered recently because his pulmonary status appeared stable. Review of systems ascertained a 10 ± 15 pound weight loss and decreased energy for 2-3 months. Physical examination was unrevealing. Laboratory data was significant for serum creatinine of 2.5 mg/ dL and calcium of 11.3 mg/dL. Renal ultrasound showed right hydronephrosis and bilateral nephrolithiasis. Computed tomography showed left staghorn calculus with caliectasis and right ureteropelvic junction stone with hydronephrosis, hydroureter and a more distal stone. A right percutaneous nephrostomy was placed and serum creatinine decreased to 1.8 mg/dL. Evaluation of the etiology of hypercalcemia included normal serum PTH, protein electrophoresis and PSA levels. Urinary calcium excretion was 252 mg/24 hours. The hypercalcemia was felt to be secondary to sarcoidosis and therefore the prednisone dose was increased. He underwent right nephrolithotomy, which was successful only for the proximal stone. Bilateral stone removal is planned for the future. Serum creatinine is back to baseline and serum calcium is normal. DISCUSSION: Hypercalcemia -an uncommon complication of sarcoidosis ± results from endogenous overproduction of 1,25 dihydroxyvitamin D. Nephrolithiasis is more common, occurring in about 10% of patients, and more frequently associated with hypercalciuria than with hypercalcemia. Nephrocalcinosis is much less common and is probably related to chronic hypercalcemia. This patient had both nephrocalcinosis and nephrolithiasis, likely due to both long-standing hypercalcemia and hypercalciuria leading to chronic and then acute renal failure. Long-term management of patients with sarcoidosis includes frequent monitoring of calcium levels and renal function.\nA PRESENTATION OF SHEEHANS SYNDROME 22-YEARS LATER. W. Whitwam 1 , D. Stuart 1 ; 1 Hennepin County Medical Center, Minneapolis, MN LEARNING OBJECTIVES: 1) Recognize complications of analgesics associated with unrecognized hypothyroidism. 2) Understand the complications of Sheehan's syndrome and panhypopituitarism. CASE INFORMATION: A 62-year-old African-American female, with a past medical history notable only for a prior cesarean section, received an uneventful laparoscopic repair of a ventral hernia. Post-operatively, she received morphine analgesia by a patient controlled analgesia (PCA) pump. After developing an unanticipated stupor, her morphine was discontinued. She received several doses of naloxone without response. A computerized tomography (CT) of the head demonstrated no acute changes. With an arterial pCO2 value of 62 mm Hg, she was placed on biphasic positive airway pressure (BiPAP) ventilation. She remained somnolent. A formalized evaluation of her delirium included a thyroid function panel and random cortisol level, both studies demonstrated abnormally low values. A magnetic resonance image (MRI) of her brain revealed a partially empty sella turcica. Her normal luteinizing hormone (LH) and folliclestimulating hormone (FSH) levels were discordant with her post-menopausal status, and her prolactin level was below the normal reference range. After receiving thyroxine and hydrocortisone replacement therapy, her depressed consciousness resolved within two days. Upon a later interview, she revealed that 22-years prior she had a cesarean delivery of her seventh child, resulting in a large blood loss and subsequent hypotension. Unlike her six previous pregnancies, she failed lactation after delivery, and subsequently never resumed menstruation. DISCUSSION: Sheehan's syndrome is an obstetric complication that occurs with excessive blood loss and ischemic shock following a complicated delivery. This results in an infarction of the pituitary's anterior lobe. As a result of her hypopituitarism, this patient developed secondary thyroid and adrenal failure as well as gonadal insufficiency. With an unrecognized secondary hypothyroidism, the exaggerated depressant effects of her analgesics resulted in a postoperative stupor. Careful attention to hypothyroidism should be noted with any patient who develops an excessive central nervous system response to anesthetics or analgesics.\nEPISODIC HYPERTENSION ASSOCIATED WITH SITTING ON HARD SURFACES. R.M. Witteles 1 ; 1 Stanford University, Stanford, CA LEARNING OBJECTIVES: 1. Distinguish between the potential causes of episodic hypertension. 2. Recognize the practical consequences of the complex relationship between pain and elevated blood pressure. CASE INFORMATION: A 76-year-old Japanese-American male was referred to the hypertension clinic for a long-standing history of baseline normotension interrupted by hypertensive episodes associated only with sitting on hard surfaces. Home blood pressure measurements (consistent with measurements in clinic) ranged between 110 ± 120/70 ± 80 mm Hg, but rose to 180 ± 190/100 ± 110 mm Hg immediately after the patient sat on a hard surface such as an ordinary chair; no change in blood pressure from normal values was observed when the patient sat on a soft surface or was in any other position. Measured blood pressure was the same in both arms, and returned to a normal range immediately after the patient stopped sitting on a hard surface. The patient experienced no symptoms when sitting on a hard surface except for bilateral blurry vision; this too promptly returned to normal after getting up from the hard surface. At various times in his life, the patient had taken reserpine, lisinopril, verapamil, enalapril, atenolol, hydrochlorothiazide, and clonidine to control his hypertensive episodes, always without efficacy; he is currently taking no anti-hypertensive medications. Of possible relevance, the patient had chronic neck pain which was exacerbated by sitting on hard surfaces; however, the change in blood pressure reliably and reproducibly occurred before the pain. The patient had normal urinary catecholamines, vanillylmandelic acid, and metanephrine values, normal thyroid studies, no evidence of panic disorder, and no evidence of end-organ damage. A trial of opioid analgesics had no effect on the patient's blood pressure. DISCUSSION: Episodic hypertension is a relatively uncommon clinical entity, most often associated with the \"white-coat\" phenomenon, pheochromocytoma, ingestion of sympathomimetic drugs, withdrawal from alcohol and other drugs, thyrotoxicosis, panic disorder, and intermittent pain. Prior studies have discovered a complex relationship between pain and blood pressure, with involvement at multiple areas of the central nervous system. This patient demonstrated an unusual temporal relationship between pain and blood pressure, with the rise in blood pressure occurring first; the hypothesis of a conditioned response should be considered.\nBELLS PALSY AND HYPERTENSION SECONDARY TO DISCONTINUATION OF ANTIHYPERTENSIVE TREATMENT. S. Yakoob 1 , K. Barnard 1 , J. Haretos 1 ; 1 UPMC Shadyside, Pittsburgh, PA LEARNING OBJECTIVES: 1. Recognition of an association between facial nerve paralysis and accelerated hypertension.2. High dose corticosteroid therapy often prescribed for facial nerve palsy has serious consequences in hypertensive patients.3.Facial palsy resulting from severe hypertension may be distinguished from Bell's palsy by lack of pain and preservation of taste. CASE INFORMATION: An 84-year-old male who presented with a two-day history of rightside facial drooping, difficulty in chewing and inability to close his right eye. His past medical history is significant for hypertension controlled on quinapril and bisoprolol/HCTZ. Two days prior to the onset of symptoms, the patient had discontinued his medications. Physical examination was remarkable for a blood pressure of 190/96 and signs of right lower motor neuron facial nerve palsy. Diagnostic evaluation included CT scan of the brain that was negative for hemorrhage and a maxillofacial CT that showed thickening of paranasal sinuses. An EMG revealed decreased conduction over the right facial nerve.The patient's blood pressure was controlled by oral metoprolol and quinapril. High dose corticosteriod therapy was started and subsequently discontinued when the patient's blood pressure increased to 205/ 105 mm Hg. This case is similar to two other case reports in the literature of abrupt withdrawal of antihypertensive treatment resulting in an acute increase in the blood pressure and subsequent facial paralysis. DISCUSSION: LEARNING OBJECTIVES: To recognize toxic methemoglobinemia as a rare but potentially lethal complication of local anesthetics so that prompt diagnosis and treatment can be initiated. CASE INFORMATION: A 37 year old white female patient with negative past medical history, presented to our clinic with a complaint of epigastric pain and was scheduled for endoscopy. Benzocaine spray was administered topically in the oropharynx during endoscopy and the procedure was uneventful, but approximately 10 minutes after completion, the patient oxygen (O2) saturation began to fall and she became cyanotic despite administration of 100% O2 via a non-rebreathing mask. Intravenous Narcan and Flumazenil were administered without improvement. The O2 saturation continued to fall with the lowest level obtained was 81%. Arterial blood gas analysis showed PaO2 of 367mmHg and PaCO2 of 23mmHg so methemoglobinemia was suspected. Methemoglobin level was sent and later showed a level of 37.6%. Methylene blue was given intravenously, the patient started to improve gradually and within 20 minutes O2 saturation rose to 96% and cyanosis was resolved. The patient was transferred to intensive care unit, where her O2 saturation rose to 99% and methemoglobin level dropped to 1% . The remainder of hospital stay was unremarkable. DISCUSSION: Methemoglobinemia is a rare complication of local anesthetics that has been reported with procedures including bronchoscopy, endoscopy, tracheal intubation and dental procedures. Methemoglobin is formed normally in the body because small amount of ferrous iron in hemoglobin is continuously oxidized to ferric iron which can't carry oxygen or carbon dioxide. Red cell defense against accumulated methemoglobin is methemoglobin NADH reductase which is responsible for 99% of in vivo reduction of methemoglobin to form normal hemoglobin. Methemoglobinemia has both acquired and congenital forms. Acquired methemoglobinemia can be associated with toxic shock or can result from ingestion or skin exposure to oxidizing agents which oxidizes hemoglobin to methemoglobin. Although cyanosis may appear with levels as low as 15%, acquired methemoglobinemia is rarely symptomatic when levels are below 20%. Lethargy, dizziness, lightheadedness and anxiety are present between levels of 30 ± 40%. Coma, seizures, arrhythmias and acidosis could be caused with levels of50 ± 70%. Levels more than 70% are fatal. We were first alerted to the presence of methemoglobinemia by decrease in O2 saturation inspite of normal O2 tension (PaO2) which is almost a universal finding. Methylene blue given intravenously 1 ± 2mg/Kg over 5 minutes acts a cofactor in the transfer of an electron from NADPH to ferric iron and methemoglobinemia should resolve within one hour, if not the dose may be repeated. LEARNING OBJECTIVES: 1) To consider dural venous sinus thrombosis in the differential diagnosis of puerperal psychoneurotic symptoms. 2) Initiating management as soon as the diagnosis is recognized can cause major decrease in morbidity and mortality. CASE INFORMATION: 21 year old female patient with no significant past medical history, had normal vaginal delivery 3 weeks prior to admission, presented with depressed mood, poor concentration and headache. Physical examination was negative. Brain computed tomography scan (CT) was done and found to be negative so the patient was diagnosed as a case of postpartum depression and was admitted to psychiatric floor. One day after admission patient became drowsy and her level of consciousness dropped. Head and neck CT scan with contrast showed thrombosis of the right jugular vein. Magnetic resonance image (MRI), magnetic resonance angiography (MRA) and magnetic resonance venography (MRV) were done and showed thrombosis of the superior sagittal sinus, right transverse and deep venous sinus. Patient developed respiratory distress and was intubated and was started immediately on Intravenous Heparin. After few days she was extubated and transferred to medical floor where she was switched to oral anticoagulants. Patient depressive symptoms improved and her mood returned back to normal. DISCUSSION: Dural venous sinus thrombosis is an unusual disorder most often attributed to hematological disorders, oral contraceptives, association with pregnancy and puerperium, Bechets disease, cardiac disease and post operative conditions. Patients usually present with nonspecific symptoms as headache, seizures, hemiplegia or other neurological symptoms. Brain swelling and bilateral involvement can produce lethargy or stupor early in the course. Diagnosis depend on recognition of impaired venous flow. On contrast CT scan a nonenhanced triangular area surrounded by contrast in the posterior sinus (the empty delta sign), should suggest the diagnosis. Cerebral angiography has been considered the gold standard for the diagnosis but MRI, MRA and MRV are becoming the diagnostic studies of choice.Management increasingly relies on the use of anticoagulation even in the presence of superimposed parenchymal hemorrhage. Venous occlusions are serios and often fatal but acute anticoagulation started as soon as the diagnosis recognized appears to lessen substantially the morbidity and mortality of the condition. Nonanticoagulated venous sinus thrombosis that is not complicated by infection carries a mortality rate of 25 to 40% . Uncontrolled series suggest that early heparin treatment can reduce mortality by more than a half. OBJECTIVES: Reflective practice may enable physicians to become more competent and successful practitioners. This technique, however, is usually developed at the faculty level. The objective of this workshop is to begin to develop this skill at an earlier level. Students may then begin to utilize this skill for success at the student/resident level as well as honing this skill for use when at the faculty level, for both clinical practice and teaching. METHODS: Students will be given a brief (approximately 1 1/2 hour)skill session in the use of reflective practice. Intervention outcome will be assessed using the following:Pre/post test for knowledge, skills and attitudes regarding reflective practice.Randomization of the same class of students (Class of 2005)À 1/2 to intervention, 1/2 no intervention. Each group will then be given a reflective practice questionnaire following an OSCE exercise, to assess utilization of the skill. OBJECTIVES: The UCSF Division of General Internal Medicine (DGIM) faculty development program trains general internists to be effective teachers in primary care settings that serve a diverse patient population. During the first year of our program, we recruited general internists from community based health centers and faculty of the DGIM at UCSF. We developed and taught a 9-month long curriculum with six core content areas: teaching skills, evidence based medicine, cultural competency/caring for vulnerable patients, psychosocial medicine, population medicine, and leadership. Sessions were 3.5 hours long, two afternoons per month. METHODS: Prior to implementing our program we conducted a baseline needs assessment among trainees. An external evaluator met with the trainees during and at the end of the year to elicit feedback and to assess if the program was meeting its' objectives and the trainee's learning goals. RESULTS: Twelve trainees, 5 from under-represented minority groups, participated in the program last year. The evaluation concluded that the most valued aspect of the program for the trainees was the opportunity to learn and practice new teaching skills. The trainees also expressed interest in developing a tangible product, such as a new curriculum, at the completion of the program. RESULTS: Primary care physicians in the SF Bay Area have adequate access to new medical knowledge, but have limited opportunities to learn and refine new teaching skills. Now in year 2, the program has been modified to put greater emphasis on learning and practicing teaching skills, and less time is devoted to teaching the trainees new medical knowledge. Trainees are now required to develop a project, with the guidance of a program mentor, that is relevant to their own teaching setting. The faculty development program has enabled community and academically based general internists to form a common bond around teaching. METHODS: Instructional methods supporting individualized learning plans were enhanced by yearlong, on-site, one-on-one educational support from faculty development specialists. Curriculum has evolved to address the changing medical education environment. The program was examined within grant objectives, historical records, and archival data obtained through written surveys and focus groups. RESULTS: Focus groups and survey results have tentatively identified that the one-on-one consultation enhanced the 59 participants' ability to provide curriculum instruction within SCS member hospitals. 88% of graduates continue to teach in SCS institutions, using skills obtained through the program. 33% of the participants have received teaching awards with two winning national poster awards. 95% of the participants have remained in primary care. Some graduates have moved to higher positions within medical education. CONCLUSION: Developing part-time, community-based faculty is a multi-faceted endeavor. Faculty are challenged between providing service versus training others. The focus of providing a yearlong stipend supported program with one-on-one consultation support by specialists has proven to be a successful model in attracting and developing part-time, community-based faculty. OBJECTIVES: Primary care physicians are challenged to be clinically productive and effective teachers. A critical review of the literature revealed limited evidence regarding effectiveness of four often cited teaching methods: 1-2 focal teaching points; priming; teaching in the patient's presence [TIPP] ; and feedback. To address this gap seven experienced clinician educator [CEs] faculty (4 general internists; 3 pediatricians), participated in a faculty development project to evaluate the effectiveness of the four selected clinical teaching methods. METHODS: During monthly faculty development sessions, CEs were trained on the teaching methods using multiple strategies including simulations and written exercises. Each CE recorded use of these methods over a 10-month period on a Palm Pilot(tm) using a specially designed form. Baseline (July 1997 ± June 1998) and study period clinical teaching evaluation ratings for CEs (treatment) and non-participating faculty in their specialties (control) were compared using ANOVA. RESULTS: CEs reported using all targeted teaching methods > 50% of time during recorded teaching sessions (N=1,176 sessions) with use of priming and feedback increasing over baseline while use of the other two methods remained constant. CEs reported that the teaching methods focused both the learner and the clinical teacher, making subsequent encounters more productive and efficient. CEs teaching evaluations were significantly higher (p < .001) on three items during the study period while control group's ratings showed no change (i.e., responded to student initiated learning issues; emphasized comprehension of concepts; instructor had sufficient data to assess learner performance). During the study period, two CEs were first time recipients of major department teaching awards with three CEs receiving highly competitive, college-wide teaching awards one-year post study period. CONCLUSION: Experienced CEs who participate in a faculty development program can improve their clinical teaching ratings using four literature-based clinical teaching methods. Recognition by learners and peers of the CEs educational excellence through awards was an unanticipated outcome. OBJECTIVES: The population of older persons is rapidly rising in developed countries like Japan. Oldest old patients aged 90-year-old and more had occasionally utilized acute care hospital emergency department in recent years. To our knowledge, there were no study evaluating clinical reasons and outcomes of these highly aged population. METHODS: To determine the short-term outcomes and predictors of outcome, we reviewed medical records of elderly patient aged over 90-year-old visiting emergency department in Okinawa Chubu Hospital, acute care community hospital in rural Okinawa, Japan, in 1999. RESULTS: Three hundred seventy one patients (95 men, 276 women: mean age 92.9 years old) were studied. Five common chief complaints were fever (65 cases),shortness of breath (46), consciousness disturbances (33), leg pain (29),and nausea or vomiting (21). Pulmonary diseases (94) were the commonest category of disorders, followed by orthopaedic diseases (43) and gastroenterologic diseases (42). Only 42(11% ) patients were dead during hospitalization following admission from emergency department except for 14cardiopulmonary arrests at arrival. In multiple logistic regression analysis, cognitive dysfunction was only significant predictor for short-term mortality. CONCLUSION: Majority of oldest old patients had survived to discharge following emergency department admission. Cognitive function was important in terms of survival prediction.\nAN INTEGRATED PARTNERSHIP BETWEEN THE UNIVERSITY OF CINCINNATI AND COMMUNITY HEALTH CENTERS. T. Redington 1 ; 1 College of Medicine, Cincinnati, OH OBJECTIVES: 1. Identify the common interests of both academic health centers and community health centers that cause them to be natural partners. 2. Demonstrate the unprecented level of support that the University of Cincinnati has shown in preserving the capacity of community health centers. 3. Describe the team approach adopted by the providers of the Cincinnati Health Network, Cincinnati Health Department,University of Cincinnati Hospital clinics, and Southern Ohio Health Network, which included a common eligibility system and data sharing thoughout the delivery system. 4. Discuss this common approach to patient eligiblity and clinical care includes disease management protocols for asthma, diabetes, hypertension and depression. This common clinical approach is expected to improve if not eliminate health disparities, consistent with the Bureau of Primary Health Care's 100% access, 0% disparities goal. METHODS: The University of Cincinnati has budgeted 2 million dollars of inkind support for all the center sites of the Cincinnati Health Network. This support includes a pharmacy with pharmacist, radiology suite, midwives, and 12 full time providers, eleven are physicians. These physicians are extensively involved in medical student education at these center sites. Also the University led the successful effort to recieve a million dollar grant (from HRSA the Community Access Program) to improve the infrastructure around the indigent and uninsured in Southern Ohio. The major partners include the University, a hospital of the Health Alliance of Greater Cincinnati, a five hospital system in southerwestern Ohio, the Cincinnati Health Network, the Southern Ohio Health Network(both Federally Qualified Health Centers),and the Cincinnati Health Department. Together, these partners serve about 200,000 lives, 90% are at or below the minimum federal poverty level. RESULTS: The quality of care standards around depression, hypertension, asthma and depression have been installed. The information technology infrastructure and eligibility systems are being developed and linked between different computing platforms. The measurement of our quality of care will begin later this calendar year. Medical education in these clinic sites is extensive, about three quarters of the medical student class are being educated in these sites.\nCONCLUSION: There is a major partnership underway in southern Ohio, linking the University of Cincinnati's Hospital and College of Medicine with four large, previously independent health care systems, that have traditionally provided for the same patients, the indigent and uninsured. By the adoption of common eligibility and disase management standards, access will be improved and disparities reduced.\nA. Rubin 1 , T. Bertsch 1 ; 1 University of Vermont College of Medicine, Burlington, VT OBJECTIVES: To retain and recruit community-based preceptors in Vermont, we developed, distributed, and analyzed a survey to understand the needs of our community faculty and the barriers to precepting students in their offices. METHODS: We mailed questionnaires to all 570 primary care physicians in Vermont. We received replies from 251, of whom 160 were preceptors. We asked questions about their needs to become better teachers, the infrastructure needed to teach, rewards for teaching, and the desired format for faculty development. RESULTS: Areas of high importance for teaching include becoming more efficient, balancing student needs with patient time, increasing abilty to teach clinical skills, givng feedback to students, dealing with difficult students, and finding ways of orienting students to practice. In terms of infrastructure, preceptors want more time and space to teach. They want rewards in the form of CME or CME credit and in hearing feedback about their students. They are happy to have faculty development sessions once a year, either at a central site or near their practice site. CONCLUSION: As with our students, eliciting feedback from community based teachers can result in programmatic change. As a result of ths survey, we have developed a teaching skills exercise using standardized students and presented itin three sites. We are also changing the ways we award CME credits.\nA TEACHING EXERCISE FOR COMMUNITY PRECEPTORS USING STANDARDIZED STUDENTS. A. Rubin 1 ; 1 University of Vermont College of Medicine, Burlington, VT OBJECTIVES: To assess the teaching skills needs of our community based teachers.To develop and present a workshop that would allow them to practice these skillls. METHODS: As part of our HRSA project we developed a needs asssessment for our community based teachers. Of the areas in which they wanted to enhance their teaching skills, we chose four to learn and practice: Teaching Efficiently, Framing a Clinical Question, Giving Feedback, and The Difficult Student. We developed scripts and videos for a four station teaching exercise using a rolling role play and standardized students. RESULTS: Preceptors rotated in groups of four through each half-hour station. In each, a brief didactic presentation was followed by a rolling role play with a standardized student, which gave each preceptor a chance to practice. A short debriefing followed. We have presented this workshop to 60 of our preceptors in three areas of the state. CONCLUSION: Preceptors rate the workshop format as effective and``couldn't be better.'' They find the topics appropriate and practical. They especially like the student participation and the ability to practice what they learn in small groups. We don't yet know whether preceptors are using the skills they have learned. This is an area for further inquiry. OBJECTIVES: Despite expansion of programs in geriatrics, it will not be feasible to train enough geriatricians to care for all elderly patients. Several organizations have suggested that improved training of primary care physicians in geriatrics is likely to have the greatest impact on improving medical care of geriatric patients. To address this need, we have enhanced several aspects of our curriculum in geriatrics to strengthen the clinical competencies of primary care residents. Our specific objectives are 1) to improve the didactic curriculum; 2) to increase the number and quality of ambulatory clinical experiences; and 3) to develop new methods for measuring the clinical competencies of residents in geriatrics. METHODS: Primary care residents spend six months both years on structured ambulatory rotations. Two half days per week are devoted to structured didactics and eight half days to ambulatory rotations. Portions of both of these experiences are now devoted to enhanced geriatric training. Didactic curriculum: We have developed twenty specific learning objectives in geriatrics. These are taught in ten seminars accompanied by a syllabus of recent and sentinel articles. Ambulatory experiences: Ambulatory geriatric training begins in the first year on a home care rotation directed by geriatrics faculty. Second and third year residents may also work on home care and/or in a variety of geriatric health centers. Selected residents spend one half day per week during each ambulatory block in geriatric health centers as a second continuity experience. We have also begun to increase geriatric teaching in the residents' own primary care continuity clinics by recruiting more geriatrics-trained faculty to serve as core resident preceptors. Enhanced evaluation: We have enhanced our efforts at evaluating resident experiences in geriatrics with the use of a web-based evaluation system. We will also evaluate resident attitudes toward the care of older patients and the extent to which learning objective have been met. Finally, we will develop new strategies in measuring resident competency in geriatrics with a geriatric clinical evaluation exercise (geri-CEX) and with structured medical record reviews. RESULTS: We hypothesize that these methods will increase the knowledge, attitude and skills of residents in geriatrics and will result in improved competency in geriatrics for primary care physicians trained in this manner. CONCLUSION: The curriculum of structured primary care residency programs can be modified to address specific deficiencies in health manpower. We have designed a strategy for specifically enhancing the competency of primary care physicians to care for the elderly. OBJECTIVES: 1. Promote community-oriented primary care. 2. Teach medical students and residents about COPC through service learning in diverse underserved communities. 3. Develop role models in public health practice. 4. Engage the academic health center in its community. METHODS: 1. We established an \"Institute for Community Health\" in Cambridge and Somerville, dedicated to research and education in health promotion and disease prevention. Three health care networks (Cambridge Health Alliance, CareGroup and Partners) found common cause in a scholarly collaboration to improve the health of these communities. 2. The Community-oriented Primary Care (COPC) curriculum was chosen as one of three priority initiatives. The curriculum contains: a) experiential (research) elements: community defined projects; teams of a preceptor, medical resident and one or several medical students; year-long longitudinal projects. b) Didactic elements include a seminar series taught by faculty drawn from collaborating institutions. 3. We expect to enroll the first students in September. RESULTS: 1. Trainees will be evaluated by faculty and their community partners -in accordance with defined competencies, the quality of their products (e.g., policies, guidelines, webpages, interventions, etc.) and the sustainability of their work. 2. The faculty and currriculum will be evaluated by the community on the basis of the contributions to the local community's health, spread to other communities (within the collaborators' catchment areas), and impact on students' careers; and by the trainees. CONCLUSION: This educational model has the potential to positively influence the health of underserved communities, the careers of health professionals, and the missions of health care institutions -in Cambridge and Somerville, and beyond.\nRESIDENT PARTICIPATION AT NATIONAL SGIM MEETINGS. D.W. Brady 1 , L.J. Schultz 1 , W.T. Branch 2 ; 1 Emory University, Decatur, GA; 2 Emory Healthcare, Atlanta, GA OBJECTIVES: 1. Expose residents to general medicine career options by having them attend National SGIM meetings. 2. Encourage residents to participate in National SGIM meetings by submitting vignettes, abstracts, and workshops. METHODS: Our residency training grant affords us the opportunity to send all 10 of our second-year primary car residents to the national SGIM meeting each year. We encourage the residents to actively participate in the meeting by collaborating with faculty on workshops and submitting their own research abstracts or clinical vignettes. By exposing them to generalists from various institutions and supporting their own scholarly activity, we hope they will pursue generalist careers and remain active in SGIM. RESULTS: Beginning with the 1998 SGIM meeting in Chicago and including this year's meeting in San Diego, we have brought 40 second-year residents to national SGIM meetings. In 1998, all 10 residents collaborated on a workshop that won the David E. Rogers Award. In 1999, two of those residents returned as senior residents to present research posters. In 2000, three third-year residents presented research posters, and one second-year resident presented a clinical vignette poster. This year, one second-year resident is co-leading a workshop while the othe 9 second-year residents are all submitting clinical vignettes. So far, 90% of our graduates have remained in general internal medicine, with approximately 50% obtaining academicallyaffiliated positions. CONCLUSION: By exposing our residents to the myriad of people and opportunities afforded them at national SGIM meetings, we encourage them to remain excited about generalist careers and to pursue their own academic interests. We believe their attendance at the national SGIM meetings is an integral part of our internal medicine primary care residency program and our HRSA grant.\nSELF-REFLECTION AS A TOOL TO ENCOURAGE CULTURAL COMPETENCY. D.W. Brady 1 , I. Genao 1 , J. Bussey-jones 1 ; 1 Emory University, Atlanta, GA OBJECTIVES: 1. To promote cultural competency through self-reflection. 2. To encourage residents to examine their own cultural biases and prejudices. 3. To provide a safe environment where residents can ask difficult questions about other cultures in a spirit of inquiry. METHODS: As part of our cross-cultural curriculum, we set aside one session to allow residents to self-reflect and ask questions they had about each other's cultures. The group consisted of 10 third-year primary care residents -six men and four women, including two African-Americans, two Asian-Americans, at least two evangelical Christians, one Jew, at least one atheist, and two homosexuals (these were some of the represented cultures known to the group). We asked them each to write down a question and place it in an envelope. They were asked to write questions that they had always wanted to ask a person of a different culture (race, ethnicity, religion, sexual orientation, etc.) but were either afraid to ask or thought was too stereotypical. The questions were not to be directed personally at any specific individual within the group. After collecting the questions, we passed the envelope back around the room and asked each person to take out one question and read it to the group. The intent was to create a safe environment where the residents could bring their \"forbidden\" questions into the open and to allow them to see the reaction of the group without being personally humiliated or attacked for asking their questions. Our hope was for each resident to become more aware of his or her own cultural competency and to learn to hear \"the other\" in a spirit of inquiry. RESULTS: The group rated this session higher than any other session in the curriculum. We originally intended for the group to just hear the questions and feel their impact. The group, however, at the beginning of the session decided by consensus to allow some discussion of each question by the group before proceeding. In evaluating the experience, the residents said that the discussion was integral to making the experience its most meaningful. They also said that late internship might be a good place to introduce this exercise as it would have helped them earlier in their training. They added, however, that introducing it too early, before the group has a chance to form, might have risked alienating members of the group, thus damaging their ability to develop cohesion. CONCLUSION: The depth of learning and the residents' evaluation of the exercise have encouraged us to retain it as a part of our multicultural curriculum. Whenever we as physicians can afford trainees the opportunity to be self-reflective, expecially in a cross-cultural context, we can enhance doctor-patient communication, learn more about ourselves and each other as human beings, and, ultimately, take better care of each individual patient and society as a whole. OBJECTIVES: To describe the efforts of one general internal medicine training program to promote the selection of generalist careers by residents. In the past couple of years, there has been a declining interest in generalist careers, yet primary care physicians play an important role in meeting the health care needs of the medically underserved. METHODS: Multiple strategies for promoting selection of primary care careers have been piloted in the Primary Care track of our internal medicine residency program: ÁMentorship: Each primary care resident is paired with a general internal medicine faculty member based on common career or personal interests and goals. Mentorship pairs meet at least twice a year to review clinical performance, research, educational plan, and career goals. ÁContinuity clinics: Primary care residents have two half days of clinic in settings which serve a large Medicaid and medically indigent population. ÁPrimary care rotations: Several primary care rotations including adolescent medicine, women's health, and outpatient HIV have been developed to improve the ability of residents to practice in a variety of outpatient settings. ÁPreceptorships: All primary care residents are required to do a preceptorship during the R2 year. Rotations in medically underserved and rural areas are strongly encouraged. ÁCareer development seminars: Topics such as Finding a Job, Evaluating Contracts, and interaction with a panel of internists who practice in various settings are incorporated into the three-year primary care curriculum. RESULTS: During HRSA funding (1997 ± 2000) , 23 of 25 graduates chose primary care careers; of the two choosing fellowships, one was in geriatrics. The career development sessions were consistently rated very positively; 4.85 to 5.00 on a 5-point scale. In focus groups, residents also reported high satisfaction with these sessions, primary care rotations, continuity clinics and the mentorship program. CONCLUSION: While we have successfully promoted primary care career choices, we have been less successful in placing graduates in medically underserved areas.In attempt to increase graduates choosing careers in medically underserved areas, we are focusing recruitment efforts on students with documented interest in care of the medically underserved and supporting this interest throughout residency training. Due to the national trend of declining interest in primary care careers, these efforts will be extended to include categorical residents and medical students.\nA REQUIRED ROTATION IN HOMELESS MEDICINE -PROMOTING ALTRUISM TO RESIDENTS. D.R. Buchanan 1 , L. Rohr 1 ; 1 Cook County Hospital / Rush University, Chicago, IL OBJECTIVES: Although the American Association of Medical Colleges (AAMC) considers altruism to be one of the basic qualities that physicians should possess, residency programs rarely incorporate required rotations aimed at meeting this goal. Our objective was to design and implement a required rotation to promote altruism and careers with underserved populations through teaching residents about homelessness. METHODS: The course was designed to provide a balance between direct patient care and didactic sessions. Clinical experiences were supplemented by visits to providers of homeless services and by a lecture series. Residents worked in a variety of clinical settings, including performing medical outreach in locations around the city. The locations selected were meant to expose the residents to different facets of impoverished communities. In addition to the sites the residents visited as part of their clinical work, they also visited a number of medical and nonmedical sites related to homelessness. Specific sites included Chicago's only respite center for medically ill homeless people, Chicago's largest homeless shelter and its adjoining health center, and a smaller homeless shelter. A lecture series was included to help provide a context for understanding the medical and social issues unique to this population. The lectures focused on causes of poverty and homelessness as well as their effects on health and access to healthcare. The series was also used as a time for discussions, allowing the residents to reflect on their experiences and on assigned readings. RESULTS: The course was evaluated by pre and post course surveys. The surveys were designed to identify resident attitudes toward caring for homeless patients. We also used the surveys to identify barriers that residents feel toward pursuing careers in which they would care for underserved populations. Results of these surveys will be presented at the meeting. CONCLUSION: This required rotation,``Caring for Homeless Patients'', was implemented in the spring of 2001 for third year Primary Care Internal Medicine residents. Initial responses to the rotation have been positive. Future directions for this program include further assessment of whether the rotation is meeting its goal of promoting altruistic behavior among residents.\nT. Cavalieri 1 , K. Welding 1 , J. Ciesielski 1 , J. Kaiser-smith 1 , H. Dombrowski 1 ; 1 University of Medicine and Dentistry of New Jersey, Stratford, NJ OBJECTIVES: To establish an inner-city community-based training site that will enhance primary care skills, build cultural competency, and improve internal medicine residents' skill in addressing the needs of an underserved population. METHODS: The University of Medicine and Dentistry of NJ-School of Osteopathic Medicine (UMDNJ-SOM) established an affiliation with an existing primary care practice founded by the Diocese of Camden and the Society of Jesus. This practice, St. Luke's Catholic Medical Services, serves the poor, uninsured and underinsured community of Camden, NJ. It has a fluent bilingual staff and attracts a predominately Latino population. The physician at St. Luke's was a primary care internist/Jesuit priest who was volunteer faculty in UMDNJ-SOM's Department of Medicine. Initially, a full-time faculty member from UMDNJ-SOM was assigned part-time to assist him in providing patient care and to establish a training site for internal medicine residents. The Department then recruited a full-time primary care internist with a special interest and prior experience in serving the underserved for this facility. As part of this collaboration between UMDNJ-SOM, the Diocese and Jesuit Order, the Order funded the physician's participation in a Spanish immersion program in Mexico to improve language skills and cultural awareness. The physician has since initiated care at St. Luke's and been named Medical Director. RESULTS: This new partnership with St. Luke's, attributable to the collaborative efforts of the University, Diocese and Jesuits, has led to the development of learning objectives for a primary care continuity experience for the residents in our HRSA funded primary care residency. The primary care residents have begun weekly half-day sessions at St. Luke's that sensitize them to the medical, cultural and psychosocial needs of this underserved community. As a result of this HRSA grant, the new affiliation with St. Luke's has created additional educational opportunities. It has provided a new training site for HRSA-funded geriatric fellows and UMDNJ-SOM's extern program as well as primary care residents. CONCLUSION: This collaboration has provided the University with a unique training site and rewarding learning experience. Internists intending to serve underserved populations must be able to appreciate cultural diversity both within and between cultures; understand the impact that psychosocial, educational, cultural and environmental issues may have on client utilization and compliance; and respect the distinct cultural needs of various client populations. Collaboration between UMDNJ, the community and church, through HRSA support, has created a primary care training site to enhance cultural competency.\nL. Christophe 1 , P. Arnaud, P. Eliane 1 , A. Anne-franc Ëoise 1 ; 1 University Hospitals of Geneva. Dep Int Medicine, Geneva, Switzerland OBJECTIVES: The first years of professional activity are critical for doctors in training. However, little is known about the difficulties encountered by residents. METHODS: We put the following open question to 24 consecutive residents in a structured one year training in internal medecine: «Please, identify 2 to 3 major difficulties/concerns related to your practice of medicine within this hospital». Each resident gave confidential written answers. These were coded into 8 categories by content analysis by 3 researchers. RESULTS: Characteristics of the residents were: female: 37% , mean age: 28 2.2 years, mean duration of postgraduate training: 2.5 1.3 years. The total number of answers was 122, with an average of 5.1 1.3 per resident. 93% of the residents expressed difficulties in their relationships with their colleagues or felt not adequately supervised. 63% judged that they were not respected or recognized by senior staff. 63% admitted feelings of severe disarray and helplessness in dealing with patients. 58% complained about work overload. 50% felt burdened by the intensity of emotional investment and responsibility towards patients. Finally, 16% admitted fearing for their professional future and only 8% cited lack of theoretical knowledge about the diseases they were dealing with. Four answers did not fit into the 8 above categories. CONCLUSION: Most of the interviewed residents in training expressed major difficulties within their professional relationships. Feelings of inadequate practical and relational training by medical school, of lack of recognition by senior staff, and of work pressure and overload were common. These elements challenge the organisation/coaching in our institution and should lead us to improve our teaching. METHODS: Internal medicine residents are exposed to curriculum in cancer prognosis using several instructional strategies including didactic/lecture, small group case discussions, roleplaying and debriefing actual patient encounters. Additionally, some of the residents participate in an elective block rotation in end-of-life issues providing them with more intensive exposure by following patients in our hospice program. Along with relevant reading assignments for background, residents review sample case studies of terminally ill patients at latter stages of illness. After learning a system of estimating prognosis they discuss how this information may be used to design care plans so that the therapies that are most appropriate in light of known prognosis are chosen. Residents review basic principles of communicating with patients about prognosis and end-of-life issues that they then implement in role playing exercises. RESULTS: Formative and summative evaluation methods are utilized to assess progress toward the objectives. These include individual faculty preceptor assessments of residents caring for terminally ill patients, knowledge and attitude questionnaires regarding end-of-life issues, scenarios highlighting communication issues, and resident self-assessment. In addition, roleplays are rated on several criteria and feedback is given to residents in a post session briefing.\nCONCLUSION: Although end of life issues are receiving increased attention and concern, it remains an area physicians tend to feel insecure in their skills and poorly prepared by their medical training. Developing curriculum to increase physician's confidence and comfort with these issues improves the quality of care to patients and family members in the dying process.\nCOMBINING OSCES WITH THE MINI-CEX FOR RESIDENT ASSESSMENT. R.S. Crausman 1 , J.P. Miskovsky 1 ; 1 Memorial Hospital of RI, Pawtucket, RI OBJECTIVES: To assure a high standard of clinical competence throughout the continuum of medical education, it is imperative that appropriate, task-relevant evaluative tools and methods be continuously developed and applied. In response to the perceived inadequacies of conventional evaluative measures, the use of test patients for structured, standardized evaluation (Objective Structured Clinical Examination, OSCE) has gained favor for the assessment of medical students and residents. However, the role of physician faculty observer, when present, in these exercises is unclear. Physician supervisors bring a clinical perspective to the encounter different from, and complimentary to that of the standardized patient. The challenge is to provide a structured evaluative framework to improve the quality and reproducibility of assessment. In 1995 the American Board of Internal Medicine developed and validated the Mini-CEX. These observed resident-physician patient encounters consist of a single faculty member observing a resident conduct a focused history and physical examination. Residents and evaluators report satisfaction with the format, and reliability of the evaluation is thought to be superior to traditional clinical evaluation exercises. Here we describe our early experience combining the Mini-CEX with outpatient OSCE's for enhanced resident assessment. METHODS: Second year medical residents (n = 10) in our Primary Care/General Internal Medicine Residency underwent a multi-station OSCE designed to assess their clinical skills. Each 20 minute encounter was observed by a faculty physician who structured observation and assessment using the Mini-CEX instrument which uses a two page form with a 9 point rating for assessment. After each encounter both standardized patient (five minutes) and faculty physician (five minutes) provided structured feedback to the resident trainee. A time keeper assured that encounters were kept to 20 minutes. RESULTS: A structured feedback session with all participants was conducted after the exercise and all reported high levels of satisfaction with the format. Standardized patients uniformly commented that the presence of a physician faculty evaluator represented an improvement and provided complimentary evaluative information. Faculty observers were pleased with the imposed structure that the Mini-CEX provided. Residents were satisfied that feedback was appropriate and valuable CONCLUSION: In response to the perceived inadequacies of conventional evaluative measures we have introduced a combination of outpatient OSCE's and the Mini-CEX towards developing a more flexible, reliable and task-relevant teaching and evaluation tool. Our early experience with this approach has been very positive. OBJECTIVES: One of the goals of our residency program is to improve residents' ability to care for the medically underserved. We expect that our graduates will not only provide excellent clinical care to individual patients, but will also integrate public health concerns into their medical care. In addition, we anticipate that our graduates will work with community residents, organizations and leaders to serve as advocates for the underserved. In order to expose our residents to this model of community-oriented primary care, we developed an eight-week course in community medicine. METHODS: The Community Medicine seminar series was held 1/2-day each week for eight weeks and was attended by first, second and third year residents. The curriculum included a community inventory exercise in which residents obtained information about the community in which they saw continuity patients, a community assessment in which residents toured through a neighborhood on foot and described the people, businesses, housing and transportation they encountered, a community treasure hunt in which residents shopped at local businesses and map exercises in which residents plotted the location of their continuity patients' homes. A key component of the curriculum was an intranet website designed specifically for the Community Medicine course. Residents used the website to participate in on-line discussions of reading material and to access public health information and resources. RESULTS: Residents were asked to complete an on-line assessment instrument at the conclusion of the course. 91% of the residents (10/11) found the seminar series to be very useful for their future practice of medicine. 91% (10/11) felt the course should be offered again in the future. 92% (11/12) felt they understood and could explain the concept of community-oriented primary care. 73% (8/11) of the residents felt the map exercises helped them understand where their patients lived and how they traveled to medical appointments. 100% (11/11) found the public health links on the intranet website useful for learning about COPC. 73% (8/11) residents found the community treasure hunt useful in terms of how to view a community from the standpoint of its people and institutions. CONCLUSION: This Community Medicine course increased residents' knowledge of the communities in which they and their patients live. Future directions for investigation include determining whether this course impacts residents' decisions to practice primary care in underserved areas and to implement community-oriented primary care concepts after graduation from the program. OBJECTIVES: Emergency contraception (EC) is an effective but underused method of preventing unwanted pregnancy. In a 1997 national study, only 25% of college-educated women and 6% of women with high-school degrees knew key facts about EC. Only 5% of women had heard about EC from their providers. While other studies have addressed barriers to EC use in the general population, few have focused on barriers in poor, urban populations. Three residents in a program designed to train physicians to care for the underserved looked at this issue in a community satellite clinic. METHODS: As part of a Quality Improvement/Research curriculum in a primary care residency program, we conducted a focus group with eight providers at an urban, publiclyfunded community health center in San Francisco. Provider beliefs/practices, perceived patient barriers, and possible interventions to increase emergency contraception use were discussed. The session transcript was then analyzed to identify a consensus surrounding specific barriers and interventions. RESULTS: Providers identified a significant problem with unintended pregnancy at their clinic, estimated at 85 ± 90% of all pregnancies. Despite this high rate, emergency contraception was prescribed only 2 ± 3 times a year at the clinic. Providers identified the following obstacles to emergency contraception use: lack of patient knowledge regarding the availability of EC; the prevalence of social and economic stressors that made contraception a low priority for patients; patients' fatalistic beliefs surrounding pregnancy; and operational obstacles to obtaining emergency contraception prescriptions. Identified interventions to increase use/access were: patient and community education about emergency contraception; arranging to have a prepackaged product on-site to give to patients; and routine counseling by providers at all reproductive health visits. CONCLUSION: In this urban, community-based primary care clinic, unintended pregnancy is common and emergency contraception rarely used. In the focus group session, providers clarified barriers and identified interventions that the clinic could employ to increase patient's access to and use of emergency contraception. Barriers to use by urban, underserved populations may need further study and targeted interventions before true access to emergency contraception becomes a reality for this vulnerable patient group. OBJECTIVES: Increase residents knowledge of common IM approaches (acupuncture, physical therapy/massage, mind-body medicine, herbal medicine, chiropractic) including what that approach entails, what conditions it is thought to be useful for treating, and any contraindications for its use; and ability to identify, access and critically evaluate evidencedbased research regarding treatment outcomes and efficacy of the particular approach. METHODS: Residents are assigned a faculty mentor who oversees and provides guidance in the month; helps to monitor and direct the residents' learning and to address their objectives. Residents spend several 1/2 days each week in the office of IM providers. Each provider prepares a reading list the resident is responsible for working through in that area. Residents see patients along with the provider and have opportunity to discuss the patients from that IM perspective. Residents also spend time with a general internist who is well versed in IM at our Center for Integrative Medicine. They learn how primary care physicians incorporate IM into their practice. Residents attend several one-on-one classes. For example, residents meet with the librarian to learn how to search the web to find IM information and research and are introduced to IM databases in the area (i.e., Natural Medicine Database). An oncologist talks with the residents about the use of alternative techniques cancer patients often use; how the oncologist approaches discussions about IM with patients and the contraindications that may exist for various IM approaches within oncology. The residents also go on several site visits (walk through a pharmacy with the pharmacist looking at OTC herbal remedies as well as visiting a wellness center providing patient education, support groups and counseling to cancer patients. RESULTS: Intensive interviews are conducted with each resident completing the rotation to evaluate quality of the experience and to elicit opinions for changes/additions to the experience. CONCLUSION: Setting up curriculum addressing nontraditional approaches in a western medical setting raises many issues regarding developing common standards for education, reimbursement for practitioner training time and credentialing, applying research standards to cross cultural scientific literature. The process is a challenging and yet a rewarding venture for both traditional and nontraditional medical learners and educators. OBJECTIVES: A one-month IM elective block rotation was developed for third year residents to more intensively, study IM approaches. It was hoped that this exposure would prepare residents to better integrate IM into their medical practice. Increase residents knowledge of common IM approaches (acupuncture, physical therapy/ massage, mind-body medicine, herbal medicine, chiropractic)including what that approach entails, what conditions it is thought to be useful for treating, and any contraindications for its use; and ability to identify, access and critically evaluate evidenced-based research regarding treatment outcomes and efficacy of the particular approach. METHODS: Residents are assigned a faculty mentor who oversees and provides guidance in the month; helps to monitor and direct the residents' learning and to address their objectives. Residents spend several 1/2 days each week in the office of IM providers. Each provider prepares a reading list the resident is responsible for working through in that area. Residents see patients along with the provider and have opportunity to discuss the patients from that IM perspective. Residents also spend time with a general internist who is well versed in IM at our Center for Integrative Medicine. They learn how primary care physicians incorporate IM into their practice. Residents attend several one-on-one classes. For example, residents meet with the librarian to learn how to search the web to find IM information and research and are introduced to IM databases in the area (i.e., Natural Medicine Database). An oncologist talks with the residents about the use of alternative techniques cancer patients often use; how the oncologist approaches discussions about IM with patients and the contraindications that may exist for various IM approaches within oncology. The residents also go on several site visits (walk through a pharmacy with the pharmacist looking at OCT herbal remedies as well as visiting a wellness center providing patient education, support groups and counseling to cancer patients. RESULTS: Intensive interviews are conducted with each resident completing the rotation to evaluate quality of the experience and to elicit opinions for changes/additions to the experience. CONCLUSION: Setting up curriculum addressing nontraditional approaches in a western medical setting raises many issues regarding developing common standards for education, reimbursement for practitioner training time and credentialing, applying research standards to cross cultural scientific literature. The process is a challenging and yet a rewarding venture for both traditional and nontraditional medical learners and educators. OBJECTIVES: In order to effectively care for patients and promote health, physicians must have a broader view of their role in society as``health promoters''. Knowledge of public health resources as well as the process of policy development is especially important when caring for vulnerable patients. Because of the crowded curriculum of a residency program, we have developed a public health curriculum that is integrated into the existing framework of the residency program. METHODS: A needs assessment was conducted via interviews with the Primary Care interns by an outside evaluator and a survey completed by Primary Care and traditional track house officers (80% response rate). Several areas within the existing curriculum were identified where public health concepts could be inserted Ð complementing, not replacing,``traditional medicine'' topics. All sessions are evaluated. Residents will complete yearly surveys and an exit interview to assess the impact of this new curriculum. RESULTS: The resident survey showed a significant level of interest in public health, greatest in the senior residents. Residents rated their own knowledge of many public health concepts as`a verage'' to``low'', and rated a broader knowledge of public health as``important'' to``very important'' to patient care. Faculty from the Boston University School of Public Health, Boston Public Health Commission and the Massachusetts Department of Public Health have agreed to participate, and run joint sessions with faculty from the Section of General Internal Medicine. Sessions include: 1) A monthly three-hour seminar during an ambulatory block, attended by all of the residents (both Primary Care and traditional track) that reviews the history of medicine and public health, highlighting areas of tension and opportunities for collaboration. 2) Ambulatory Morning Report: topics relevant to ambulatory medicine are presented by the Primary Care chief resident and internal medicine faculty, with monthly participation by public health faculty, adding the public health perspective with a 10 ± 15 minute discussion. 3 ) A threehour session in the Primary Care seminar, focused on the role of Departments of Public Health and an introduction to health policy and government. 4) An elective rotation at the Massachusetts Department of Public Health provides primary care residents with the opportunity to work directly with public health staff, participate in projects and learn how programs and policies are developed. CONCLUSION: Our survey found that the residents were interested in public health and felt that it was relevant to clinical care. Public health faculty were very willing to participate in resident education and collaborate with curriculum development. Important public health concepts can be incorporated into a Primary Care residency curriculum without displacing existing``traditional topics''. OBJECTIVES: Since Hepatitis B (HBV), Hepatitis C (HCV) and HIV share the same high-risk behaviors, coinfection is frequent in clinical practice. In the US, 30 ± 50% of HIV patients are coinfected with chronic HCV. Almost 95% of HIV patients have serological evidence of HBV exposure. In the era of highly active antiretroviral therapy, death from rapidly progressive viral hepatitis is increasingly common in HIV patients. Since early detection and treatment of HIV and viral hepatitis has been shown to improve the outcome of patients, reactive serologies to HBV and/or HCV should provoke discussion of HIV testing in a general medicine clinic to reveal coinfection. METHODS: Retrospective chart review of patients with serological exposure for viral hepatitis, identified or followed at a hospital based general medicine clinic in East Harlem, NY. A query was done on hepatitis serologies sent from 1/1/99 to 12/31/99 from the clinic to create 3 patient groups : 1) HBV exposure (Reactive HBc only) 2) HCV exposure (Reactive HCV only) 3) Both HBV and HCV exposure (Reactive HBc and HCV). Charts were reviewed to determine whether house staff physicians documented discussions of high risk HIV behavior after obtaining the hepatitis serologies. The number of HIV tests requested and results were noted. Patient return rate for HIV results was also noted. OBJECTIVES: Most guidelines for prevention and treatment of osteoporotic fractures are neither evidence-based nor well-suited to the needs of the diverse patient population found in urban public health settings. This Quality Improvement project aimed to create a clinical guideline adapted to San Francisco's Community Health Network and its diverse population with limited resources. The Guideline aims to be based on current evidence wherever possible, and is based in part on existing evidence-based guidelines such as the National Osteoporosis Foundation (NOF) Guideline. Implementation of the guideline will follow published effective approaches to changing practice patterns. METHODS: 1. Adaptation of currently existing evidence-based guidelines, such as the NOF guideline, and review of recent relevant literature to create a guideline appropriate for and tailored to San Francisco's Community Health Network (CHN.) 2. Distribution of these guidelines to CHN providers, ideally targeting those who see our at-risk patients, using a creative media approach with paper and electronic formats (including distribution of a file transferrable to handheld organizers such as the Palm OS) and additionally promoting the guideline in a didactic format. 3. Improving awareness of and adherence to these guidelines with such techniques as Clinical Alerts on the CHN computer record, and easy Web access on the CHN website. RESULTS: We designed a guideline that was tailored to meet the needs of the CHN patient population. This guideline was evidence-based. Distribution was achieved via CHN website, computer-based reminders and triggers where appropriate, handheld organizer files, and didactic sessions in local clinics and conferences. CONCLUSION: Few guidelines for prevention and treatment of osteoporotic fractures currently exist that are both evidence-based and tailored to meet the needs of an underserved urban public health community. Using good existing guidelines such as that published by the NOF, current literature can be adapted to design a guideline that is both based on good evidence and appropriate to the limited resources of an urban public health setting. Novel uses of computer-based and didactic tools can enhance distribution and awareness of clinical practice guidelines. OBJECTIVES: To develop and implement a curriculum in women's health for internal medicine residents in a community hospital based university program. METHODS: Participants are first year internal medicine residents in their ambulatory block month (N=16). The curriculum content was determined by a needs assessment conducted on previous and current residents. It consists of the following six units covered during three hourly didactic sessions: 1) Pretest and Introduction to Women's Health, 2) Selected Gynecological Conditions, 3) Obesity and Eating Disorders, 4) Menopause, Osteoporosis and Hormone Replacement, 5) Psychosocial Issues, 6) Resident Presentations and Posttest. Also included in the curriculum is a three-hour visit to the local domestic violence prevention center. All necessary reading materials are handed out during the first session after the pretest. The didactic sessions start with a brief case presentation by the participating resident(s); this is followed by an in-depth discussion of the topics of the day with expected resident participation. Each session except the introductory and resident presentation session ends with role-plays where the instructor plays the role of the patient and the resident the doctor. This enables the resident(s) to put into immediate practice what they have just learned. Clinical practice of women's health is expected to occur when residents see female patients in their primary care practices. To expand the scope of this curriculum beyond these limited topics both residents and faculty have been encouraged to incorporate into everyday teaching and learning how various conditions may differ or are unique in women. Participating residents are expected to make a formal thirty to forty-five minute presentation on a topic in women's health not covered in this curriculum. Learners are evaluated by the completion of a standard evaluation form used in the residency program. Evaluations are based on the residents' performance on the posttest, participation during the didactic sessions and their presentations. Learners evaluate the curriculum by completing a survey. RESULTS: The curriculum was well received by all residents. All residents surveyed either agreed or strongly agreed that they would now be able to evaluate, manage or appropriately refer female patients as a result of knowledge gained during the month. The mean posttest score was 81%, an improvement over the mean pretest score of 66%. Residents stated particularly that role-plays helped improve their understanding of the topics. A survey of these residents in their third year regarding their perceptions on the women's health education they received in this program and how well they think it has prepared them for their future practices year is planned. CONCLUSION: The curriculum improved the knowledge of residents in selected women's health topics. Using the same curricular model, topics can be adapted to meet the learning needs of residents in other residency programs.\nIMPROVING THE HEALTH CARE RESPONSE TO DOMESTIC VIOLENCE: A SEMINAR SERIES FOR PRIMARY CARE RESIDENTS. K. Riordan 1 , S. Love 1 , C. Warshaw 1 , S. Glick 2 ; 1 Chicago Abused Women Coalition, Chicago, IL; 2 Cook County Hospital, Chicago, Illinois OBJECTIVES: Violence against women has been increasingly recognized as a major public health problem. Despite this, many physicians still find it difficult to ask patients about abuse. A major barrier that health care providers face in effectively identifying and treating victims of abuse is a lack of training on the prevalence and impact of domestic violence, ways to intervene appropriately and how to access available resources. We integrated an innovative 8-week seminar series on domestic violence into our primary care residency training. The goals of our curriculum were to enable residents to: 1) understand the prevalence and dynamics of domestic violence; 2) identify the impact of domestic violence on women's physical and psychological health; 3) develop culturally respectful intervention skills to identify and assist abused patients; and 4) identify resources available to victims of abuse.\nMETHODS: The seminar series was led by the Hospital Crisis Intervention Project (HCIP), a collaborative program of the Cook County Bureau of Health Services and the Chicago Abused Women Coalition (CAWC). The seminars were held 1/2-day each week for eight weeks. Topics included An Overview of Domestic Violence; Identifying, Assessing and Intervening with Domestic Violence Victims; Working with Diverse Populations; The Batterer; Intervening with Sexual Assault Patients; Community Violence; and Child Abuse, Elder Abuse and Legal Issues. A highlight of the course was a site visit to CAWC's domestic violence shelter where the primary care residents were able to speak candidly with survivors of abuse. Seminars were led by domestic violence advocates, physicians, social workers and attorneys. Instructional format included lectures, interactive discussions, videotape reviews, problem-based case discussions and a community site visit. RESULTS: Residents assessed each session and the course overall with written evaluations. 100% of the residents (9/9) found the course very effective at helping them learn more about the dynamics of domestic violence. 100% (12/12) found it very effective at helping them learn more about the impact of domestic violence. 100% (8/8) found it very effective at helping them work effectively with diverse populations. 92% (11/12) found the course very effective in helping them learn more about the resources available to victims of domestic violence. CONCLUSION: This 8-week course on domestic violence improved residents' understanding of the dynamics and impact of domestic violence, culturally appropriate interventions and available resources. Future directions for evaluation include the use of simulated patients to determine whether this course improves residents' ability to identify and care for victims of violence in the clinical setting. OBJECTIVES: We designed a 2 week rotation in addiction medicine to promote the role of the primary care physician in prevention, early identification, and treatment of substance abuse. The objectives are to: 1) support positive attitudes towards patients with addiction disorders, 2) define the responsibilities of the primary care physician for substance abuse intervention, 3) practice new skills and apply new knowledge of substance abuse in a general medical setting, 4) provide access to physician role models. METHODS: We adapted the structure and content of a successful 4 week elective rotation in addiction medicine into a 2 week core curriculum for all second year, primary care residents. Previous experience from the longer rotation informed us that residents unanimously chose as core learning objectives: screening and assessment, discussing substance abuse with a patient, and making a referral to community resources for substance abuse problems in general medical practice; and half also chose as a focus: developing a non-judgmental approach to addicted patients, providing preventive education, and management of withdrawal. We therefore made these the learning objectives of the brief course, adding: how to monitor and support patients in recovery. Modeled on the longer elective, the two weeks are structured around the residents' ambulatory primary care clinics, where they are responsible for practicing their new skills. They attend 4 half-day workshops, including case discussions of core topic areas and role play of office-based skills. Working with hospital-based substance abuse counselors, they perform substance abuse assessments and referrals for medicine and trauma patients; observe treatment groups of an out-patient treatment program for substance abusing persons with HIV; visit community-based residential treatment programs, a social-detox program, and a 12-step meeting. RESULTS: We used one experienced faculty-member to organize the curriculum and make community contacts for the 2 week rotation. Workshops are delivered by a group of faculty including internists, psychiatrists, and chemical dependency professionals. We found that community-based, substance abuse treatment programs were willing to sponsor visits by small numbers of residents, and several would permit residents to observe treatment groups. Treatment programs were generally willing to arrange for residents to interview individuals clients. CONCLUSION: A 2 week rotation in addiction medicine can be created that focuses on the role of the primary care physician in the prevention, early identification and treatment of substance abuse. Residency programs that do not have substance abuse services within their teaching hospitals or clinics can seek cooperation from community-based, substance abuse treatment programs to provide residents with clinical experiences. OBJECTIVES: Establish an ambulatory care morning report for residents, medical students, and other trainees in a multidisciplinary clinic, and encourage the integration of evidence based information into clinical practice. METHODS: McClennan Banks Adult Primary Care Center at the Medical University of South Carolina serves as an ambulatory teaching site for internal medicine residents, medical students, physician assistant students, and pharmacy graduate students. Clinic faculty have appointments in general internal medicine, pharmacy, the physician assistant program, and psychiatry. We organized a forty-five minute, twice-weekly morning report for all residents and students on their monthly ambulatory rotations. The report takes place prior to clinic hours and is attended by four to eight residents and students and two to three faculty or fellows. Each student or resident is assigned a date to give a presentation describing one of their patients from the monthly rotation. The presenter is encouraged to focus on a common primary care topic, and to search for available evidence (guidelines, systematic reviews, clinical trials) on topics such as screening, diagnosis, prognosis, or treatment of the condition being discussed. The report begins with a brief history and physical, followed by a discussion of the diagnostic evaluation and management alternatives, and concludes with an evaluation of the evidence presented. Informal discussions, observation and exit interviews at the conclusion of the ambulatory rotation have assessed educational value and trainee and faculty satisfaction.\nRESULTS: The program has been functioning for one year, and is a valued educational experience according to feedback from the trainees. The students have had excellent attendance, while residents have needed greater encouragement to attend the sessions. Faculty participation has been excellent, as the sessions provide greater opportunity to teach than is possible during busy clinic hours. An additional benefit has been the increased interaction of learners from the variety of disciplines represented. The timing of the report has not interfered with clinic operations. CONCLUSION: Our ambulatory morning report enhances the quality of clinical education and is a valued addition to the ambulatory rotation. Plans are being discussed to include trainees from other clinical teaching sites. Future implementation should include prospective evaluation of impact on knowledge, attitudes, and the use of evidence in patient care. OBJECTIVES: As part of our HRSA-funded primary care grant, we increased the longitudinal ambulatory care requirement for our categorical internal medicine residents. With an increasing proportion of our residents having this ambulatory care experience in the community, we felt the need to determine whether the educational experience for these residents is comparable to that of residents in a more traditional hospital-based clinic setting. We developed a new evaluation tool to assess the experiences of residents in these two settings. METHODS: In developing our Evaluation of Continuity Preceptor and Site instrument, we sought to assess those aspects of the resident's experience most important for optimal ambulatory education in both community and hospital-based sites. The evaluation instrument is divided into a group of questions concerning the preceptor and another group of questions regarding the ambulatory site. The 15 preceptor items include questions about role modeling, supervision, teaching and an overall rating. The 18 site items include questions about office systems (patient flow, medical records, billing), patient characteristics, continuity of care and an overall rating. Items were rated on a 5-point Likert scale, assessing frequency of preceptor and site attributes, using terms varying from never/almost never to always/ almost always. RESULTS: The instrument was piloted and yielded good internal consistency (Cronbach's alpha .88 for preceptor items and .83 for site items). The survey was then distributed to all categorical residents semiannually for two years. The response rate was 82%, and 132 surveys were analyzed. When tested for differences using the Mann ± Whitney test, the group mean scores for site items were significantly higher for the community sites as compared to the hospital-based clinic site (4.08 vs 3.72; P=0.001). The group mean scores for the preceptor items were higher for the clinic-based faculty as compared with the community preceptors (4.58 vs 4.44), but the difference was not significant (p = 0.08). Site items rated higher by residents in the community included questions about exposure to a broad range of patients (p = 0.001) and to a full range of patient problems (p = 0.07). Residents in the hospital-based clinic felt they had a higher level of responsibility for their patients (p < 0.001) and they rated their preceptors slightly higher in providing explanations and asking questions to enhance learning (p = 0.13). CONCLUSION: Internal medicine residents are being increasingly assigned to community sites for their longitudinal ambulatory care experience. In our survey, we found that residents in the community rated site items higher and residents in the hospital-based clinic rated preceptor items slightly higher. Awareness of the differences between the two types ambulatory care experiences is important for planning future ambulatory educational activities. OBJECTIVES: A web-based modular curriculum for information literacy was developed to support evidence-based clinical practice and refine residents' skills for preparing professional conference presentation materials. This curriculum was designed to fill the gaps in informatics knowledge necessary to subsequent assignments supporting an Evidence Based Medicine (EBM) residency curriculum. METHODS: To assess readiness and assure curricular relevance, baseline data is gathered concerning essential EBM and information literacy skills. The initial one month curriculum consists of four two-hour class sessions supported by hands-on instruction and maintained on the open residency website. Content focuses on current and future informatics practice issues, orientation to significant information resources, emerging technology developments related to clinical practice, EBM searching strategies for the general Internet and quality medical sources, overview of quality patient education materials, government websites, and time saving shortcuts for common technology applications including browsers, word processing and presentation programs. The team of people who support and maintain this curriculum include a variety of Internal Medicine content experts, the medical librarian, a nursing informatics webmaster, and a research specialist. RESULTS: The initial series of courses is completed within the first three months of the first post-graduate year. Continued exposure to EBM skill usage and the website is required during all three years of the resident's outpatient clinic via topic sessions. Final assignments of the second and third residency years include a digital presentation of a clinical topic in an Internal Medicine research forum conference. CONCLUSION: Integration of information literacy skills into a residency curriculum must consider variations in learning curves and the need for changes in approaches to traditional problem solving techniques in clinical practice. Clinical faculty and residents need support and encouragement to adapt existing processes to include EBM approaches in daily practice. Traditional dogma states that a shift in institutional thinking may be needed to provide conveniently accessible Internet resources on the practice units and in the residency work areas. However, providing a readily accessible set of instructions and relevant EBM resources via a residency website removes barriers to implementation of evidenced based clinical practice for residents and attending physicians. The ACGME/ABMS vision is the integration of life-long learning for physicians with the maintenance of Board certification. Their collective goal is to ensure that physicians are better prepared to provide compassionate, appropriate, and effective patient care in the changing health care environment. DESCRIPTION OF PROGRAM/INTERVENTION: Purpose: Using this background as a framework, the ABIM Clinical Competence Program is exploring ways to foster self-reflection, self-assessment, and continuing professional development among internal medicine residents and subspecialty fellows throughout their training. Through a newly developed tool and template, the use of a portfolio designed and maintained by the physician, may facilitate achieving this goal and serve to chronicle medical professionalism. FINDINGS TO DATE: What Comprises A Portfolio?: Multidimensional, qualitative and quantitative, portfolios encompass a broad spectrum of activity from definitive learning goals, performance assessment and reviews, to personal narratives and community service. KEY LESSONS LEARNED: Conclusion: The professional portfolio is the tangible culmination of one's acquired and applied knowledge, learning experiences, and unique opportunities for self-assessment and self-reflection. The portfolio remains the practical and intellectual property of the creator. Through the creation of a portfolio, physicians are empowered to gain better insight into their own goals, personal and professional growth. and career development. We summarize our needs assessment of unique MD/MBA competencies and the integrated 5-year curriculum created in response to these needs. OBJECTIVES OF PROGRAM/INTERVENTION: Our goals were to 1) identify unique MD/MBA competencies through the input of private sector and academic physician leaders; and 2) develop new curricular opportunities to meet these needs. DESCRIPTION OF PROGRAM/INTERVENTION: Through a structured process, an advisory board comprised of 14 health care leaders identified six competency areas critical to MD/MBA graduates: clinician leadership skills in business environments, common business components of public and private health care delivery, a historical perspective on the US health care delivery infrastructure, the role of public policy in shaping health care delivery, an internal framework for ethical organizational decisionmaking and a population perspective in health care planning. These MD/MBA competencies represent additional skills required by graduates beyond the core competencies of each degree independently. During the first three years students complete all the standard MD curricular activities. MD students apply for the MD/ MBA Program during their third year. Once accepted, students spend the fourth year completing the entire core and some elective requirements in the Business school. The fifth year curriculum was developed as a summation year, integrating the two Schools' curricula while explicitly addressing the unique dual degree competencies. Fifth year curricular elements include a management component integrated into standard clinical electives, a broadened range of electives in other Schools within the University such as Public Health, an evening Physician Leadership Colloquia Series, and a clinically oriented field study/research experience. Students completed an evaluation that covered both School's core competencies as well as the unique dual degree competencies using a Likert scale. Information was also elicited about students' short and long term plans. FINDINGS TO DATE: All six members of the first cohort completed evaluations. Overall the curriculum addressed the majority of the unique MD/MBA competencies with a mean score of 3.0 or higher. Two competencies need further emphasis: developing an ethical framework for organizational decisionmaking (2.8) and an application of physician leadership skills in business environment (2.5 STATEMENT OF PROBLEM OR QUESTION: Statement of the Problem: Professional behavior is often not a structured topic in the standard medical school curriculum. Much of the socialization for medical professionals occurs in what we refer to as the hidden curriculum. Frequently medical students may not have the understanding of what is considered appropriate professional behavior, yet they are exposed to positive and negative role models from which they may choose to emulate. OBJECTIVES OF PROGRAM/INTERVENTION: Objectives: Our goal was to develop curriculum that begins to address some issues around the socialization that occurs for medical students during their clinical years of training. The purpose is to provide an informal, safe environment for students to discuss issues of professional behavior to which they are exposed during their third year of medical school. Information obtained from these gatherings is used to modify the experiences the students have during clinical training. In addition it serves the purpose of allowing students to share experiences with their colleagues. DESCRIPTION OF PROGRAM/INTERVENTION: Description: In a course entitled``The Hidden Curriculum'', junior medical students are divided into groups of 12 and have 6 scheduled meetings during the academic year with two facilitators (a faculty member and a senior medical student). For each session, the facilitators are provided with 2 to 3 questions concerning a variety of topics. Some of the topics include balance of professional and personal life, perceived medical student abuse and career goal exploration. A written summary report is generated from all of the data gathered from the small groups. The summary report is distributed to the clerkship directors, students and several of the Deans involved in medical student education. FINDINGS TO DATE: Findings to Date: Medical student experiences are generally positive but they are continually exposed to some negative role models. The results from the curriculum thus far have lead to creating a more formal evaluation of professional behavior. In some instances specific individuals have received feedback concerning egregious behavior. Students view this as a very positive experience, finally allowing them a format that facilitates critical feedback about their experiences. KEY LESSONS LEARNED: Key Lessons Learned: Medical students rarely receive formal training on what appropriate professional behavior entails. In many circumstances they do not feel comfortable providing honest, critical feedback to their superiors. Evaluation of professional behavior should be done in a more formal manner, addressing appropriate knowledge, skills and attitudes just as we do with clinical performance. OBJECTIVES OF PROGRAM/INTERVENTION: Conduct a workshop with residents using patients with life-threatening illness in order to: 1) educate residents about how to communicate bad news to patients and 2) change residents' attitudes about communicating bad news and maintaining hope for patients. DESCRIPTION OF PROGRAM/INTERVENTION: Four patients with cancer from the Wellness Community were recruited to participate in a 2 hour resident workshop on giving bad news to patients. The curriculum included patients using their own diagnoses and histories as the role play scenarios. Residents were instructed to give bad news as they usually do, then feedback from the patients, discussion about the role play and about the method as described by Buckman ensued. A second role play using the learned concepts was conducted and final debrief occurred. Prior to the workshop, residents completed an 11 item questionnaire about actions to be taken during the communication of bad news, with a similar post-test being used at the end of the workshop. FINDINGS TO DATE: Twenty-five residents participated, with 15 (60%) completing pre and post tests. Attitudes toward ensuring hope is conveyed to patients (p < 0.05), starting the discussion by ascertaining the patient's understanding of the condition (p < 0.01), and encouraging the patient to express his/her feelings (p < 0.01) all significantly improved with the use of the workshop. Most of the improvement occurred in residents with previous training. KEY LESSONS LEARNED: A role play workshop using untrained patients with cancer can significantly improve residents' attitudes about giving bad news. Volunteers are without cost to the institution, and have a positive influence in such workshops. Residents who are previously trained may hold counterproductive attitudes, and therefore all residents should participate in workshops using role play by actual patients about giving bad news. is predicated on the belief that humanism and professionalism come to students and others through understanding a number of core concepts and relationships complemented by self-reflection. The concepts, which have been described by ABIM's Project Professionalism, include ideas of integrity, respect and others; the relationships include those with patients and families and with other health care professionals. Talking Medicine offers a consistent (every other week for 10 weeks) opportunity to share experiences in small groups (6 ± 8), facilitated by two preceptors in a format driven by students' experiences. Although the focus is on students' experiences, readings are provided on basic topics and contexts in humanism and professionalism (e.g. end of life care, mistakes, spirituality in medicine, and boundaries between patients and doctors). FINDINGS TO DATE: Talking Medicine began in summer, 2000 and half of the third year class has taken it. We surveyed students (total 54, response rate 63%) and found 94% felt \"very\" or \"somewhat\" comfortable in the course. 73% of students reported that the course increased their \"connectedness\" to classmates and 61% favored it occuring during all rotations. 59% reported that their interest in caring for patients improved and 53% reported their interest in internal medicine as a field improved. Answers to open-ended questions highlighted the importance of Talking Medicine as a forum to connect with others Ð both students and faculty. KEY LESSONS LEARNED: These results suggest Talking Medicine may be most effective in helping classmates connect to and learn from each other, thereby setting a foundation for changes in how they interact with patients. STATEMENT OF PROBLEM OR QUESTION: End-of-life care is assuming increasing importance within medical education curricula. Traditional teaching methods may be inadequate to address the emotional, interpersonal, and spiritual aspects of palliative care. The performing arts offer an innovative approach to teach medical trainees in these essential but under-emphasized domains. OBJECTIVES OF PROGRAM/INTERVENTION: We instituted a program using the dramatic arts to foster empathy for the terminally ill person's experience of illness, and selfreflection with regard to personal practices in caring for dying persons. DESCRIPTION OF PROGRAM/INTERVENTION: The Wit Educational Initiative uses professional readings of the Pulitzer-Prize winning play``Wit'' at medical training sites throughout North America. \"Wit\" narrates the personal and medical care experience of a patient dying from metastatic ovarian cancer. Medical students, housestaff, and ancillary providers attend readings of the play followed by structured discussions of the play's themes. Program participants are asked to complete a survey evaluating the program's acceptability and relevance. FINDINGS TO DATE: To date, 9 of 14 program sites have returned surveys. An estimated 44% (614/1385) of program attendees completed the survey and 43% of respondents were medical students or residents. Eighty-four percent of trainees were emotionally moved``a great deal'' or``pretty much'' by the performance, (mean = 4.3; 1 =``not at all'', 5 =``a great deal'') and 64% reported that the play portrayed the emotions of dying patients in an``entirely real'' or`v ery real'' manner (mean = 4.3). Of trainees who provide direct patient care, 75% reported that the program was``extremely relevant'' or``very relevant'' to the care they provide (mean = 4.0). The program encouraged attendees to reflect on specific aspects of palliative care including helping patients live as long as possible (mean = 3.8, 1 =``not much at all'', 5 =``very much''), talking about prognosis (mean = 4.4), addressing physical pain (mean = 4.4), addressing emotional and spiritual suffering (mean = 4.4), and talking with patients about treatment wishes (mean = 4.4). The majority of trainees rated the program a more useful training tool than didactic lectures on palliative care (83%), journal article readings (85%), and bedside rounds (57%). KEY LESSONS LEARNED: The dramatic arts foster awareness of the patient experience of illness that is relevant to the care trainees provide terminally ill persons. Many trainees feel such experiences are more useful approaches to palliative care training than traditional educational methods. Medicine has partnered with a not-for-profit managed care organization, AvMed Health Plan, Inc. Students visit the administrative offices of AvMed where presentations cover general concepts of MC, physician profiling, practice guidelines, outcome management, and disease management programs. Students also``round'' in AvMed's four major departments; Pre-Authorization, Disease Management, Member Services, and Physician Services where they witness the daily operations of the health plan. A 14 item survey (5-point Likert scale) was completed by students at the beginning of the 2nd year, the beginning of the 3rd year and again immediately before (pre) and after (post) the day at the AvMed MCO. FINDINGS TO DATE: The program has been evaluated favorably by more than 200 thirdyear medical students. There were no significant differences between mean responses at the beginning of the 2nd year of school and the beginning of the 3rd year (t-test). Of the 14 items, 8 responses were neutral and 6 were negative towards MC. The responses to the pre-seminar survey were also not different from the responses at the beginning of the 2nd or 3rd years. However, post-seminar responses showed significant (p < .05) changes favorable towards MC for 7 items and > 20% of respondents abandoned their negative attitude towards MC for 5 survey items (e.g. MCO's have potential to improve quality, MCO's provide better care than the traditional system, I understand how MC functions). STATEMENT OF PROBLEM OR QUESTION: America's health care system is undergoing dramatic transformation in care management. Most medical schools, however, have not updated their curricula to prepare graduates for practice in this changing environment. OBJECTIVES OF PROGRAM/INTERVENTION: Strategies to enhance student acquisition of the knowledge, skills, values and attitudes needed to practice in intensely managed and integrated health care systems; fostering partnerships between academic centers and appropriate health care organizations; enhancing interdisciplinary primary care education in ambulatory/ community-based settings. DESCRIPTION OF PROGRAM/INTERVENTION: Project requirements include: leadership among departments/divisions of general medicine, general pediatrics and family medicine; cooperation of participating partner organizations; and establishment of learning objectives in nine content areas: health systems financing, economics, organization and delivery; evidence-based medicine; ethics; communication skills; leadership; quality measurement; systems-based care; medical informatics, and wellness and prevention. Instruction is required for all students in their clinical years, and must begin no later than the third year. FINDINGS TO DATE: The UME-21 projects within internal medicine include: Incorporating medical students in continuous quality improvement (CQI) measurements in community practices; students' use of palm pilots to monitor disease prevalence and adherence to practice guidelines in office settings; office-based instruction in physical diagnosis; web-based learning integrated into medical clerkships; utilization of simulated patients into medicine clerkships; interdisciplinary primary care clerkships; incorporation of managed care organization physicians and evidence-based medicine into medical clerkships; and epidemiology in community settings. KEY LESSONS LEARNED: With strong leadership from general internists and their colleagues, U.S. medical schools are responding creatively to the challenges of curricular innovations in this national demonstration project.\nTEACHING RESIDENTS THE IMPORTANCE OF COMMUNITY TO HEALTH. E. Jacobs 1 , C. Kohrman 2 , D. Vickers 1 , M. Lemon 1 ; 1 Cook County Hospital, Chicago, IL; 2 Westside Health Authority, Chicago, IL STATEMENT OF PROBLEM OR QUESTION: Many residents care for patients from communities that are very different from their own, yet they rarely have any introduction to the lives, families, backgrounds, and cultural and social contexts of their patients. This is a crucial issue at Cook County Hospital (CCH) where many residents are international medical graduates who have had little exposure to the lives and socioeconomic struggles of our predominantly disadvantaged African American population. OBJECTIVES OF PROGRAM/INTERVENTION: All first-year internal medicine and pediatrics residents participated in the course with the goals of (1) increasing understanding of the US system of health care for disadvantaged groups; (2) improving communication with patients from diverse cultures; (3) increasing appreciation of the complexity of patients' daily lives and how it impacts their health and ability to access health care; (4) enhancing awareness of community and local services' impact on patients' well being. DESCRIPTION OF PROGRAM/INTERVENTION: The curriculum consists of 5 teaching modules taught by faculty from CCH and the Westside Health Authority (WHA), a community health advocacy organization, in 4 half-day sessions over a month-long ambulatory rotation. The first module is a case-based overview of the financing of care in the US with a focus on Medicaid, Medicare, and the impact of insurance status on health. The second module addresses problems language or culture can create in patient encounters. The third module uses a case study of opening an ambulatory clinic in a diverse neighborhood to teach about community based primary care. The fourth and fifth modules focus on the community and its citizens and are taught by citizen leaders and a sociologist. In the fourth session, the citizen leaders lead a discussion about their community and their experiences with the health care system. In the fifth session, residents have lunch with citizen leaders in the community and are given an introduction to the neighborhood and to the mission and activities of WHA. FINDINGS TO DATE: More than 50 internal residents participated in the curriculum. The evaluations of the program have been uniformly positive, with many residents indicating that it has changed the way they think about their patients and their health care. A majority has also asked that more teaching time be dedicated to these topics. An unexpected benefit has been the education and empowerment of the citizen leaders. They came to see physicians in a new light, as accessible human beings, and learned so much about health and health care that they became community health advocates. KEY LESSONS LEARNED: Residents are eager to learn about the lives, cultural and social contexts of their patients and value this kind of teaching. Communities and residency programs can create mutually beneficial relationships in the common pursuit of improved patient care and community health. national demonstration project Ð and the acquisition of a large regional primary care network (Clinical Care Associates) provided the opportunity to introduce students to the community practice environment. Our primary goal is to develop a cohesive academic primary care network for both clerkship and residency education. The specific objective of the present component of this broad initiative was to assess medical students' satisfaction with the community training environment by comparing their evaluations of newly-recruited community preceptors with those of more experienced hospital-based preceptors. DESCRIPTION OF PROGRAM/INTERVENTION: The core clinical clerkships in medicine (12 weeks) and pediatrics (6 weeks) are both equally divided between inpatient and outpatient experiences. Three weeks of the medicine clerkship are spent in a general internal medicine practice, three weeks in a family medicine practice, and six weeks in an inpatient setting. Pediatrics is comprised of three weeks in an outpatient setting and three weeks on an inpatient service. Outpatient preceptors work in 1:1 relationships with students. FINDINGS TO DATE: In the 1995 ± 96 academic year, primary care clerkship students spent a total 483 half-days in an outpatient environment, all as part of the internal medicine clerkship. Today, students spend 10,720 half-days annually in physicians' offices (internal medicine=3840, family practice=3840, and pediatrics=3040 half-days, respectively). Every student receives a minimum of 72 half-days of ambulatory (predominantly community-based) primary care training during their core clinical clerkship year. Using 9-point Likert scales in up to 15 different categories, students' ratings of newly recruited community internists and pediatricians compared very favorably with those of more experienced hospital-based preceptors. Community family physicians were more highly rated than a core group of residency-based family physicians. KEY LESSONS LEARNED: Despite the increasing demand for clinical productivity, community faculty accepted medical students with enthusiasm, and students were delighted with the opportunity to work closely with community primary care preceptors. The positive responses from students indicate that the integration of community practice experiences into the undergraduate curriculum will provide medical schools with the opportunity to expose students to diverse role models and a wide spectrum of patients in very favorable settings. The SP is a free health clinic that was started in 1996 by Tufts University medical students and volunteer physicians. The SP's mission is to improve the Chinatown community's access to health care by providing initial care and then facilitating referrals to ensure continuity of care. The clinic space is donated by a local church and laboratory services are donated by a local laboratory. A grant from the Massachusetts Medical Society, technical support from the New England Medical Center, and an annual, student-run auction, allows the SP to provide services free of charge. First and second year medical students handle the clinic's management and administration; they act as physician's assistants, case managers and medical interpreters. Students are supervised by volunteer physicians. Since 1996, the clinic has continued to expand and now offers the following: General medical care every Tuesday, 6 ± 10 PM, year round; Psychiatric and gynecological services; Onsite EKG machine and phlebotomy; Free medication samples; Referrals to primary health care providers, specialists, dentists, and optometrists; Adult and child immunizations; Anonymous HIV counseling and testing. FINDINGS TO DATE: Over the past 5 years, more than 400 medical students have volunteered at the SP. First-year medical students take over project management mid-year. The SP's success is a result of the dedication and innovation each class brings to the project. Medical students from Boston University and Harvard, as well as residents from the New England Medical Center have become involved and now contribute to the project. The following are demographics for the 1528 patient visits from 02/97 ± 12/00: Ethnicity(%) Chinese=81.7, White=9.8, Black=2.7, Hispanic=1.3; Age of Patient(%) 0 ± 19=4.7, 20 ± 39=29.2, 40 ± 59=32.2, 60+=33.9; Health Insurance(%) Yes=20, No=76. KEY LESSONS LEARNED: The SP provides an educational model for medical schools around the country. It is an example of how medical students can establish and manage a free health care clinic and actively participate in patient care during their pre-clinical years. We have learned that it is important to stress the mission of the SP to its patients. The SP is not a substitute for primary care delivered to Chinatown residents. The SP is a place where medical students can begin to take responsibility for patient care and patients can begin to access the American health care system. In an ideal teaching environment medical interns and residents are expected to assume total care of teaching patients. When the residents are bypassed in the decision making process, it is likely due to a communication problem which leads to attending physicians writing orders directly. Despite repeated efforts to dissuade attendings from writing orders at our community based program we decided to look at what other factors may contribute to this. OBJECTIVES OF PROGRAM/INTERVENTION: The goal was to improve communication between house staff and attendings and to ultimately decrease attending order writing on teaching patients. In addition we wanted to see an increase in satisfaction by the house staff, nursing staff, and attendings with the communication process. DESCRIPTION OF PROGRAM/INTERVENTION: A team consisting of house staff, nursing staff, and attendings met on a regular basis to identify key issues impeding good communication. Using an interrelationship diagraph the most important issue was identified as the nursing staff directly calling attendings on management issues, due to difficulty in identifying the house staff carrying their patient. The following interventions were developed to facilitate better identification and location of house staff. First, the intern's name and beeper number were entered on the nursing station board, as well as in the computer system which printed it daily on the nursing cardex. Second, the interns were issued personal non-cellular phones to provide direct communication with nurses and attendings. Third, the nursing station board was updated daily identifying the house officers on call. Fourth, the interns and residents were required to twice daily update their status on their beeper voice messages. Outcome measures used were orders written per patient by attendings as identified by a weekly log kept by the house staff. Second, there was a monthly survey of nursing staff, house staff, and attendings of their satisfaction with the communication process. FINDINGS TO DATE: The average number of orders written per chart by attendings was 1.7 and showed no substantial variation on a run chart since the beginning of the intervention. Poor return on the satisfaction surveys has limited any useful interpretation at this time although efforts are being made to increase their return. KEY LESSONS LEARNED: KEY LESSONS LEARNED: First, we learned that the major barrier to the communication process was the difficulty in identifying or locating the appropriate house staff. Second, we learned that the motivation for writing orders by attendings is multi factorial and often system related. We will continue to identify those other components that may contribute to order writing as we track the above outcomes. This program serves as a model that provides a proven approach to community-based learning for medical students which is translatable to traditional Western medical educational systems. DESCRIPTION OF PROGRAM/INTERVENTION: The COBES program implemented at MUFHS over the past ten years provides a valuable model for community-oriented education. The current program comprises approximately 27% of the curriculum for medical students at this nascent medical school in Eastern Africa. Under this program, students spend classroom time developing knowledge of research methodology, epidemiology and public health initiatives. During three years of the curriculum, they spend three to six weeks per year in rural communities learning community interaction skills, public health intervention, and health administration skills. In addition, they spend two years designing and implementing research projects within an urban community of their choice. FINDINGS TO DATE: Focus groups with students and faculty have demonstrated overwhelming support for the value of this community-based educational program in Kenya. Students have gained valuable training and experience within communities, experienced crosscultural interactions and have been able to conduct relevant descriptive and interventional research projects. COBES projects have won international awards for student research, but more importantly, the findings have been useful in creating public health initiatives for a variety of communities at a local level. KEY LESSONS LEARNED: The COBES program at Moi University provides an important model for community-based education that can also be applied to the Western system of medical education. The community interaction skills, public health knowledge and research techniques gained by the students will benefit them in any future endeavors, whether in a subspecialty, research or in community practice. While the immediate value of these types of programs is obvious, further observation and research will be needed to demonstrate the longlasting effects of this model of community-based education on the makeup and practice of medical practitioners in the future. Texas, provides educational services for clinicians. The medical education director of TOAETC served as consulting physician (CP) for this project. In July 2000, a 1800 number was established to accept requests for patient specific consultations from providers in Texas. The number was advertised by: 1. Mailing brochures to potential HIV medication prescribers (n=17,000); 2. Distribution of brochures at TOAETC sponsored lectures (n=421); 3. Two day clinical trainings in which the CP engaged in hands on patient care with community based HIV providers, and personally distributed the TOAETC brochure (n=24). Electronic consultations involved Email, faxes, websites, digital photography, as well as phone conversations. Examples will be displayed. All consultations were completed within 24 hours of the request. FINDINGS TO DATE: Forty-six patient specific consultations were performed between July 5, 2000 and January 5, 2001. The vast majority (87%) of requests for consultation came from community providers with whom the CP had engaged in a two day clinical training (n=40). Six requests came because of referrals from the CP's colleagues. No patient specific consultations stemmed from either the mass mailing or the direct appeals at TOAETC sponsored lectures. Total cost for the mass mailing was $36,545. Total cost for the clinical trainings was $6232. Interestingly, all requests came via the CP's private office number or his private email. The 1800 number was not utilized for its intended purpose of facilitating consultation. KEY LESSONS LEARNED: Community HIV providers are willing and able to link with providers at tertiary care centers via electronic consultation. However, our experience suggests that they are not likely to utilize electronic consultation methods without first establishing a professional relationship between consultant and consultee, as can be accomplished in a cost effective two day clinical practicum. STATEMENT OF PROBLEM OR QUESTION: Community-based educational programs pose challenges that include providing a high quality consistent curriculum across teaching sites and reducing student isolation. OBJECTIVES OF PROGRAM/INTERVENTION: To provide an interactive all day educational experience for students in our community-based medical school. Education Day is designed to: 1) Provide consistent high quality instruction for all students in key areas of the clerkship curriculum; 2) Allow students to learn from our best teachers; 3)Provide a recruiting opportunity for our community-based residency programs; and 4) Demonstrate the fun and intellectual challenge of internal medicine. DESCRIPTION OF PROGRAM/INTERVENTION: Students at our institution receive their clinical training in one of six community campuses located throughout the State. Students in the Medicine Clerkship travel to one campus for an all-day educational experience. The program includes three large group sessions and five small group workshops. Master teachers chosen from each community conduct the sessions. The three large-group sessions include: 1) Acid-Base Interpretation, an interactive lecture; 2)``Thieves Market'' where students try tò`s teal'' the diagnosis in a series of progressively disclosed cases; and 3)``Stump the Chump'' where the department chairman``thinks aloud'' working through a series of difficult clinical cases in a CPC-like format demonstrating the thought processes of an internist. Small groups of students rotate through each of five workshops, which are interspersed between the large group sessions. They include: 1) The Bedside Cardiac Exam using a Harvey (r) mannequin; 2) Starting IVs with Confidence using models and an interactive computer program; 3) Dynamic ECG Interpretation; 4) Chest X-Ray Interpretation; and 5) Utilizing the Peripheral Blood Smear. FINDINGS TO DATE: We have conducted two iterations of Education Day that included 70 students. Written and oral feedback has been overwhelmingly positive with some suggestions for minor changes. Students expressed specific appreciation for the quality of the program, a chance to see their colleagues at other campuses, and a break from the tedium of their clinical responsibilities. Several students requested expanding the program to two days. KEY LESSONS LEARNED: Education Day is one approach we have used to address the challenges of community-based education. Although it is not the entire answer, we are pleased with the initial results. While the feedback from students has been overwhelmingly positive, we look forward to assessing whether Education Day has achieved other desirable outcomes including improved shelf exam scores and an increased interest among our students in a career in internal medicine. STATEMENT OF PROBLEM OR QUESTION: Our Internal Medicine ambulatory rotation hosts a variety of learners with separate calendars and needs, and our many faculty members had their own sets of performance expectations. We had no consistent orientation process for these learners. OBJECTIVES OF PROGRAM/INTERVENTION: 1) to have an orientation program for our ambulatory site listing expectations for medical students, physician assistant students, Internal Medicine residents and Gynecology residents 2) to develop an orientation program using a minimum of faculty time and available to learners at any point during the rotation DESCRIPTION OF PROGRAM/INTERVENTION: Working with the Educational Technology Lab at MUSC, we developed an electronic, on-line orientation program for our Internal Medicine ambulatory block that can be navigated in a learner-specific fashion. This includes a virtual tour of the clinic, an overview of the patient population and expectations for medical students, physician assistant students, Internal medicine and Gynecology residents. We will present the orientation on computer notebook for perusal by attendees, and will discuss the development of the program. FINDINGS TO DATE: The orientation has been well received by faculty, staff and students. The amount of faculty time spent in orientation has been minimal. We are currently gathering formal feedback that will be available for the presentation. KEY LESSONS LEARNED: Learners must be given a set of expectations that should be communicated to them in a timely and consistent fashion. This on-line orientation is engaging, readily available and easy to update. STATEMENT OF PROBLEM OR QUESTION: With the increased demands on teaching faculty time, the need for standardized methods to assess the acquisition of auscultatory and observational skills of students is required. OBJECTIVES OF PROGRAM/INTERVENTION: This module was developed and administered to test clinical skills for medical students as part of their introduction to clinical medicine course, with focus on cardiac and chest auscultation as well as determination of radiological images findings. DESCRIPTION OF PROGRAM/INTERVENTION: A computer software (Question mark Perception) was used to present five clinical scenarios to the students. Each followed by five to six multiple choice, one-best answer questions. Radiological images (. jpg format) and sound files (. wav format) were used to present common cardiac and period in the fall of 2000. A total of nine computers were available in the Learning Resource Center of the library. Library staff members lung findings. The exam was administered to 143 second year medical students over a one-week proctored the exam. The exam was scored with the same software (Question Mark Perception) FINDINGS TO DATE: The average time the students used to complete the test was 47:35 minutes. Minimum time was 4:13 minutes and maximum time was 2 hours and 21 minutes. Of the total class, 84 % of the students took the exam in the last two days. There was virtually no waiting time, although one student where he had to wait for 5 minutes for a computer to be available. The maximum score obtained was 97%; minimum score was 58%. The software program reported the mean score, the standard deviation and degree of difficulty of each question. One question was eliminated from scoring because the sound quality was poor. KEY LESSONS LEARNED: Computer based testing of clinical skills needs further development, but is currently feasible. We plan in using the same software for an exam in spring 2001. This method of testing can also be expanded for use in clinical clerkships as well as assessing clinical skills of house staff. STATEMENT OF PROBLEM OR QUESTION: Clinical practice in community sites is an integral part of our primary care internal medicine training program. Despite this, many of our residents remained unfamiliar with the practice of community-oriented primary care (COPC). In order to teach them about and encourage them to practice COPC, we developed an eightweek course in community medicine. In conjunction with this course, we designed an intranet website to facilitate rapid learning of COPC. OBJECTIVES OF PROGRAM/INTERVENTION: The goals of our intranet site were to: 1) provide residents with readily accessible information about community medicine; 2) prompt residents to consider, discuss and learn about COPC on a near-daily basis; and 3) introduce distance learning concepts into the residency program. DESCRIPTION OF PROGRAM/INTERVENTION: We located an internet website (http:// intranets.com/) that supported the creation of free intranet sites. Once created, access to the intranet site was restricted to the primary care residents and selected faculty. The intranet site (http://pcimcommunitymed.intranets.com) featured discussion groups, documents and readings, links to important public health internet sites and a weekly course calendar.\nResidents were oriented to the intranet site during the first session of the community medicine course. Each week, residents were asked to respond to an on-line discussion question. Homework exercises and additional reading assignments were also posted on-line. Residents were required to use several internet links to gather neighborhood public health data and to propose a COPC project. FINDINGS TO DATE: 73% of the residents who participated in the community medicine course (11/15) responded to an anonymous post-course evaluation posted on the intranet website. 73% of the respondents (8/11) found the intranet site enhanced their learning of community-oriented primary care. 9% (1/11) found it neither enhanced nor detracted from their learning experience and 18% (2/11) felt it detracted from their learning. 40% of the respondents (4/10) used the intranet site more than once per week; 50% (5/10) used it once per week; 10% (1/10) used the site less than once every two weeks. The median number of visits to the intranet site during the eight-week Community Medicine course was 11 per resident (range 3 to 60). Seven residents did not participate in the on-line discussion groups. Key reasons for lack of participation included the amount of work assigned for the course (33%), lack of internet access at home (27%) and difficulty navigating the intranet site (20%). KEY LESSONS LEARNED: Use of an intranet website shows promise as a tool to facilitate rapid learning of concepts in community-oriented primary care. STATEMENT OF PROBLEM OR QUESTION: A significant portion of graduate medical education is based on exposure to a variety of clinical experiences. For residents training in internal medicine the majority of these interactions occur in the inpatient setting. It is important not only to document the range of diagnoses that residents encounter, but also to track length of stay and readmission rates, two important markers of the quality of inpatient care. Quality markers may be linked to attendings supervising the residents and may indicate topics for faculty development. OBJECTIVES OF PROGRAM/INTERVENTION: We developed a computerized database to 1) track inpatient diagnoses that IM residents encounter on a general medical service; 2) track length of stay, readmission rates, and other performance quality measures for particular diagnoses; and 3) link resident practice measures with supervising attendings to identify areas for faculty development. DESCRIPTION OF PROGRAM/INTERVENTION: We created a computerized database in Microsoft Access. The database documents every patient admission to the internal medicine teaching service; the medical team (residents and attending) responsible for the patient's care; primary and secondary diagnoses listed for the patient (from the discharge summary); admission and discharge dates. The database user interface is organized with multiple data tables supporting``drop boxes'' to click on the appropriate physician names and diagnoses. An administrative assistant enters the data, and any diagnoses not listed in the data tables are referred to a physician for clarification. FINDINGS TO DATE: This computer-based information system allows tracking of all diagnoses seen by IM residents on a general medical service. The data can be used to compare each resident's exposure to specific diagnoses with his/ her performance on in-training exam sub-scale scores; educational interventions can be developed to enhance medical knowledge in those areas identified as having low exposure and low in-training score. The documentation of residents' experiences also fulfills the Residency Review Committee's requirement for documentation of IM residents' diagnostic experience. The database is used as part of hospital-wide disease-specific quality improvement initiatives to review length of stay and readmission rates on the teaching and non-teaching services, and to target physician education at all levels to improve patient outcomes. Future links between this database and pharmacy and laboratory utilization measures will permit even more detailed analyses of physician performance. KEY LESSONS LEARNED: An accurate computerized tracking system is a useful educational and quality improvement tool for residents and attendings in internal medicine residency programs. This database recognizes the value of integrating measures of quality improvement into residency training, and may be used by training programs in other disciplines to track clinical performance. STATEMENT OF PROBLEM OR QUESTION: When faced with teaching physical examination skills to a large class of medical students (as well as coordinate the teaching efforts of a diverse group of physician teachers), it is a challenge to ensure that students are exposed to a common set of physical exam findings and find techniques that optimize their opportunity to develop physical exam skills. OBJECTIVES OF PROGRAM/INTERVENTION: To expose students to certain common abnormal physical exam findings and enhance learning of physical examination skills through the integration of new technologies and traditional educational strategies. DESCRIPTION OF PROGRAM/INTERVENTION: Computer-based technology is becoming an integral part of curriculum innovation at Ohio State University. As a part of this pedagogical effort, an innovative, highly interactive website was developed to promote active learning at a level appropriate for undergraduate medical students and to be compatible with the current Physical Examination Course curriculum. This web-site makes use of Macromedia Flash, video streaming, and SMIL technologies to bring to life portions of the physical exam and to make available common abnormal findings often difficult to demonstrate to medical students at any specific time. It emphasizes learning through reading, hearing, seeing, and doing in a sequence designed to capture the learner's attention and to make information available through multiple sensory modalities. There are 6 core modules (HEENT, pulmonary, cardiac, abdominal, neurologic, musculoskeletal) under development. Each module includes the following: a Flash-based interactive component highlighting important anatomy and physiology; web text and video clips discussing and demonstrating essential physical examination skills; Flash-based interactive components where students hear findings or practice selected physical examination skills (e.g. taking a blood pressure and hearing Korotkoff sounds); a glossary of clinical terms; links to clinical pearls and additional explanations of techniques or abnormal findings; and clinical correlates where students practice integrating physical examination findings. This exhibit will showcase interactive portions of the modules via a CD-ROM presentation. FINDINGS TO DATE: Pilot data were collected and of the nine respondents, all reported that this site was an excellent learning tool, desired further development of similar technology, and would recommend this site to other students. Eight of the nine students reported being able to navigate the site and use the interactive Flash components without difficulty, and stated they would be more likely to use this site than the textbook to prepare for practice sessions with faculty tutors. Overall, students found it helpful to hear findings, liked the links built into the site, and enjoyed the clinical correlates component. The only weakness identified was the time to download the video clips if access was obtained from modems as opposed to a high-speed internet connection. KEY LESSONS LEARNED: Next Steps: Data are currently being collected with a larger sample concerning the use and effectiveness of this site to guide subsequent revision of current modules and to plan the design of future ones. Also being considered is the development of patient cases representing core disease presentations, as well as modules specifically designed for educators in this area. STATEMENT OF PROBLEM OR QUESTION: Advances in evidence-based research, together with unprecedented access to original sources of information, provide a unique challenge to medical decision-makers that may improve the quality of care provided to their patients Ð if they can find and correctly interpret this readily available information. OBJECTIVES OF PROGRAM/INTERVENTION: Our aim was to instruct clinicians accessing and interpreting online sources of evidence-based research using a cognitive theory that has been applied to clinical decision-making. DESCRIPTION OF PROGRAM/INTERVENTION: Decision-making tasks concerning chest pain evaluation in women were developed for medical students and internal medicine residents. The cognitive theory Ð fuzzy-trace theory Ð guided the selection of online sources (e.g. target articles) and decision-making tasks. FINDINGS TO DATE: Thiry-four participants (12 students and 22 internal medicine residents) attended didactic conferences emphasizing search, evaluation, and clinical application of evidence. A 17-item Likert scale questionnaire assessed participants' evaluation of the instruction. Ratings for each of the 17 items differed significantly from chance in favor of this alternative approach to instruction. KEY LESSONS LEARNED: Fuzzy-trace theory was found to be a useful guide for developing internal medicine learning exercises in medical informatics and decision making. However, studies with more learners and behavioral evidence of improved clinical decision-making skills and, ultimately, patient outcomes are needed. STATEMENT OF PROBLEM OR QUESTION: Scheduling conflicts prevent many residents, especially those on``non-ward'' rotations, from attending morning report. Even when able to attend, however, residents may miss salient learning points presented during those conferences. In order to circumvent these problems, we created eReports, electronic summaries of morning report distributed to all of the internal medicine residents by E-mail. OBJECTIVES OF PROGRAM/INTERVENTION: The goals of our intervention were: 1) to provide all of our residents with the learning opportunities of morning report even if unable to attend; 2) to model the practice of asking and answering patient-centered questions in an evidence-based way; and 3) to strengthen the educational impact of morning report by cogently synthesizing information presented, further emphasizing salient teaching points, and providing additional relevant information. DESCRIPTION OF PROGRAM/INTERVENTION: Each day, we developed a summary of the material presented in morning report and distributed it to all of the residents by Email. These summaries, which we called eReports, mirrored the structure of our morning reports. Each eReport began with follow-up information that included clinical follow-up to previously presented cases and literature-based answers to questions generated by those cases. We then summarized the two new cases discussed in morning report. At the end of each eReport, a task (e.g., view the peripheral smear) or a central question (e.g., how helpful would a positive result of your planned diagnostic test be?) was generated for each new case. FINDINGS TO DATE: Residents were asked to complete an anonymous survey prior to the dissemination of eReports. 81% of the residents responded. Of these, 71%``almost never'' or only``occasionally'' knew the content of morning report (cases or follow-up) when they were unable to attend. 88%``strongly agreed'' that eReports would be educationally helpful. Further investigation is underway to determine if eReports were, in fact, helpful. For example, did they successfully clarify teaching points, stimulate self-directed learning, or contribute to improved memory of cases and information presented in morning report throughout the rotations studied? KEY LESSONS LEARNED: eReports are a promising way to facilitate case-based learning among internal medicine residents. Not only do they ensure the dissemination of the material presented in morning report, they also afford a synthesis that strengthens the quality and content of that material. STATEMENT OF PROBLEM OR QUESTION: The ACGME has endorsed competencies in 6 core areas as a first step to emphasize educational outcome (rather than process) assessment for residency program accreditation. The guidelines require the residency programs to provide an appropriate educational experience in these areas, evaluate the residents' performance, provide feedback and achieve progressive improvements in residents' competence and performance. In addition the effectiveness of the educational program itself also needs to be evaluated. To achieve the ACGME goals, residency programs must develop a new educational model that will engage residents in a time efficient manner. In addition to their areas of clinical training, the new curriculum should cover areas such as doctor patient communication, medical ethics and professionalism. OBJECTIVES OF PROGRAM/INTERVENTION: We have designed and are now implementing a web-based interactive curriculum that would meet common and specific needs of various residency programs at our institution. The objectives of this web-based program are: 1. The curriculum is available at a convenient time and place convenient to the residents. 2. The residents' decide the order and pace of the curriculum. 3. The curriculum utilizes existing teaching materials as far as possible. 4. The new curriculum makes efficient utilization of educators' time and skills. 5. An efficient and effective evaluation and feedback system is an integral part of the new curriculum. DESCRIPTION OF PROGRAM/INTERVENTION: We designed the web-based curriculum to be based on interactive, question and answer scenarios. The key features of this program are: 1. The curriculum is on a password protected web site 2. The curriculum consists of brief interactive case scenarios that are grouped in the 6 core competency areas endorsed by the ACGME. 3. These case scenarios can be created by any educator by copying and pasting text from a text document into the text box of a separate development web site. Content is automatically converted into interactive HTML format and placed on the web site. Audio, video and graphic files can also be included. 4. Pretests, posttests, multiple-choice questions and free text entry areas can be included to capture the users responses, track performance and assess competency. In order to simulate a real-life scenario, the residents have an option to order consults and clinical tests to solve the cases. 5. The program provides extensive feedback including tracking amount of money spent while resolving a case scenario, amount of questions answered correctly at first attempt, number of attempts and the ability to compare themselves with other residents who have tried that particular scenario. 6. The program creates a customized recommended reading material based on the questions answered incorrectly. 7. If a user leaves a case scenario incomplete, the progress is saved and can be continued from that point when the user logs on the next time. 8. The program uses a database of house staff to create individualized reports for program directors on the participation and performance of their residents, and also is used to assign particular cases to a particular group of residents. FINDINGS TO DATE: We plan to formally implement the program beginning in the July 2001 academic year. Pilot testing has shown this system to be acceptable to the house staff, attractive to the program directors and effective and efficient method of teaching for the educators. KEY LESSONS LEARNED: As new requirements are placed on a medical education system strained for resources and funding, creative solutions are needed that will make efficient use of existing education material, to meet the needs of the students, educators and organizations using current technological advances in computers and the Internet. Using appropriate technology it is possible to create stimulating and educational material that also seamlessly combines tracking, feedback and evaluation and thus efficiently meet the ACGME goals.\nWEB-BASED INTERACTIVE CURRICULUM FOR THE ACGME OUTCOMES PROJECT. N.B. Mehta 1 , A. Hull 1 , J.H. Isaacson 1 , A. Jain 1 , 1 Cleveland, Cleveland, OH STATEMENT OF PROBLEM OR QUESTION: The ACGME has endorsed competencies in 6 core areas as a first step to emphasize educational outcome (rather than process) assessment for residency program accreditation. The guidelines require the residency programs to provide an appropriate educational experience in these areas, evaluate the residents' performance, provide feedback and achieve progressive improvements in residents' competence and performance. In addition the effectiveness of the educational program itself also needs to be evaluated. To achieve the ACGME goals, residency programs must develop a new educational model that will engage residents in a time efficient manner. OBJECTIVES OF PROGRAM/INTERVENTION: 1. Implement a web-based interactive curriculum that would meet common and specific needs of residency programs 2. The curriculum be available at a time and place convenient to the residents. 3. The residents' decide the order and pace of the curriculum. 4. Utilize existing teaching materials as far as possible. 4. Make efficient utilization of educators' time and skills. 5. Incorporate an evaluation and feedback system seamlessly in the new curriculum. DESCRIPTION OF PROGRAM/INTERVENTION: 1. The curriculum is on a password protected web site and consists of brief interactive case scenarios (ICS) grouped in the 6 core competency areas. 3. The ICS can be created by an educator without programming knowledge by automatically converting text into interactive HTML format on the web site. Audio, video and graphic files can also be included. 4. Various question formats can be included to capture the users' responses and assess competency. The residents can order consults and clinical tests to solve the ICS. 5. Extensive feedback includes amount of money spent while resolving an ICS, number of attempts and questions answered correctly at first attempt and comparison with peers. 6. The program creates a customized recommended reading material based on performance. 7. If interrupted, the user's progress is saved for subsequent sessions. 8. The program creates individualized reports for program directors on the participation and performance of their residents. FINDINGS TO DATE: We plan to formally implement the program beginning in the July 2001. Pilot testing has shown this system to be acceptable to house staff, attractive to program directors and effective and efficient method of teaching for the educators.\nKEY LESSONS LEARNED: As new requirements are placed on a medical education system strained for resources and funding, creative solutions are needed that will make efficient use of existing education material, to meet the needs of the students, educators and organizations. Using current technological advances in computers and the Internet, one can create stimulating and educational material that combines tracking, feedback and evaluation and thus efficiently meet the ACGME goals. STATEMENT OF PROBLEM OR QUESTION: There is interest in using the Internet to assist in teaching medical clerks ECG reading skills, but multiple technical barriers have impeded efforts thus far. These include inability to place high quality ECG images on the Internet and failure to utilize software that allows interactivity yet is supported by most web browsers. OBJECTIVES OF PROGRAM/INTERVENTION: To develop an internet-based, comprehensive ECG tutorial that is interactive and can be viewed by standard web browsers. DESCRIPTION OF PROGRAM/INTERVENTION: We developed a new method for digitizing paper ECG files. We then digitized our complete ECG library used for our four session, clinical clerkship ECG tutorial. These high quality images were placed in web pages using multimedia development software that can be viewed with most web browsers. FINDINGS TO DATE: An online ECG tutorial is now available to all internal medicine clerks. It covers 4 chapters of material and includes over 30 ECG cases. The interactivity allows users of various skill levels to choose the amount of assistance they receive in interpreting the cases. KEY LESSONS LEARNED: A large amount of effort was required to overcome technical barriers. Once these issues were overcome, further work was very efficient. The solutions to these technical issues can be easily shared. STATEMENT OF PROBLEM OR QUESTION: Evidence-based medicine and Internet resources have become increasingly important in the organization and philosophy underlying morning report. Electronic media is easily available to house officers in real time and this often supercedes the limited, outdated content in standard textbooks. Managing that data and integrating it into an educational culture is a new challenge for residents and faculty alike. OBJECTIVES OF PROGRAM/INTERVENTION: Prior to implementation of the new paradigm, the chief resident conducted morning report and the post-call senior resident presented the case. Faculty, other senior residents, interns, and medical students were present in the room. Qualitative surveys of residents at the time revealed concerns of knowledge stratification between interns and senior residents, need for more current handouts, and a desire for more involvement by the interns and medical students in discussion. In the new model, two chief residents conduct morning report and the two-day post call intern presents the case. No senior residents or faculty are present in the room. DESCRIPTION OF PROGRAM/INTERVENTION: We describe a novel morning report format that:\n1. Separates intern and senior morning reports to alleviate pressures of knowledge stratification and to enhance the learning experience for each group. 2. 3. Facilitates learner interaction by optimizing learning climate and geography (e.g., location,\nfood, and open table settings). 4. 5. Implements two active facilitators to engage the group, maximize participation, and utilize complementary skill sets. 6. 7. Applies educational and computerized resources including electronic databases, networked computers, and data projectors to develop an efficient and educationally driven case selection process. 8. 9. Incorporates electronic tools (``E-tools'') to adapt quickly to learner goals and needs, based on time of year, learner experience and learner interest. 10. FINDINGS TO DATE: Anonymous intern evaluation data suggest that the new model has effectively addressed these issues. Based on current academic year data of almost 100 responses, all interns consistently felt either``challenged'' or``interested''. Almost 25% of interns in fact preferred a longer morning report. The use of electronic handouts has met with universal acceptance: 77% thought the handouts were``very good'' and 23% considered them``good''. Only one resident had a neutral opinion and there were no negative responses. Almost 96% of respondents agreed with exclusion of faculty and senior residents in morning report. Interns who were exposed to the morning report prototype (n=47) had a significant 9.7% increase in average ITE percentile scores (p = 0.015, unpaired t-test) compared with interns not exposed to the model (n=96). KEY LESSONS LEARNED: We conclude that our morning report model has increased the level of participation among interns, alleviated pressures of knowledge stratification between interns and residents, and successfully utilized electronic tools to enhance and adapt to a learnercentered environment. Areas for further study include successful adaptation of the model to the dynamic needs of report participants, correlation with the ITE, development of more specific outcome metrics, and impact on patient care.``I The third year of medical school can be particularly stressful for students. Some students are reluctant to access their school's mental health resources for fear of appearing``weak'' or unable to cope in a highly competitive environment. OBJECTIVES OF PROGRAM/INTERVENTION: To help students cope with the stresses of their clinical clerkships, the Program for the Humanities in Medicine at Washington University School of Medicine instituted the Clerkship Counseling Hotline. DESCRIPTION OF PROGRAM/INTERVENTION: The Clerkship Counseling Hotline was a confidential, anonymous service intended to provide third year students with a short-term mental health resource aimed at easing stress. The hotline was staffed by a masters level counselor around the clock. FINDINGS TO DATE: 17 calls were received by the Hotline from August 1999 through November 2000. Issues prompting calls, in order of frequency, included a) disillusionment with medical environment; b) anxiety over performance/evaluation; c) personal/relationship problems; d) requests for information/referral; and e) anxiety over decision to go into medicine. 15 of the 17 callers were female. A year-end survey assessing the Hotline was returned by 83% of the students. 75% of all students said that continuing the Hotline was``somewhat important'' or``very important,'' and 75% of students described the availability of the Hotline as`s omewhat reassuring'' or``very reassuring.'' 88% of women found the hotline reassuring, compared to 58% of men (P=.002). KEY LESSONS LEARNED: This Clerkship Counseling Hotline was regularly used by our 3rd year students, who strongly endorsed its continuation. Female students used the Hotline substantially more frequently than did male students. Such a Hotline may provide a service that is complementary to the traditional mental health resources offered by most medical schools. Comply with the ACGME-RRC scholarly activity requirement (SAR). 3. Evaluate the program. DESCRIPTION OF PROGRAM/INTERVENTION: Key components of our SAP include: precise delineation of requirements; dedicated time of a research director and coordinator; core curriculum with didactics (trial design, test interpretation, meta-analysis, bioethics, medical writing, informatics, computer graphics), journal club focused on trial design, optional clinical teaching seminars, and an elective 10-hour biomedical writing course; optional research modules; SAP manual; staff mentors and institutional support. The SARs include: participation in didactic program and journal club; completion of a pre-approved written project; completion of a (staff evaluated) 30 ± 60 minute oral presentation using computer-generated slides; and a 1:1 literature search session with a librarian. An annual research day includes: residents' poster presentations (in 2000: 33 posters) with a reception and a visiting professor lecture. Two selected research projects are presented by the residents at a medical grand rounds. Our 2001 budget of $26,340 includes: resident travel to present at meetings, printing of SAP manual, biomedical writing course, Research Day expenses, and general operating costs. The budget does not include salaries for Research Director (10%) or Coordinator (50%). FINDINGS TO DATE: Since inception of our SAP, resident publications and presentations have increased (in 2000: 10 published articles, 12 abstracts, 42 presentations at national or regional meetings). The SAP and the research mentor's department co-support residents' travel to present at meetings. The most common written project is an original research abstract; case reports and simple literature reviews are not acceptable. A survey of our graduating residents indicates acceptance of the SAP (20 of 29 PGY-3 residents felt the SAP to be``very valuable''). KEY LESSONS LEARNED: We believe the success/acceptance of our SAP are due to an established core curriculum, supportive personnel, dedicated funding (in particular for resident trips to meetings), availability of staff mentors, strong institutional resources, and meticulous tracking of residents' progress.~50% of our residents take 1 ± 2 research modules, which must be approved in advance. No relation exists between the type of project and whether residents took a research module. and an audience response system was also used to administer the questionnaire during a noon conference FINDINGS TO DATE: Thirty five HS participated in this survey; (48% GL-I, 24% GL-II, 14% GL-III, 7% GL-IV and 7% GL-V). Most of them thought the book was trustworthy (87%), accurate (86%), user friendly (57%), and overall useful for the management of patients (87%). Although most HS always carried the CCF book (73%), most also carried other commercially available antibiotics recommendations book(s) (68%). There was no consensus which of these books was more useful for them. The CCF Book sections on guidelines for specific diseases and dosing information were referred to most often (37% & 40%; respectively). Requests were made to add more microbiology information and other clinically important antimicrobial drug interactions. KEY LESSONS LEARNED: The CCF Guidelines for Antimicrobial Usage book is well respected among the HS. The book serves it's purpose well in helping HS with their daily care of hospitalized patients. This survey helped us in implementing some changes that we believe will improve these guidelines and consequently, improve the quality of care for our patients.\nDIVING FOR PERLS: USING PORTFOLIOS FOR EVALUATION AND REFLECTION ON LEARNING. K. Edwards 1 , L.E. Pinsky 1 ; 1 University of Washington School of Medicine, Seattle, WA STATEMENT OF PROBLEM OR QUESTION: Lifelong learning is an important goal of medical training. However, objective tools for teaching skills of self-assessment and for giving feedback on professional development are lacking. Medical training is an experiential process and reflection is necessary to develop expertise and professionalism from the practical experiences. OBJECTIVES OF PROGRAM/INTERVENTION: Residents participating in the portfolio program will: 1) learn how to set achievable learning goals; 2) identify learning experiences that will facilitate achieving learning goals; 3) develop skills of self-assessment, identifying strengths and weaknesses 4) receive structured peer and supervisor feedback in a range of skill areas 5) develop important professional skills that support life-long learning, physician-patient communication, and excellence in patient care. DESCRIPTION OF PROGRAM/INTERVENTION: Portfolios constitute an integrated system of learner-directed evaluation. The primary outcome of the evaluation project is for each resident to create a portfolio that includes a comprehensive collection of different types of feedback and self-assessment. The components of the portfolio may vary, but are based on specific activities to assure broad coverage of skill areas. They include: worksheets on goal setting, tracking and self summary of learning, teaching self-evaluation, colleague feedback; critical incident type narratives; mini-CEX observations; CD-ROM compilation of the resident's videotaped encounters with patients, directed self-evaluation, a summary of resident/attending meeting, and overall department evaluation. There is a Working Portfolio for the resident's use and work-in-progress, and a Performance Portfolio for highlighting core competencies and strengths at the end of the year. FINDINGS TO DATE: After a pilot program with the internal medicine residents, a surprising number of the portfolio skills and objectives needed significant attention by the mentor faculty. Simple goal setting exercises presented challenges for some residents. This finding highlighted the need for the portfolio exercise within the residency program. KEY LESSONS LEARNED: We identify 5 elements critical to the success of a meaningful and effective portfolio intervention: 1) distinguishing the goals of the Working and Performance portfolios; 2) establishing a collegial educational climate; 3) teaching and refining selfassessment acumen; 4) promoting reflection on goals and progress over time; and 5) supporting structured autonomy via iterative discussions with mentors. Without these elements, trainees may view a portfolio simply as another tedious exercise and we risk trivializing an innovative tool. STATEMENT OF PROBLEM OR QUESTION: Community based ambulatory educational experiences have been increasingly emphasized in medical education due to the need to prepare medical students and residents to become more knowledgeable about primary care medicine in the era of managed care. At our institution, students start working in community based practices in the first year of medical school. Effectiveness of the students' educational experience in the community has frequently varied depending on the skills and interests in teaching of the physicians participating in the program. All physicians in our network are required to teach medical students at least 1/2 a day a week but little faculty development in teaching has been provided. How to effectively address the needs of these faculty is addressed in this innovative peer teaching program. OBJECTIVES OF PROGRAM/INTERVENTION: In order to improve the overall experience for both students and faculty, our institution has developed a``Teach the Teachers'' program. The primary objective of the program is having community based faculty associated with our institution's primary care network teach their peers skills in effective teaching and mentoring of medical students. It is also an objective to develop a core of community based faculty who will be able to provide leadership in the faculty development arena for our community based teachers of medical students. DESCRIPTION OF PROGRAM/INTERVENTION: Over the course of a year, the university staff collaborated with four affiliated community based primary care faculty to guide them in selecting the curriculum modules they felt would be most useful to office based teachers. The team also participated in the nationally organized General Internal Medicine Faculty Development Project (GIMGEL) where additional skills and training techniques were acquired. In order to develop a quality program, the community faculty selected four curriculum areas as well as a kick-off session for their program activities. They also worked with the Sr. Associate Dean for Medical education to make sure that the chairman of the Department of Medicine supported the program and would strongly encourage all of the university's primary care network of physicians (total of 91) to participate in at least 20 hours of faculty development over a two year time period. CME was also offered for all faculty development. The program selected includes the following modules:\n1. The kick off program( one time session for all participants), Developing Teaching Skills for the Primary Care Physician A nationally known physician educator, Gary Ferenchick, MD, MS, was selected to present the basics of community based teaching to the entire group of primary care network physicians at one of their required dinner meetings. The speaker was chosen for his enthusiastic hands-on approach to office-based teaching. The speaker at the end of his talk then introduced the community based faculty development program and the four peer faculty. Each of the four peer faculty developed an interactive module to be presented to each of four groups of teaching physicians at dispersed sites around our large urban service area. They selected these topics based on a needs survey and their own personal experiences in teaching. These topics include the following:\n2. Orienting and Organizing the Student Experience 3. The One Minute Preceptor-Teaching on the Run 4. Arrows in the Quiver: Using Multiple Strategies to Reach Your Student 5. Developing Effective Techniques for Student Evaluation and Feedback FINDINGS TO DATE: In an earlier program of training of community based faculty by our institution, training of community based faculty was very successful but the lack of departmental support for the training led to smaller numbers of faculty participating. This program reviewed the prior one and developed a different strategy. The four community based faculty have spent two hours each month for the past year in preparation for the training program in addition to attending the three day GIMGEL conference on faculty development. They have reviewed major articles and materials on curriculum development in order to select those they felt most appropriate based on the initial needs assessment of their peers. They have obtained the Chair of Medicine's support for the program as well as for the publication of the four curriculum articles. The program times are scheduled and the kickoff session is set. Success of the program will be reported on at the conference. KEY LESSONS LEARNED: Community based faculty need to be involved in leadership roles in faculty development for their peers. The topics may not be new to the field but having peers teaching peers is innovative and an effective strategy for encouraging the acceptance and adoption of new teaching skills. Materials Produced and Available A series of four curriculum articles written by the four community based faculty and workshop handouts will be available for those interested in implementing a similar type of peer training program. A video will also be available to demonstrate the interactive modules. For this reason medical schools are devoting time and resources to prepare faculty for providing students with effective feedback. In an effort to reach more faculty and allow members to learn at their own pace, we designed a web-based faculty development module to address the relevant skills. OBJECTIVES OF PROGRAM/INTERVENTION: -Improve the observation and feedback skills of faculty.\n-Design an interactive web-based teaching module utilizing multimedia. DESCRIPTION OF PROGRAM/INTERVENTION: The web-based module is the first of a series of teaching modules planned to support the Macy Initiative in Health Communication at NYU (http://endeavor.med.nyu.edu/macy/nyumacy/). The Macy Initiative is a three school (New York University, University of Massachusetts and Case Western University Schools of Medicine) project funded by the Macy Foundation to improve the communication skills of physicians. Part of the faculty development efforts at NYU include plans for several on-line teaching modules. The observation/feedback module contains three sections. In the first section participants are asked to watch a 1st year student interview and then to complete a checklist. During the second section, the participant's checklist data is compared with that of several expert faculty. In the last section, participants are taken stepwise through the feedback process. They begin by orienting the student, then by asking for the student's own self-assessment, followed by giving feedback and finishing with the closing. At each step participants are asked to enter their own comments and then watch a video clip of the same 1st year student receiving feedback from an expert faculty member. Seminar leaders for the current Physician, Patient and Society Course as well as Primary Care Residents will be asked to take the module. Participants fill out a pre-post survey to assess changes in attitudes and confidence in giving feedback and to comment on the module. FINDINGS TO DATE: It is feasible to design an interactive web-based faculty development module. We are able to track which faculty have completed the module using a log-in page and retrieve faculty responses anonymously by having programmed the module to send them to a separate web-site without identifiers. Data on the changes in attitudes and confidence as well as feedback on the module will be available. KEY LESSONS LEARNED: -Helpful collaborations can take place among Clinical and Academic Computing Departments. -These collaborations often require learning each others`l anguages'' and involving project managers. -Interactive digital media is a very exciting, innovative way to teach the medical interview. DESCRIPTION OF PROGRAM/INTERVENTION: Our program was comprised of an interactive discussion entitled``What Every Clinician Needs to Know About Quality'' led by an international expert in QI, followed by a series of observed structured teaching experiences (OSTEs). These OSTEs were case-based teaching modules utilizing role-playing to address QI issues encountered in typical clinical cases. The program was evaluated through a formal discussion with participants at the conclusion of the seminar along with pre and post-program surveys. FINDINGS TO DATE: Twenty faculty from the Departments of Medicine and Family Medicine participated in the program. The faculty was surveyed pre and post-program on their knowledge, competency to lecture about, and competency to teach clinical applications of QI. A 10-point scale was utilized, with 0 indicating no knowledge or competency, 5 being neutral, and 10 indicating superior knowledge or competency. In addition, attitudes towards teaching about QI were surveyed pre and post-program, using a 5-point Likert scale. With an 85% response rate, perceived knowledge of QI increased from below neutral to above neutral in 29% of respondents. Competency to lecture on QI increased from below neutral to above neutral in 41% of respondents. Most importantly, competency to teach clinical applications of QI increased from below neutral to above neutral in 59% of respondents. The perceived value of teaching about QI also increased, (p = 0.04) and faculty uniformly commented on how this program reinforced the importance of (or value of) teaching this essential clinical competency. KEY LESSONS LEARNED: QI issues are seen in daily clinical practice and need to be addressed with medical students and residents. Faculty development through interactive role-playing and discussions can be influential in changing attitudes and self-perceptions of teaching abilities. Objectives of Part I were to enable faculty participants to understand the purpose of incorporating genetics into primary care teaching and practice, identify specific teaming needs for home faculty and available resources, develop a 6-month implementation plan and acquire teaching techniques to incorporate genetics into primary care teaching and practice. DESCRIPTION OF PROGRAM/INTERVENTION: Part I of the two-day GPC Training Program held in October 2000 included didactic and interactive plenary sessions, content-based breakout sessions, a poster session, and computer demonstrations. Twenty competitively selected faculty teams were paired to enhance interaction and to facilitate discussions with advisors (members of the GPC Advisory Committee or Genetics Education Consultant Committee) regarding specific implementation plans for faculty development. A draft curriculum manual, largely case-based in its organization with information concerning electronic and written resources, was prepared for use by participants and faculty in the course. FINDINGS TO DATE: Findings from the external evaluation of Part 1 of the GPC Training Program show that the greatest self-reported increase in knowledge and skills are in the areas of resources, development of a specific implementation plan, and in applying teaching techniques for addressing ethical, legal, and social implications of genetics in medicine. Participants say they need more help with genetics content and with around specific methodologies for delivery of genetics-based faculty development to primary care faculty. KEY LESSONS LEARNED: Meaningful incorporation of additional genetics content into Part 2 of the GPC Training Program is indicated. Additionally, more interaction time and opportunities for team members to observe and practice specific methods of delivering training may enhance participant teaming. students were randomly assigned to participate in a 2-week ambulatory block during their 2 month required IM clerkship; the remaining 6 weeks were spent on inpatient wards. The remaining 17 students completed 2 months of inpatient wards. The settings included: community private clinics, VA Clinics, and faculty practices. The 2-week period was comprised of clinic 5 half days per week, and 4 half days of small group interactive sessions covering general medicine topics. Students were required to present an interesting case they were exposed to during the ambulatory portion of the clerkship. FINDINGS TO DATE: Compared with inpatient students there was no difference on their performance. However, 60% were more likely to go into Internal Medicine, while only 33% stated it had no effect on their career choice. They enjoyed working in the outpatient setting, and saw on average 3 ± 4 patients per 1/2 day. Approximately 50% watched procedures, while 20% were able to participate in procedures such as pelvic exams, joint injections and mole removal. They saw a wide range of problems including chronic medical diseases, skin diseases, smoking related issues, as well as preventative medicine. Overall, the students enjoyed their outpatient experience and would have preferred more time in the clinic setting. KEY LESSONS LEARNED: Even though there was no statistically significant difference in how well the students performed on their examinations, they did view this two-week ambulatory block as a positive experience. In addition, we feel it is significant that 60% of the students reported they were more likely to go into Internal Medicine despite this very brief exposure to outpatient medicine. STATEMENT OF PROBLEM OR QUESTION: The most common deficiency among our graduating residents was a lack of preparation for dealing with the business side of medicine. What is the best way to introduce third party payment systems and the intricacies of running an office practice to residents? OBJECTIVES OF PROGRAM/INTERVENTION: We hope to create an ambulatory experience that would combine patient care with an educational focus on medical business. The residents will have an introduction to billing and coding. They will become familiar with the different payer sources and how they relate to patient care. The curriculum will allow residents to apply the basics of managed care (including formularies and referral) while taking care of patients. The residents should also develop a basic knowledge ethical conflicts that exists within insurance entities. DESCRIPTION OF PROGRAM/INTERVENTION: The educational curriculum consists of one month rotations in ambulatory clinics with significant managed care exposure. The clinic experience is supplemented with lectures on billing, coding, insurance basics, and ethics. Residents provide input on deficiencies in their knowledge and which topics should be expanded. Residents also receive hands on experience by observing ancillary office staff in their different duties (billers, referral staff, etc.) in order to help solidify their learning experience. FINDINGS TO DATE: Residents show improvement in their ability to bill and code appropriately. They also are satisfied with their learning experience and think it should be expanded. KEY LESSONS LEARNED: Knowledge of the business aspect of medicine has become an essential part of medical practice. Residency programs need to address this need in educating their residents. Residents are very receptive to lectures on medical business and see these topics to be an important supplement to medical education.\nT. Houston 1 , R. Connors 1 , N. Cutler 1 , M. Nidiry 1 ; 1 Johns Hopkins University, Baltimore, Maryland STATEMENT OF PROBLEM OR QUESTION: Although a highly rated Rheumatology rotation exists, our residents rated their training in``primary care'' musculoskeletal medicine (knee pain, biceps tendonitis, epicondylitis, ankle sprain, injections, etc.) as less than optimal. OBJECTIVES OF PROGRAM/INTERVENTION: Success in``Primary Care'' Musculoskeletal Medicine training was limited by lacking primary care preceptors trained in musculoskeletal problems and by not having a concentrated collection of patients with musculoskeletal problems. Our OBJECTIVE was to establish an innovative primary care musculoskeletal medicine clinic precepted by general internists with additional training in joint issues. DESCRIPTION OF PROGRAM/INTERVENTION: In the planning year, faculty attended special musculoskeletal training sessions sponsored by SGIM and Rheumatology organizations, attended orthopedic clinics, and assembled a musculoskeletal patient population. Triage nurses were instructed to only schedule patients with acute and chronic joint problems to a special clinic session at a community-based practice site. One half-day per week, residents interviewed patients, discussed diagnosis and management with faculty, and then were supervised during procedures. The curriculum also included group discussions focusing on diagnosis and treatment (medications, physical therapy, etc) of musculoskeletal problems not covered in the Rheumatology rotation, skills practice with injection models, and a reference syllabus. FINDINGS TO DATE: Our evaluation of the musculoskeletal clinic within the first six months focused on the success in achieving a concentrated experience for the first six residents to participate in the rotation. Residents averaged seeing 4.3 patients with musculoskeletal complaints per half-day clinic. The most common musculoskeletal complaints were knee pain(25%), back pain(19%), shoulder pain(16%), and hip pain(9%). In treating these patients, residents performed a mean of 2.0 procedures per half-day clinic. The most common procedures included injections of the : knee, subacromial region, and trochanteric bursa. Residents have uniformly rated the usefulness of the clinic as very good/superb. An additional pre-post knowledge and skills assessment of residents will be completed at the end of the two-year curriculum. KEY LESSONS LEARNED: Our needs assessment indicated that residents wanted additional training in musculoskeletal medicine, but preferred to learn in a primary care setting. Despite the logistic challenges, our curriculum has demonstrated the feasibility of a concentrated primary care musculoskeletal medicine experience. STATEMENT OF PROBLEM OR QUESTION: Medical advice and nicotine replacement are effective smoking cessation interventions. However physicians have a limited impact as they miss many opportunities to help smokers quit and often lack the necessary counseling skills. As smoking cessation is a stepwise process, physicians should tailor counseling to each smoker's motivation to quit. OBJECTIVES OF PROGRAM/INTERVENTION: We designed a program based on active learning methods and the``stages of change model'' to train physicians in smoking cessation. At the end of training, physicians should be able to: (1) assess each smoker's motivation to quit; (2) advise all smokers with strategies matching their motivation to quit; (3) prescribe pharmacotherapy to smokers ready to stop. DESCRIPTION OF PROGRAM/INTERVENTION: In two 4-hours sessions, active educational methods enable participants to progressively learn these new skills. In the first session, learners use a checklist to observe 3 videotaped encounters with smokers and identify the 3 main stages of motivation to quit. An interactive workshop follows for presentation and discussion of basic concepts and strategies of smoking cessation in relation to videotaped cases. Then participants are involved alternatively as physician, patient and observer in role plays with smokers at various motivation stages. In the second session, learners practice counseling skills with trained standardized patients who portray smokers with different profiles and readiness to quit. Trainees also receive a reference document, pocket algorithms, a record sheet for smokers, 5 stage-matched brochures for patients and instructions to patients about use of nicotine replacement. FINDINGS TO DATE: We tested this training program in a randomized trial among residents in primary care clinics. Compared to the control group, trained residents provided smoking cessation interventions of higher quality (mean score: 4.0 vs. 2.7, p=0.01, range: 0 ± 14) and expressed higher confidence in their skills 3 months after training (mean score: 7.7 vs. 5.2, p=0.002, range: 0 ± 10). Moreover, 1-year smoking abstinence almost doubled among smokers visiting trained residents (13% vs. 7%, p=0.04). KEY LESSONS LEARNED: We could develop a physician training program in smoking cessation based on active and progressive learning of counseling skills. This program was effective to improve physicians' practices and smokers' cessation rate. If this training is implemented at a large scale, physicians could contribute more effectively to reduce smoking prevalence and its major health consequences. ; 2) To allow interested PGY-2 and -3 residents an opportunity to work closely with faculty in developing publicspeaking, advocacy, and teaching skills. DESCRIPTION OF PROGRAM/INTERVENTION: Two PGY-2 residents were identified who had an interest in DV. These residents helped to design a 3-hour curriculum which was implemented on a monthly basis for all interns during their Ambulatory Medicine rotation. The curriculum consisted of: 1) A portion of a video about DV survivors; 2) A lecture on the dynamics, screening, and management of DV; 3) A handout including articles on DV and an annotated bibliography; 4) A talk by a social worker on principles of counseling and local resources for survivors of DV; 5) A role play demonstrating screening strategies; 6) An interactive discussion of three cases of``positive``results of screening; 7) Small group sessions in which the interns were given case scenarios and asked to try the screening and management techniques they had learned. The lecture was given by the lead author, who is a faculty member. Elements 5 ± 7 of the curriculum involved the PGY-2 residents to an increasing degree over the course of the spring. FINDINGS TO DATE: The curriculum was well-received by the interns. Both the lecture and the portions led by the PGY-2's were consistently rated highly in feedback obtained at the conclusion of the sessions. The interns commented frequently on the benefits of the role playing. We were also able to identify several interns interested in DV who have been recruited to participate in the curriculum this spring. KEY LESSONS LEARNED: Residents respond well to learning skills of outpatient medicine from their peers, whom they may feel have a better sense of the limitations and realities of their own practice. Many housestaff have an interest in both the care of vulnerable patients and advocacy that is rarely put to use in the traditional IM residency. Curriculum design and implementation can be made easier and more appealing by involving residents with specific interests. Faculty can serve as mentors in order to facilitate resident academic growth and development. The educational intervention consisted of a 3-hour interactive seminar on DV, including discussion of video material, case discussion, an evidence-based literature review, and role-playing. The seminar was designed to heighten awareness of abuse as a problem in the primary care setting, and to teach the importance of screening and useful techniques for screening. After the seminar, residents were asked to screen all of their female patients for DV for the next 2 weeks, and return to discuss their findings in a follow up seminar. Six to 12 months after completing the intervention, all second and third year residents received a questionnaire containing eight modified essay questions concerning clinical scenarios. All PGY3 residents surveyed (intervention group) had received the educational intervention while none of the PGY2 residents (control group) had received it. The residents were unaware of the purpose of the questionnaire, and all responses were anonymous. Three of the eight cases described scenarios in primary care which were designed to stimulate a suspicion of DV, for which residents were asked to list up to five leading diagnoses. A response was counted as correct if it contained the words``violence'' or``abuse.'' Questionnaires with at least 2 correct responses (to the 3 suspicious questions) were graded as positive for demonstrating adequate suspicion of DV. FINDINGS TO DATE: The program was well-received, with an average rating of 4.6 on a 5point scale (n=33.) As of 12/29/00, response rates were 70% (26 of 37) in the intervention group and 51% (19 of 37) in the control group. The rates of \"positive questionnaires\" were 21% (4/19) for the control group and 58% (15/26) for the intervention group (p=.014) KEY LESSONS LEARNED: An educational intervention in domestic violence using video, case discussion, evidence-based review and role-playing was well-received, changed physician awareness of domestic violence in primary care, and had a lasting effect six to 12 months after the intervention. DESCRIPTION OF PROGRAM/INTERVENTION: We will demonstrate the session plans of two EBM courses. Course 1 consists of an introduction session in which staff present the fundamentals of asking clinical questions, searching for the best evidence, and critiquing articles. Afterwards, one resident prepares a patient case and presents it to the group. The resident then formulates a clinical question and searches for the best evidence to answer it. The resident must individually meet with a staff preceptor to review the answer. Each week a different resident presents a case, formulates the question, meets with a staff preceptor, and presents the findings to the group. EBM Course 2 consists of five didactic sessions. The first session is the same as for Course 1. The subsequent sessions include staff presenting methods on how to evaluate articles of diagnosis, therapy and prognosis. The students and staff then discuss cases, formulate clinical questions and go to the library together to search for answers. The group reconvenes to critique the search strategies. FINDINGS TO DATE: Residents (N=12) in both courses completed the same 32-item questionnaire on the first and last sessions. Residents were asked the amount of searching done, comfort in searching/critiquing findings, applying findings to patient care, and what data source they'd search first to answer a particular clinical question. Pre and post differences were tested with the Wilcoxon Signed Ranks test. Comments from Course 1 regarding the need for more time to learn how to search prompted formal didactic sessions for Course 2. Course 2 residents significantly increased their frequency of searching for answers to clinical questions/applying EBM(Z = 2.02, p = .028). They reported more confidence in their ability to conduct quality searches (Z = 2.21, p = .027) and to critique the articles(Z = 2.23, p = .026). Residents from both courses mentioned that they learned about many new sources of medical information on the Internet and library databases. KEY LESSONS LEARNED: These courses may be effective means to increase residents' confidence to conduct EBM searches and to evaluate results. These courses are important ways to recruit staff to actively further their own education and application of EBM. Subsequently, they spent an afternoon session in groups of four students, one faculty coach and four standardized patients (SP's). Each student interviewed a patient, received verbal and written feedback from his/her peers, from the coach, and from the SP. Then they would re-do parts of the interview based on suggestions. At the end of the year, students interviewed a different SP case for a final exam, and were rated by instructors, the SP and themselves. The videotapes of these evaluations are then reviewed one on one with a faculty member trained in this process. FINDINGS TO DATE: In contrast to group lectures and small group sessions, students were most highly engaged in the group practice sessions with the SP's. Many students used a high control interview, but with feedback, rapidly learned to use more patient-centered interview techniques. KEY LESSONS LEARNED: After trying several didactic methods (lecture, small group workshop of 10 ± 15 students, optional videotape review sessions), we found that students responded best to a carefully facilitated SP session within a very small group (4 students) with immediate feedback from student colleagues, coach and SP. STATEMENT OF PROBLEM OR QUESTION: The first 2 years of medical school are filled with a seemingly endless stream of new information. While the basic science classes are similar to prior learning experiences, clinical learning, which may be more satisfying (i.e.,``feeling like a doctor''), requires skills that are relatively new and different. History taking skills are often evaluated by Observed Structured Clinical Exams (OSCE) or by direct observation and feedback. The traditional method of understanding the medical history is often a static list of questions and categories that fail to direct the flow of the student-patient interaction. In addition, students often desire more feedback than is possible to provide in a busy office setting. OBJECTIVES OF PROGRAM/INTERVENTION: This project seeks to create and implement a system by which medical students can understand, self-evaluate, and improve their history taking skills. DESCRIPTION OF PROGRAM/INTERVENTION: The subjects were 8 first-year medical students at Dartmouth Medical School enrolled in the On Doctoring course ± a longitudinal clinical care experience for all students in years 1 and 2. The students alternate one half-day per week with a facilitator in a small group setting, then one half-day one-on-one with a preceptor in an ambulatory setting. In the small group, the facilitator (GSO) and the students together built a process flow diagram of the medical history placing a strong emphasis on the transitions of the process. This process diagram was then used as a template for students to evaluate and improve their history taking skills.``History scorecards'' were given to the students for use in their subsequent preceptor visits. At the preceptor's office, immediately after taking a history, the student rates several aspects of the interaction (e.g., introduction of self, agenda setting, use of silence) on a 10-point scale for each patient encounter. FINDINGS TO DATE: On the subsequent preceptor visits, 25% of students used the``history scorecards'' to evaluate their performance. Those who used the system found it easier to track their skills and to ask for directed feedback from their preceptor. Those who did not use the system either forgot to bring the scorecard, found it cumbersome, or did not realize that it was an``assignment.'' KEY LESSONS LEARNED: In addition to learning history taking skills, students design a process diagram, analyze the process to find aspects that can be changed/improved, and collect and analyze data so as to monitor improvement. This novel way of teaching and learning the medical history incorporates elements of evidence based decision making for directed feedback and quality improvement skills that are important for students to understand. Graduate Medical Education, and the results of needs assessments of internal medicine residents at our institution. Using Medline, an extensive literature search was performed on the following topics: osteoporosis, breast cancer, hormone replacement therapy, domestic violence, coronary artery disease in women, menopause, headaches, substance abuse in women, urinary incontinence, dementia, sexual dysfunction, and evidence-based medicine to create a bibliography of readings for residents who rotate through our women's health center. Peerreviewed journal articles were compiled. Priority was given to data published since 1990, and randomized, double-blinded, placebo controlled studies. Faculty and residents review and analyze two to four articles weekly on a given subject. Content experts provide context and clinical expertise to discussions. Clinical questions, such as``What is the risk of my patient developing breast cancer on hormone replacement therapy?''; and``Should I prescribe hormone replacement therapy to my post-menopausal patient to help prevent coronary artery disease?'' are addressed in each session. Evidence-based medicine core concepts are reviewed and applied, including the number needed to treat, absolute risk reduction, and relative risk. FINDINGS TO DATE: Previous work from a needs assessment of residents at our institution found a discrepancy in perceptions and actual knowledge in women's health. Ninety-one percent of the residents rated inadequacies in the women's health curriculum. Our evidencedbased curriculum serves to bridge this gap of knowledge. Residents participating in our curriculum have expressed increased knowledge in the subjects discussed in our weekly conferences. Also, residents have stated that much of what is taught in this curriculum has not been covered elsewhere in their residency curriculum. KEY LESSONS LEARNED: A gap exists to be filled between perceptions of curriculum adequacies in women's health and actual knowledge. Our curriculum serves as a forum for an update in women's health literature, an exchange of ideas for the improvement of women's health as it is taught in internal medicine, and for further elucidation of the evidence behind what we practice and teach. ; 2) individualized educational tutorials using the assessment videotapes and published guidelines; and 3) measurement and feedback of practice patterns relative to standards of care. The efficacy of this model was assessed through a randomized trial of educational interventions for two independent guidelines. After initial assessments with standardized patients, 28 internal medicine residents were randomly assigned to receive either the guideline intervention for elders at risk for depression or the guideline intervention for patients with diabetes. All residents in the study are reassessed through direct observation with standardized patients and through chart review of a panel of clinic patients, thus providing independent comparative measures of competence. The two randomized groups are being compared on a measure of the difference in performance with diabetic patients and elderly patients. This study design, using two interventions, has the potential to double the effect size producing a more powerful study than one with a single intervention for research involving small numbers of residents. FINDINGS TO DATE: To date, half of the residents in the intervention have been reassessed using standardized patients. These results show that residents with the diabetic intervention performed better with their diabetic patients than with their elder patients, and the reverse was found for the residents trained in elder care. Difference between the diabetes trained (n=7) and elder care trained (n=6) residents on their difference in performance on two diabetes cases versus two elder care cases was large. The effect size of the two interventions (combined) was .72 standard deviation units (+/-.27 standard errors) and significant (p = .02). These results were consistent for the component score differences for history taking, physical examination, and diagnosis, but inconsistent for management plan. KEY LESSONS LEARNED: The overall advantage of the intervention is an educational experience that involves faculty and residents in an interactive learning and evaluation setting. Added benefits are the development of new tools to measure quality and physician compliance with guidelines for patient care. Videotape review of standardized cases, tutorial outlines and chart review templates used in the study will be available at the exhibit. sheet``to model searching behavior that will assist the learner in executing effective searches for clinical decision-making activities for the case studies. To conclude the topic, the nursing informatics educator/webmaster identifies quality patient education resources for the specific outpatient topics. An online submission form sends case study answers via e-mail to the clinical faculty or chief resident for evaluation prior to the outpatient conference. The case study submission is also filed on the website server for future reference. FINDINGS TO DATE: This approach has a strengthened the preparatory behavior of Internal Medicine residents in relation to the outpatient curriculum. Year 1 of a 3-year follow-up prepost resident self-report suggests mosest changes in attitudes and EBM behaviors (p < .10). The lead resident submits information prior to the group presentation. The monitor receives the residents' answers via e-mail and addresses significant issues confidentially. KEY LESSONS LEARNED: The outpatient website has increased the use of EBM clinical decision-making tools in the clinical practice settings. It has provided opportunities for improving residents' information literacy skills and knowledge of quality patient education resources. Attending physicians and monitors are requesting additional education related to information literacy and evidence-based medicine. Clinical activities take place in 3 ambulatory settings: a primary care clinic serving mainly people of lower socio-economic status and migrants, a geriatric clinic delivering home care to fragile elderly patients, a unit caring for patients with alcohol or illicit drug abuse. Students attend 8 tutorials for clinical reasoning and problem solving regarding common ambulatory conditions. A workshop based on role plays sensitizes students to counseling for smoking cessation or alcohol use. All students must also present and comment a clinical case to their colleagues. Time is allocated for self-directed learning to prepare some activities. Students receive formative assessment based on their behavior in clinical activities and their ability to solve a new clinical problem. FINDINGS TO DATE: At the end of the clerkship, students evaluate its various aspects on a 5point scale. After the first year, 108 students expressed a high global satisfaction with the clerkship (mean score: 4.2). Mean scores were also high for key elements like achievement of learning objectives (4.3), organization (4.5) and quality of teachers (4.6). KEY LESSONS LEARNED: We successfully developed a structured clinical clerkship in community medicine enabling students to learn about ambulatory care and health of vulnerable populations. This program met students' expectations and was highly appreciated. Further research should assess the impact of this clerkship on clinical competences in ambulatory care and career choices at the end of undergraduate training. Medicine residents lack self-confidence and competence in end-of-life patient care, including breaking bad news, patient goal-setting, DNR discussions and death pronouncement. In 1999, we formed a working group to teach and evaluate these core communication skills in our Internal Medicine residency program.\nÁ Demonstrate how to give unwanted news Á Demonstrate how to conduct a discussion with thè`a ngry patient'' Á Demonstrate how to lead a discussion to establish goals/patient preferences, including DNR orders and changes in treatment from curative to palliative approach DESCRIPTION OF PROGRAM/INTERVENTION: All interns attend 2 required afternoon retreats (8 hours total contact time). The first retreat focuses on basic communication skills such as empathy and attentive listening and breaking bad news. The second retreat addresses goalsetting and DNR decisions with the terminally ill patient and death pronouncement. We used a variety of formats: large group role-plays and debriefing discussions, brief handouts, pre-tests and videotaped, standardized patient stations with immediate, individual feedback from faculty observers. FINDINGS TO DATE: 20 interns completed the first retreat training and 22 completed the second. Intern pre/post self-assessment shows significant improvement in degree of competency and knowledge. On a scale of 1 to 4 (4 being most competent/most knowledgeable), interns report greater knowledge in discussing goal setting (2.5 to 3.5; p < 0.05) and discussing DNR orders (2.4 to 3.5; p < 0.05) and self-perceived increased competence in giving bad news (3.1 to 3.7; p < 0.05 ) and death pronouncement procedures (2.9 to 3.7; p < 0.05). Interns praised the retreat for the open interaction with faculty experts, the opportunity to role play patient care situations and the exposure to topics not previously discussed in medical training. A few mentioned distaste for being videotaped. KEY LESSONS LEARNED: A retreat format, using large group role-plays and individual standardized patient encounters with feedback, is a useful method of teaching and evaluating important end-of-life communication skills. Interns who needed extra help in communication and interpersonal skills were identified and individually reviewed these skills with faculty mentors.\nA RESIDENT-AS-TEACHER CURRICULUM DURING AMBULATORY BLOCK. T.L. Simon 1 ; 1 Mount Sinai School of Medicine, New York, NY STATEMENT OF PROBLEM OR QUESTION: Residents are expected to teach interns and medical students, yet receive no training to help improve their skills as teachers. An effective resident-as-teacher (RAT) program needs to utilize protected time and provide sufficient opportunity for skills practice, reflection, and reinforcement. OBJECTIVES OF PROGRAM/INTERVENTION: (1) To prepare residents for their role as teachers in the medical setting. (2) To enable residents to examine their own teaching skills and behaviors, and take active steps to improve their teaching effectiveness. (3) To improve clinical teaching in the Department of Medicine. DESCRIPTION OF PROGRAM/INTERVENTION: The RAT curriculum consisted of four consecutive 2.5 hour weekly workshops, repeated monthly, during the PGY2 ambulatory block rotation in the first half of the academic year. A group of 5-6 residents met with one faculty member for sessions utilizing interactive techniques such as facilitated discussion, videotape review, and role-play. The workshops covered the following content areas: (1) Introduction to Teaching Principles/Diagnosing the Learner, (2) Microskills of Clinical Teaching/Giving a Microlecture, (3) Resident as Team Leader, and (4) Evaluation and Feedback. Teaching homework assignments were completed between sessions and reviewed as part of the next workshop. Reinforcement materials were sent to the residents 1, 2, and 3 months after completion of the curriculum. FINDINGS TO DATE: The program was well-received, with a mean``overall rating'' score of 4.5 on a 5 point scale (n=33). A pre-test rating scale of teaching behaviors was also administered at the beginning of the curriculum, and a post-test will be distributed to each group four months after completion. Qualitative data was collected during workshop discussions, on topics such as`O vercoming Barriers to Teaching on the Wards'' and``Characteristics of the Ideal Resident''. In addition, residents completed self-assessment checklists of their own performance as clinic preceptors. Finally, there is a long term plan to analyze resident teaching evaluations from interns and medical students before and after the institution of this curriculum. KEY LESSONS LEARNED: RAT programs have traditionally been done in either a single 1 ± 2 day block, or``longitudinally'' with discrete sessions in each year of training. Utilizing the ambulatory rotation allows for protected time with small groups of residents in a more relaxed setting. Weekly workshops provide the opportunity for practice teaching exercises, review, and reflection. Residents are eager for this type of training, participate enthusiastically, and rate the program highly. Whether this will translate into measurable improvement in teaching months to years later will be assessed. Traditionally, faculty observe residents interviewing and examining patients and discuss further care with the resident. Time pressures have led to difficulty recruiting adequate numbers of faculty to participate in the traditional CEX at one institution. An alternative format became a necessity. OBJECTIVES OF PROGRAM/INTERVENTION: Our primary objective was to develop a more reliable and reproducible test using standardized patients that successfully assessed the same skill areas as the traditional CEX. We also wanted to obtain data on resident competency that would be better suited to use for program evaluation. DESCRIPTION OF PROGRAM/INTERVENTION: Four SP cases (chest pain/unstable angina, headache/tension, chest pain/pulmonary embolus and abdominal pain/dyspepsia) were developed. The residents were instructed to obtain a history of present illness, past medical and psychosocial history, perform a comprehensive physical examination (PE) and write an assessment and plan (AP). SPs completed the history (Hx) and PE checklists (yes/no, yes = done and done correctly, no = omitted or done incorrectly). The SP rated PE technique (6 questions) on a 5-point Likert scale e.g.``the resident minimized patient movements'', interpersonal skills (5 questions) on a 5-point Likert scale, e.g.``the resident demonstrates a professional demeanor'', and nonverbal communication skills (16 questions) e.g. \"the resident's tone of voice was 1=unfriendly, cold, 7=friendly, warm\". AP score was based on key developed by 5 faculty. FINDINGS TO DATE: 50/52 intern and senior residents participated. Mean (SD) percent sub-scale scores were Hx 58.4 (7.0), PE 72.2 (15.8), AP 42.7 (18.5), technique 94.0 (4.9), interpersonal skills 94.9 (5.1), and non-verbal communication skills 83.8 (16.7). The scores on the Hx, PE, and AP were lower than expected and ratings of technique, interpersonal and nonverbal communication skills were acceptable. KEY LESSONS LEARNED: The SP CEX is a feasible alternative to the traditional CEX. However, a considerable amount of time and effort is required to establish an examination using this format. A pre-existing SP program is a helpful to this process. The SP CEX enables residency programs to standardize the CEX and avoid variability from different raters (faculty). The SP CEX provides more detailed information on resident performance than the traditional format, which will be useful in providing more specific feedback to examinees and directing curricular development within a program. STATEMENT OF PROBLEM OR QUESTION: In the care of patients with chronic diseases, no doctor is an island. This is particularly true in group practices, clinics and health centers where a number of professionals and support staff interact with each patient. When a large resident component is added to the provider base, there is increased demand on all staff to understand the content and process of caring for individuals with common chronic diseases. Educating residents in the care of multisystem chronic conditions in outpatients is also vital to successful care, particularly with evolving changes in management.\nHealth Center (MSNHC) is a hospital operated health center with a staff of 9 attendings, 8 RN's, 3 NP's, 32 med/peds residents, and 8 MA's. It serves 600 diabetic patients and employs a \"staged diabetes management\" guideline for care. MSNHC is challenged with keeping staff knowledgeable in the many facets of diabetes in order to improve care and teamwork. It must do so without a devoted diabetes educator to run such an effort. DESCRIPTION OF PROGRAM/INTERVENTION: The authors developed a monthly diabetes lunch conference for all health center staff. Drawing on the resources of the hospital, each conference centered on one diabetes related specialty and featured a guest presenter. Topic areas have included vascular disease, pharmacology, renal, nutrition, etc. The format is casebased with 15 minutes left at the end for prepared comments in the presenter's field. Attendees are asked to complete a feedback form highlighting what they have learned in the session and listing questions for the next month's presenter. FINDINGS TO DATE: Over 1/2 years, the average attendance has been 17 (3 attendings, 2 NP's; 4 residents, 2 RN's, 2MA's, 1 student). The most often cited key elements of the programs have been: lively discussion, a practical handout, good visual aids, and lunch. Most attendees have been able to specify 1 ± 2 practice process changes they would engage in as a result of attending the conference. At one conference the group developed quality improvement goals for lipid management in diabetics. KEY LESSONS LEARNED: ISSUES AND LESSONS FOR CONSIDERATION: 1) We have brought specialized knowledge to an interdisciplinary health center staff with a minimum investment of any one person's time.\n2) Time pressures make staff involvement a challenge to sustain.\n3) Our next step is to measure the long term effects of this intervention. introductory workshop on the history and physical of the female patient using videotaped interviewing and surrogate patients, continuity clinics in gynecology and mental health, and clinical experience in metabolic bone clinic. A yearly community month at an interdisciplinary women's health care center and a breast health month with clinical exposure to oncology, pathology, radiology, and plastic surgery provide additional women's health training. Didactic components include a journal club on landmark women's health studies, women's health seminars and grand rounds, a primary care/women's health didactic month, and a CME women's health conference. Clinical research and community outreach projects are encouraged. A 36-month reading curriculum provides knowledge of core women's health topics. This consists of review articles from reputable journals, now accessible as a web-based curriculum. Clinical and didactic components are integrated into the Internal Medicine curriculum and all Women's Health residents are board-eligible in Internal Medicine at the completion of the program. FINDINGS TO DATE: In July 1997, the program was piloted. Subsequently, highly qualified applicants have filled all 4 annually allotted positions. The ratio of applicants to positions continues to increase with a current ratio of 15:1. Of the graduating residents, 3 are pursuing fellowships, 2 are joining community practices, 1 has become an academic faculty member, and 1 has become a chief resident. KEY LESSONS LEARNED: With both institutional support and interdepartmental collaboration, this program continues its success. It attracts highly motivated and qualified applicants, it is a vehicle of awareness for the medical community through the recognition of Women's Health residents, and it has begun to foster future leaders for the continued advancement of women's health research, writing, and education. This program is a model for integrated and comprehensive women's health training. national demonstration project ± provided the opportunity to introduce medical students to the foundations of population-based medicine upon entry to medical school. Our primary goal is to give students this fundamental skill set prior to their entry into the core clinical clerkships. In sequential fashion, students are introduced to: validity, uncertainty, resource allocation, financing and access, and managing care to enhance quality. DESCRIPTION (3) Managing Care. FINDINGS TO DATE: Students rate the curriculum in terms of content, presentation and teaching materials. Some courses are as highly rated as more traditional biomedical science courses. Unfavorable ratings correlate best with poor course organization. However, perceived``inappropriate'' course content also plays a role. Student assessment maps changes in knowledge, values and attitudes, and includes a variety of traditional and more innovative modalities. Overall, students perform as well in these courses as in biomedical science courses. Preliminary data suggests that students attitudes about managing care may also be changing. KEY LESSONS LEARNED: Interdisciplinary curriculum design, implementation and oversight ± and extensive and ongoing input from students ± have been critical elements in the success of this broad-based initiative. Additional interventions are planned during the clinical curriculum, where the principles introduced in the preclinical years will be reinforced and expanded. Future studies will assess whether this approach influences how students perform as clinical clerks and residents. -The evaluation team has completed the pre-and post-test of the control group and is collecting data on the intervention group. KEY LESSONS LEARNED: -It is possible to collaborate on a large curriculum development project across three medical schools. It is important to define which aspects of the project the schools will collaborate on and which will be done individually.\n-The competency document has helped drive specific curriculum at each school and has emphasized the evidence behind our curriculum.\n-The teaching of communication skills is best done if it is integrated into clinical content and it provides active``hands on'' experience.\n-Rigorous, comprehensive evaluation is possible and integral to the success of the project.\nTEACHING CROSS-CULTURAL COMMUNICATION. S. Mutha 1 , C. Allen 1 , M. Evaluations of the trainings have revealed high levels of agreement that the content was highly relevant to clinical practice and high satisfaction with the training sessions. A six-month followup evaluation is underway to assess the ways in which attendees have incorporated the contents of the training into their practices. KEY LESSONS LEARNED: The curriculum content and design allow successful interaction among multidisciplinary health professionals. Participant evaluations underscore that simulations and experiential exercises are an especially powerful way to increase sensitivity to and awareness of diversity in the clinical setting. The sensitive nature of issues surrounding diversity such as prejudice as well as discrepancies in power and authority emphasizes the need to develop a cadre of clinician/educators who are committed to providing this type of training in clinical settings. medical students between the M1 and M2 years, with each student linked directly to a senior administrator of a major academic medical center or private managed care organization. Each fellow is assigned a project, which is summarized into a presentation to all program faculty. Weekly journal club (focusing on health policy, administration, and financing) is also required. FINDINGS TO DATE: Evaluations by students of experience in the program have yielded uniformly positive results when asked to rate the quality of the program, as well as its importance to their careers as physicians. 6 of the 16 students participating in the program have applied for admission to UCLA's combined MD/MBA program upon completion of the fellowship. Students have gained increased understanding of the administrative aspects of health care organizations. KEY LESSONS LEARNED: 1) Dedicated and experienced mentors are critical. 2) Students must tailor work projects to normal operations of organization; 3) Feedback and discussion with students during program is a critical success factor; 4) Most medical students could benefit from some type of exposure to the administrative STATEMENT OF PROBLEM OR QUESTION: Current fiscal reality has resulted in increased demands for clinical/ economic productivity on the part of all physicians. This is perhaps most acutely felt in academic practice groups that devote part of their activity to nondirectly reimbursed teaching activity. With pressures to see more patients while teaching students and residents, time and excellence in teaching and research may suffer.\nEducation and Research Foundation (BMERF), at the strong urging of its membership, endeavored to develop a financial incentive program to reward and encourage excellence and productivity in research and teaching . As a multispecialty group, it was important to the group to treat all specialties fairly and equitably. DESCRIPTION OF PROGRAM/INTERVENTION: The chairs of each academic department (Emergency Medicine, Medicine, OB/Gyn, Pathology, Pediatrics, Psychiatry and Surgery) nominated one member from each department to a Committee on Academic Excellence. Committee members identified a set of achievement criteria and then weighted them to form a quantitative measurement scale.\nIn the first year, major weighted criteria included but were not limited to: article publication; editor or author of text or journal; receipt of large research grants; achievement awards from national organizations. Junior faculty received additional consideration for more modest grant receipt; advancement to associate professor; and chairmanship of major committees in medical societies or at the medical school. Criteria were further refined the second year based on BMERF member feedback. FINDINGS TO DATE: In the first year of 197 eligible members, 47 (24%) submitted material for consideration for compensation. 31 awards were given (66%), ranging upwards from $1,000, few going to junior faculty (defined by the committee as 7 or fewer years out of residency). The second year there were 77 submissions (39%) and 41 awards (53%), with a minimum award of $625. Three General Medicine Division members received awards, from a division membership of 24. KEY LESSONS LEARNED: LESSONS AND ISSUES FOR CONSIDERATION: 1) The concept and execution of the program were well received.\n2) There will be pressure to allocate more money in future years.\n3) We need to further refine criteria for junior faculty and generalists. 4) It can be difficult to identify and reward excellence in teaching. HCA serves 32,000 patients, 40% capitated, with 91,000 annual visits and is the primary ambulatory teaching site for 130 medical housestaff. We reward our clinicians with added compensation for high patient visit volume, panel growth, severity of illness, commitment to teaching (comanagement) and, most recently, panel management. DESCRIPTION OF PROGRAM/INTERVENTION: Funded annually by the hospital, the incentive pool derives from an estimated payout per faculty FTE for practice-wide projected increases in visit volume and panel size. The core clinical payout is based on a point system. Promoting both visit volume and panel growth, new patient encounters earn 3 points, while all others earn 1 point. Incentive is paid for all points above a threshold of 8 patients/session, averaged over the year. To promote teaching, we incentivize comanagement sessions more heavily than individual practice sessions. As a proxy for severity of illness, we pay a third component based on the inpatient activity of a faculty member's panel. We are now adopting a more diversified model, with 25 ± 35% of total incentive dollars linked to panel size, age and gender adjusted, to reflect the realities of clinical practice more accurately. We are also moving to include provider specific quality measures in the incentive system, and to extend incentive payments to non-physician and non-clinical staff. FINDINGS TO DATE: Faculty visit volume productivity increased 30% in 3 years and overall panel has grown at a rate of over 400 new patients/month. In academic year 99 ± 00, the faculty incentive paid a total of $310,000 for an average payout of $8,000 per faculty member, with a range of $0 ± $19,927, and we have been able to fill our comanagement teaching slots easily. System is a multi-site county health care system, with significant variability in clinical background and practice styles in primary care providers. System wide disease management initiatives and clinical practice guidelines have been created in an effort to standardize care but have been difficult to disseminate and implement. Traditional CME is individually chosen to accommodate the provider's preferences regardless of the system's needs. Furthermore, there is evidence that this approach rarely modifies physician behavior resulting in a delay of standard of care practices implementation. A program that combines disease management, continuing medical education, and peer review was designed to foster the creation, dissemination and evaluation of clinical practice guideline implementation for primary care issues, and improve system-wide provider communication.\nProviders to exchange clinical interests and expertise.\nTo provide a forum to develop, implement and monitor quality improvement initiatives.\nTo develop a curriculum of clinical material relevant to primary care.\nTo promote collaboration among adult providers across the healthcare system.\nTo promote development of one standard of care throughout the system, that is both evidence based and cost effective. DESCRIPTION OF PROGRAM/INTERVENTION: Description: An initial curriculum was developed based upon a survey of provider's interests and system needs identified by the quality management department. Prior to meetings planning committee reviews or creates disease specific standard indicators for appropriate measurement of guideline implementation. Monthly meetings consist of a lecture given by a recognized expert and a discussion of the perceived barriers to achieving that care in our institution. The indicators are then presented followed by a review of charts of identified patients with the discussed medical illness. Results of previous peer reviews are presented with a discussion of what could be done to improve them. Individualized provider specific feedback is distributed confidentially. FINDINGS TO DATE: Results: Re-measurement of disease specific indicators have shown significant increase in provider compliance with established standards of care: a 24% increase in the use of ACE-Inhibitors in CHF, a 45% of increase in screening for microalbuminuria in diabetics. Yet, some of the newer disease management knowledge, such as use of beta-blocker in patients with CHF showed no significant change. KEY LESSONS LEARNED: Key Lessons: A program that combines disease management and a continuing medical education involving self-evaluation through peer-reviews more effective to disseminate and implement system based clinical practice guidelines, also improves providers communication and involvement in those initiatives. identified and a multi-disciplinary team consisting of a hospital pharmacist, dietician and discharge planner visit and educate the patient and family. Standardized education materials are provided. The GHC home-visiting nurse service, called Family Health Workers(FHW) also visits the CHF Project patient while in hospital. At discharge, the hospital pharmacy provides an updated medication list to the patient, the FHW, the patient's pharmacy and the GHC electronic medical record. The same FHW visits the patient within 48 hours of discharge, and arranges for further follow-up as needed. FINDINGS TO DATE: [1] Over the first six months, the CHF Project has been well received by all. A crucial and well-appreciated role of the FHW is to sort out discharge medications at the initial home visit. [2] There have been a total of 174 CHF patient admissions, of which 57 belong to the GHC and were exposed to the intervention. [3] There has been a 68%, [95% confidence interval (CI) 9% ± 96%] decrease in readmission rates compared to historical controls and a non-significant 57% [95%CI -5 to 89%] decrease compared to concurrent non-GHC controls. [4] There has also been a non-significant trend towards decreased mortality. KEY LESSONS LEARNED: A community-based collaborative to help CHF patients in the crucial discharge transition period can be effective in a non-tertiary care setting. Further efforts are needed to expand access to the program for all patients, to study and improve upon separate components of the program and to sustain the improvements already achieved. (range 23 ± 87); 69% of patients were African American; 60% were Female; 58% had less than a high school education; over 50% relied on Medicaid or pharmacy assistance for medications. Average duration of disease was 10.4 years (range 0 ± 42). Although 81% of patient had hypercholesterolemia and 88% had hypertension, only 30% were on a lipid lowering agent and only 60% were on or had ever taken an ACE-inhibitor. Baseline HgbA1c at enrollment was 10.8% and did not differ significantly from values obtained 6-12 months prior to enrollment (mean 10.2%). At three to four months follow-up, the mean reduction in HgbA1c was 1.7% points (p < .0001).Baseline diabetes knowledge score on our 11 question diabetes knowledge test (DKT) averaged 36%. Of 69 patients who have retaken the DKT, there was a 23% point improvement from baseline to follow-up (p < .0001). Blood pressure and total cholesterol did not change importantly from baseline to follow-up. Multiple regression analysis shows that higher educational status, when adjusted for baseline HgbA1c, is associated with a significant improvement in HgbA1c. Other socioeconomic factors, such as race and gender were not significant predictors. KEY LESSONS LEARNED: A pharmacist-assisted care program can improve patient's knowledge of diabetes and significantly reduce HgbA1c. We did not see improvement in blood pressure or lipid control -two areas that we did not specifically intervene upon. To further improve care and reduce the risk of macrovascular complications, we plan to test pharmacistassisted, algorithm-based, hypertension and cholesterol management in a randomized controlled trial. STATEMENT OF PROBLEM OR QUESTION: Nursing staff in VA primary care clinics are required to document numerous patient education and screening activities, and they are using progressively more advanced software in the electronic patient record to accomplish this documentation. However, time limitations make it difficult to accomplish all these educational and screening mandates while the patient is in clinic, and staff may miss documenting activities which were previous accomplished but not quickly found in the record. OBJECTIVES OF PROGRAM/INTERVENTION: We experimented with the process by which nurses documented that patients were taught the proper use of metered dose inhalers (MDIs). We chose to update our electronic documentation of this activity (1) at a time other than the patient visit and (2) by using a population based approach. DESCRIPTION OF PROGRAM/INTERVENTION: At our facility, two teams of similar provider and support staff composition provide primary care in non-teaching clinics. Of the 6129 patients in group practice A, 640 use MDIs, and 572 of 5596 group practice B patients use MDIs. Information about this population of MDIs users was compiled, and their names and telephone numbers were listed in the chronological order of their next appointment. Starting in May 2000, clerks began calling each patient as their next appointment approached, in order to remind patients to obtain MDI instruction when they attended clinic. In group practice B, nurses also used the list during lulls in routine clinic screening activities to contact patients, ask them about prior MDI education, and then update the electronic record accordingly. Five months later, electronic documentation of MDI patient education was extracted from the computer patient database. Documentation data was also obtained about one education/ screening activity not targeted in this study (depression screening) because this activity applies to all group practice patients and are electronically documented in the same manner as MDI teaching. FINDINGS TO DATE: In group practice B, MDI inhaler education and depression screening were documented on 84% and 60% of the 572 MDI patients. In group practice A, the same percentages were 52% and 65% of their 640 MDI patients. Chi square comparisons were made of patients receiving only MDI instruction or MDI teaching and depression screening. In each comparison, group practice B performance was significantly (p < .05) higher. Logistic regression also showed that group practice B was associated with a higher likelihood of MDI teaching documentation (odds ratio 3.54, p < .001). KEY LESSONS LEARNED: As the number of routine patient education guidelines expands, there is not sufficient time during each clinic visit to complete and document them all.\nDocumentation may be better accomplished when a population of patients is targeted for review at times when they are not actually in clinic. Americorps volunteers who serve as case managers, a part-time lay health worker, and four medical students who coordinate service-learning activities. We developed an Access database to monitor clinical services, pharmaceutical utilization, and case management with a focus on preventive care, referral data, Medicaid enrollment, and pharmaceutical costs and utilization. Students in pharmacy, medicine, and nursing conducted quality assurance projects, including a patient satisfaction survey and chart reviews to assess hypertension management and antibiotic usage. FINDINGS TO DATE: On-site dispensing was promoted over vouchers at a cost saving of $21.34 per prescription with a total saving of about $20,000 a year. Blood pressure control was achieved in 46% of hypertensive patients, but almost half failed to return for follow-up. Narrowspectrum antibiotics were selected as first-line therapy 66% of the time, but over-prescribing for URI was also noted. In a predominantly uninsured (73%) clinic population, 27% of all patient encounters resulted in referrals with 30% of all referrals for dental care, 17% to primary care providers and medical specialists, and only 1% to behavioral health. Sub-optimal documentation was noted in chart reviews. KEY LESSONS LEARNED: Based on our findings, we implemented an appointment reminder system, progress note templates, and a mechanism for tracking referrals. Providing routine feedback to volunteer providers on findings such as mental health referrals may promote more aggressive screening and counseling. In conclusion, quality assurance projects incorporated into volunteer clinic activities can identify unmet health needs and areas requiring improvement. Services Administration is the second and current stage of program development. Focus groups and advisory work groups with pharmacists, physicians and nurse practitioners, and counselors are being held to identify potential barriers to participation. Actuarial consultants are developing cost estimates and financial models for the proposed program. Project leaders work with state and federal regulatory and legislative officials to facilitate program planning and implementation. Phase III: Implementation of a pilot project is targeted for the summer of 2001. FINDINGS TO DATE: The phase I community planning created program framework with the San Francisco Department of Public Health as holder of a central Narcotics Treatment Program (NTP) licensure for the OBOAT Program, in which interested and eligible public and private-sector physicians, counselors, and pharmacists would be trained to offer methadone and other approved medicines to treat opiate addiction in the setting of their regular clinical practices. Pharmacists view the opportunity for professional development as an incentive for potential program participation. They view lack of lack of methadone education in pharmacy school and inexperience with methadone dispensing to addicts as barriers to potential program participation. The planning process has challenged physicians in the narcotic treatment arena and those in the primary care arena to learn about each other's`c ulture'' and communicate effectively to develop an innovative program that will serve patients in new way. KEY LESSONS LEARNED: The planning process has involved reframing opiate addiction treatment as a medical model, rather than a regulatory model. Close collaboration with existing methadone clinics has been critical in moving program planning forward. Close work with state legislature has resulted in recent passage of enabling legislation as well as allocation of additional resources. (CTI) is a computer-based application that automatically calls patients to remind them of their appointments. This program is interfaced with the scheduling system to automatically download patient and appointment information. It uses an interactive voice response technology to call patients 24 ± 72 hours before their appointment. If there is no answer, the patient will be called back up to 5 times. All patients with appointments scheduled at least 72 hours ahead of time at several Community Health Clinics over a period of 6 weeks were eligible for our study. 5,696 patients had appointments during this time and thus could have been called by the CTI system. During randomized alternating one week periods, patients at each of the participating clinics were assigned to receive automated appointment reminders. Patients not receiving calls were used as the control group. Kept and missed appointment rates were recorded for each of these groups. FINDINGS TO DATE: 5,696 patients (3, 038 in the appointment reminder group, 2,617 of whom consented to being called by the system; 2,658 in the control group) were prospectively followed over a 6 week period. The kept appointment rate in the appointment reminder group was 69.2%. The kept appointment rate in the control group was 64.1%. This result translated into an 8% increase in kept appointments when the automated system was used (p < 0.01). The increase occurred despite the fact that only 1,192 of 2,617 eligible patients were actually contacted. KEY LESSONS LEARNED: A computerized telephone reminder system increased the kept appointment rate in our Community Health Clinics. This increase may have a considerable economic impact. Assuming an increase in the kept appointment rate of 8% translates into 8% more patient visits, this could represent 9,000 more visits a year to our clinics. If the average charge for an outpatient visit is $177.35, 9,000 more visits represents $1.5 million in increased charges. Implementation of such a system could have a large positive economic and health impact in a public health system such as ours.\nIMPROVED GLYCEMIC CONTROL IN DIABETIC PATIENTS UNDERGOING CORONARY ARTERY BYPASS SURGERY. N. Ashri 1 , R. Lippe 1 , N. Rao 1 ; 1 UPMC Shadyside, Pittsburgh, PA STATEMENT OF PROBLEM OR QUESTION: Management of blood sugar in the metabolically stressful postoperative period in a Diabetic patient undergoing major surgery such as coronary artery bypass (CABG)is often a challenging task. However, adequate glycemic control is necessary to prevent postoperative infections such as mediastinitis and delayed wound healing.Conventionally, the management involves insulin sliding scale and despite multiple calls to physicians and inconvenience to nursing staff, the glycemic control is not adequate. OBJECTIVES OF PROGRAM/INTERVENTION: 1) To develop a simple, effective insulin infusion protocol, which will maintain Diabetic CABG patients capillary blood sugars (CBS) below 200 mg/dl in the first 48 hours postoperatively. 2) To decrease the rate of mediastinitis in postoperative period. \nconducting end-of-life discussions is presented. The four steps are based on structured interviews at a major university hospital with five faculty clinicians experienced in the care of dying patients. Their experiences and actual words have been synthesized into this 4-step approach. The four steps are: 1) Initiating Discussions 2) Clarifying Prognosis 3) Identifying End-of-Life Goals 4) Developing a Treatment Plan FINDINGS TO DATE: Using the 4-step approach with your patients will result in improved communication. Good communication results in patient fears being allayed, pain and suffering minimized, and facilitates developing a treatment plan that is medically sound and concordant with the patient's wishes and values. KEY LESSONS LEARNED: Providing good end-of-life care requires both an understanding of how patients and famlieis experience the dying process, and a sensitive communication style. With these skills, physicians are able to conduct thoughtful discussions in which most decisions evolve comfortably and without controvery. Providing care for a dying patient is challenging, and when done well, is a meaningful and gratifying experience for the physician. To help someone die in comfort, in peace, and with dignity is to give one final gift of life. and an audience response system was also used to administer the questionnaire during a noon conference FINDINGS TO DATE: Thirty five HS participated in this survey; (48% GL-I, 24% GL-II, 14% GL-III, 7% GL-V). Most of them thought the book was trustworthy (87%), accurate (86%), user friendly (57%), and overall useful for the management of patients (87%). Although most HS always carried the CCF book (73%), most also carried other commercially available antibiotics recommendations book(s) (68%). There was no consensus which of these books was more useful for them. The CCF Book sections on guidelines for specific diseases and dosing information were referred to most often (37% & 40%; respectively). Requests were made to add more microbiology information and other clinically important antimicrobial drug interactions. KEY LESSONS LEARNED: The CCF Guidelines for Antimicrobial Usage book is well respected among the HS. The book serves it's purpose well in helping HS with their daily care of hospitalized patients. This survey helped us in implementing some changes that we believe will improve these guidelines and consequently, improve the quality of care for our patients. On an average there are close to 1,500 patients seen in this unit annually, with a wide variety of medical, surgical and rehabilitation problems requiring skills of a physician trained in the care of these patients. This presentation is based on our experience and observation. The Lecture format will be as follows: Introduction: Advances in technology and an increase in life expectancy for the general population have resulted in an increasing demand for medical services. In the early 1980s, for example, Medicare introduced the Diagnosis Related Group (DRG ) based system of payment for inpatient care. The DRG system created financial disincentives for the hospitals by imposing a fixed reimbursement rate for each condition unrelated to patient acuity or the actual costs of care incurred by the hospital. In response, many hospital systems created alternative discharge sites, thus shifting care to subacute units, nursing homes, and home-based care. Principles of subacute care The philosophy of Subacute or Transitional care units is the management of patients after acute exacerbation of an illness, providing a more healing environment and a change of focus away from high technologic interventions. While these patients do not need intense diagnostic work-up or invasive procedures, they still require frequent physician monitoring, nursing care, and rehabilitation. Patient Selection: The decision of appropriate referral to subacute unit is a key to both continuity of care and financial viability. Appropriate subacute care candidates must have a definitive goal and identified skilled needs. It is important to identify these patients as early as possible during their acute hospitalization in order to reduce the length of stay. Examples of patients appropriate for subacute units will be discussed in detail. Also difference between these patients and nursing home patients requiring custodial care will be discussed. Financial challenges: As the number and type of alternate care sites have increased, federal efforts at cost containment have shifted to limit financial reimbursement to post-acute care facilities as well. The Balance Budget Act of 1997 introduced the Prospective Payment System for these units, shifting the reimbursement from per-diem rate to a fixed rate which would include all the ancillary services received by a patient except physician visits. This fixed rate is calculated based on the Resource Utilization Group per Medicare guidelines. In many cases, the cost of providing care for medically complex patients far exceeds the reimbursement. Mounting economic pressure to contain treatment costs may threaten the financial health of many subacute units especially as hospitals seek to admit only the sickest of the sick and treat them in the shortest possible amount of time. This section will include discussion on the problems created by these changes in reimbursement and the possible solution to maintain quality of care. Educational opportunities: With the decrease in length of stay on the acute inpatient site and increasing medical complexity, subacute units continue to provide care for a variety of patients. Members involved in their care require both medical and surgical skills. Our subacute unit is a site for residency training program and continues to offer wide variety of experience ranging from routine postoperative care to caring for the elderly and medically sick patients. Residents also learn the importance of maintaining functional integrity in elderly patients, concepts of rehabilitation working in an interdisciplinary team model and dealing with end of life decisions. They also learn to provide care in face of financial cut-backs, without compromising on the quality of care. Outcomes research continues to remain an unexplored area in this arm of health care. The value of this cannot be underestimated, especially in view of financial cut-backs, with the goal of maintaining a high standard of care for an enlarging, vulnerable patient population in this new environment. FINDINGS TO DATE: KEY LESSONS LEARNED:\nPHYSICIAN RECORD KEEPING WAS ALTERED BY FOCUSED PROFILING. R.E. White 1 , D. Gray 1 ; 1 Albuquerque VAMC, Albuquerque, NM STATEMENT OF PROBLEM OR QUESTION: Since 1998, attending physicians in our general medicine, continuity clinics have instructed resident practitioners to enter patient problem lists into the electronic medical record. After eighteen months, however, only 45% of patients had an electronic problem list (EPL). OBJECTIVES OF PROGRAM/INTERVENTION: We implemented a profiling system based exclusively on the electronic medical record database. It allowed one person to review 100% of patient and resident records and then provide peer compared feedback. DESCRIPTION OF PROGRAM/INTERVENTION: Data about resident entry of EPLs was extracted from the computerized, medical record database and recurrently fed back to them between February and June 2000. Profile reports were posted in clinic, and all practitioners could compare their patient panel sizes and EPL completion rates with their peers. Several times during the intervention, each resident also received a list of his or her assigned patients who needed EPL action. The profiling intervention was suspended July through September 2000. Since all practitioners in the teaching group practice were profiled, no control group existed. Therefore, the presence of an EPL for each patient and the entry dates of individual problems contained in those lists were analyzed and compared across three time periods: the seven months before profiling (July 1999 through January 2000), the five months during the profiling intervention (February through June 2000), and after profiling ceased (July through September 2000). FINDINGS TO DATE: During the five months of profiling, residents added Electronic Problem Lists to 1087 patients, raising the percentage of patients with EPLs from 45% in January to 88% in June. An unintended, side product of profiling was an 11% increase in the number of patients which residents identified as belonging to their panels, from a total of 2048 in January to 2272 in June. Besides starting more EPLs during profiling months, residents also entered significantly more individual problems into those lists during profiling. During the seven pre-profiling months, they entered an average of 321 individual patient problems per month, and during profiling they entered 757 per month (a 136% increase). Not only did individual residents increase their number of electronic problem entries, but also more residents entered problems. Prior to profiling, 18 of 71 residents did not enter a single problem over seven months, and only 30 residents each month entered problems. During profiling all 71 residents entered problems and 50 did so each month. Forty-two residents were present during all data collection months (July 1999 through September 2000). These 42 residents entered individual problems into EPLs at monthly rates of 220 before, 526 during and 285 after profiling.``Before'' and``after'' periods differed significantly from the profiling period (ANOVA p < .005) but not from each other. The number of residents adding problems each month averaged 20 before, 31 during and 26 after profiling (Chi-square p < .02 for the``before''/`d uring'' comparison). KEY LESSONS LEARNED: Profiling was associated with increased compliance with record keeping policy, and affected all residents. Repeated feedback to practitioners about their specific performance and with peer comparisons is more engaging and motivating than reminders by supervising attendings. Using a computer patient database to review 100% of all patient and physician documents offers an efficient means to enhance physician performance.\nTHE USE OF PALM HANDHELD COMPUTERS TO CARE FOR NURSING HOME PATIENTS. J.M. Previll 1 ; 1 East Carolina University, Greenville, NC STATEMENT OF PROBLEM OR QUESTION: Our medical school nursing home service has had difficulty monitoring the care of our nursing home patients because different attendings and residents were involved in the care of these patients. These difficulties included not having access to the patient's diagnoses, code status, current medications, pending labs, imaging, or procedures scheduled for the patient, or the subsequent results. There was also the question of when patients must be seen to comply with Medicare and institutional requirements. OBJECTIVES OF PROGRAM/INTERVENTION: The objective was to develop a system that would enable attendings and residents to track all the data on patients and provide better care with this information. DESCRIPTION OF PROGRAM/INTERVENTION: A Palm Pilot database called HandBase was used to develop a database that had information on the nursing home patients concerning their location, dates seen, diagnoses, code status, allergies, medications, reports on labs and x-rays, and CPTand ICD-9 codes for billing. HandJet, an interface program, linked the database in the Palm Pilot to a Microsoft Access database located in the Brody School of Medicine computer network. Patient data is protected by passwords in the Palm Pilot and the firewall in the Brody School of Medicine computer network. Changes to patient records can be made during time of visit or by telephone and disseminated to others by infrared beaming between Palm handhelds or hotsyncing to the computer network. FINDINGS TO DATE: This system allows database backup, printing of work lists, and patient data notebooks. It also enables all physicians and staff involved in patient care to have access to information needed in the ongoing care of the nursing patients. Having a current and complete profile on each patient has given the doctors the ability to respond and care for these patients in a more timely manner. Paperwork is simplified for admissions, discharges and scheduling. Documentation is immediate and more detailed, making billing more efficient and accurate, as it is now a part of the report generator. KEY LESSONS LEARNED: Maintaining a database that is mobile improves effectiveness and provides more timely patient care in the nursing care setting.\nPATIENT PROFILE: A PRACTICE MANAGEMENT SYSTEM DEVELOPED USING AN OFF-THE-SHELF RELATIONAL DATABASE PROGRAM. T. Yackel 1 , B. Slater 2 ; 1 Oregon Health Sciences University, Portland, OR; 2 George Washington University Medical Center, Washington, DC STATEMENT OF PROBLEM OR QUESTION: The George Washington University Department of Health Care Sciences is part of a multi-specialty faculty practice with departmentally centralized paper medical records. An internal study revealed that patient chart availability at the time of care was as low as 66%. The use of electronic resources to obtain patient specific information was limited to laboratory results retrieval and patient scheduling. Laboratory results were available online only for tests drawn in the past 60 days. OBJECTIVES OF PROGRAM/INTERVENTION: 1) Improve availability of patient information at the time of care. 2) Implement an electronic disease management system for patients with HIV and diabetes mellitus. 3) Accomplish these objectives in a short period of time with little funding. DESCRIPTION OF PROGRAM/INTERVENTION: Using an off-the-shelf relational database product, Access 97 (Microsoft, Redmond, WA), we implemented an advanced clinical information system over a six-month period. The program was written during spare evenings and weekends by the authors, neither of whom have had formal training in database programming. The system includes sophisticated features such as a problem list linked to ICD-9 codes; a prescription manager that provides drug-drug interaction checking and patient-specific formulary information; templates for HIV and diabetes disease management; automatic importing of laboratory information with longitudinal views, including results from the past 3 years for HbA1C, PSA, cholesterol, HIV viral loads, and CD4 counts; a reminder system; the ability to import dictations; user-level security and encryption of data; and an easy-to-use interface. FINDINGS TO DATE: Patient Profile has been in continuous use since June 1998 by a group of 3-5 users and has required little maintenance. The availability of the system exceeds 99%. Due to the program's simplicity, low-cost, and popularity among care providers, it became a prototype for a practice-wide electronic medical record project. KEY LESSONS LEARNED: Using commercially available tools, clinicians with minimal experience in computer programming can develop and implement their own practice management systems for a fraction of the cost of commercial products. Because these programs are infinitively customizable, they may serve the specific needs of providers better than a system purchased from a vendor. All students were mailed a questionnaire requesting they rate the importance of various factors in choosing a residency program using a 5 point Likert scale (1 = not important, 3 = somewhat important, and 5 = very important). Underrepresented minority (URM) applicants were defined as African American, Latino, or Native American. RESULTS: Response rate was 36% (1005/2820), 55% were men, 30% were married and 61% self-rated in the top 25% of their class. The factors most important to all applicants were good housestaff morale (mean SD= 4.54 0.72), the academic reputation of the program (4.46 0.78), a positive interview experience (4.10 0.99), variety of clinical experiences by residents (4.05 0.72) and location near spouse/significant other (4.04 1.43). URM applicants (N=92 or 9%) were significantly more likely to identify ethnic diversity of the patients ( CONCLUSION: Applicants rate location and program-related factors as most important in influencing their decision to chose a particular Internal Medicine Residency Program. However, URM applicants also place significant importance on the ethnic diversity of patients, housestaff and faculty, and the support provided to ethnic minorities within the academic environment. Residencies must place an emphasis on improving these factors if they wish to recruit highly qualified minority applicants. Although there is some literature on violence in the medical workplace, little has been written about threats physicians experience on the job. Our study examines the epidemiology of threats to resident physicians in internal medicine by patients, their families, and non-physician staff. METHODS: We implemented a mass-administered survey to internal medicine residents in two large urban training programs. One program is in a public county hospital which serves predominately uninsured patients. The other is in a private, tertiary care, academic medical center whose patient base is predominately insured by Medicare or managed care entities. We defined a threat as an actual or implied action intended to harm or torment someone. The survey measures the frequency, type and setting of threats. It examines the support structures residents use to deal with threats. We also assess resident attitudes towards receiving threats, particularly as they relate to patient care. RESULTS: Our preliminary data analysis shows 24 of 69 (34.8%) residents at the public hospital and 15 of 40 (37.5%) of residents at the private hospital have experienced one or more threats (total number of threats 66, mean 1.7). The threats were most commonly from patients assigned to that resident (47%), 16% from patients they covered on call, 19% from family members of patients, and 18% from other non-physician staff. Eighty five percent of the threats were verbal in nature and 15% were physical. The location of the threat occurrence was usually on the wards (70%). The remainder occurred in the emergency department (24%), and 6% occurred in outpatient clinics. Residents reported that the threats made them feel angry, nervous, afraid, and helpless. Approximately three-quarters of residents (74%) sought some support for these episodes. This support came from a variety of sources including peers, family, attending physicians, and security. Of residents reporting a threat, one in five (21%) said the experience impacted on the care of the patient involved, while only 2 of 39 (5%) said the experience impacted on how they care for other patients. Eleven of 39 (28%) said that following the threat they are more likely to avoid certain situations at work to feel more safe. Residents strongly disagreed with the statement that being threatened by a patient or their family members is a part of their job. CONCLUSION: Residents commonly experience threats from patients, their family members and non-physician staff in the workplace. The prevalence of threats was similar at both a public and private hospital. The most common reactions residents reported to these experiences include negative emotions and an impact on their care of the involved patient. An in-house support system needs to be developed to prevent and handle threats residents experience in the workplace. Residency programs have used support groups and Balint groups to address some of the stresses faced by trainees. We developed a monthly, faculty facilitated, hourlong session for residents on a ward or coronary care unit rotation. The goal of this session was to promote an opportunity and a safe environment for residents to explore clinical issues that had an impact on the feelings, attitudes, and challenges of becoming a physician. In each session, ground rules for safety were reiterated, as was the voluntary nature of this session. The group was held during one half of a scheduled attending rounds session in the third week of the month. The teaching attending (different from the facilitator) was welcome to participate. METHODS: We developed a confidential and anonymous survey given to all medicine and medicine pediatrics residents at the end of the 1999-2000 academic year. The survey was designed to access resident attitudes towards participation, their acceptance of this discussion format and any potential behavior change as a result of participation in those groups. RESULTS: Overall response rate was 75% (65% medicine and 35% medicine pediatrics). 62% of respondents were under age 30; 27% were age 30-35. 62% of respondents were female. Residents had attended an average of 1.6 sessions on the wards, and 1.1 sessions in the CCU. 90% of respondents indicated the timing was appropriate. Residents rated their comfort in these discussions with fellow residents on a 1 (low comfort) to 5 (high comfort) scale, with the mean response of 3.83. Residents were asked the following: The issues discussed in the balance groups are ones you may have thought about or discussed in the past. As a result of the discussions this past year, please rate your current activity from 1 (less activity) to 3 (same activity) to 5 (more activity) in: self reflection (mean response = 4.41), discussions with peers (mean response = 4.33) and discussions with family and friends (mean response = 4.17). CONCLUSION: Balance groups in this Internal Medicine residency training program were well received regarding their timing, and the comfort felt among participants. Importantly, residents indicated that they continued to reflect privately and with peers, family, and friends. Timing and attendance are significant barriers to successful groups; we found that scheduling this during a regular educational session, reinforcing appropriate ground rules and the voluntary nature of this session were important to its success. We recognize that some groups are longitudinal and benefit from the increased trust gained by the group. We chose to focus on the small group of each ward or unit team, and planned this discussion later in the month when some group identity and cohesion was more likely to have taken place.\nPREDICTORS OF EFFECTIVE PHYSICAL DIAGNOSIS TEACHING. K. Barnard 1 , D. Lescisin 1 , N. Armistead 1 , D. Elnicki 1 ; 1 University of Pittsburgh, Pittsburgh, PA PURPOSE: Interns play an important role in the teaching of medical students. We sought to identify demographic characteristics and teaching behaviors that predict effective teaching of physical diagnosis skills. METHODS: Self-administered surveys were completed by third year students at the end of their internal medicine clerkship at two medical schools from 7/00-11/00. Questions included demographic characteristics of interns and students, and occurrence of teaching behaviors, such as observation and feedback, reviewing cases, and establishing a comfortable learning climate. We also asked students to assess to what degree the intern contributed to their learning physical diagnosis skills (diagnosing heart murmurs, distinguishing pulmonary consolidation from pleural effusion, detecting hepatomegaly, examining the thyroid gland, detecting a joint effusion and lymphadenopathy). These items were selected from the CDIM-SGIM curriculum, and were combined to form a grouped physical diagnosis variable. A forward, step-wise linear regression model was built to determine which of the independent variables predicted effective physical diagnosis teaching. Adjusted p-values are shown here. RESULTS: A total of 70 students were surveyed (95% response rate). The teaching behavior,`m aking helpful suggestions to improve performance'', was most important in predicting effective teaching of physical diagnosis, as measured by the grouped variable, (p = 0.006). The addition of two other variables,``corrected mistakes without making you feel belittled'', and intern gender created a model with R2= 0.35. When the physical diagnosis variables were analyzed separately, the teaching behaviors,``observed you elicit physical examination findings'' and,``demonstrated a breadth of knowledge in internal medicine'' were important in predicting the ability to distinguish pleural effusions (p = 0.087 and 0.075 respectively). Observing students eliciting findings was important to examination of the thyroid gland (p = 0.07). The models for the individual physical examination skills explained 23%-59% of variances. CONCLUSION: Teaching behaviors involving giving feedback and female gender were most important to students learning physical diagnosis skills from interns. Other teaching behaviors were important for individual physical examination skills. The reasons for the gender differences remain unclear. PURPOSE: In order to prepare residents for primary care practice an understanding of what factors influence their satisfaction with the outpatient experience is necessary. However, few studies have addressed determinants of resident satisfaction in their continuity clinics. This study was conducted to determine these factors. METHODS: Over a 2-month period, resident satisfaction was assessed through a selfadministered questionnaire completed by each internal medicine resident in their outpatient continuity clinic. All 68 residents working in the clinic completed the questionnaire. The number of questionnaires completed by each resident ranged from 1 to 60 with a median of 23. The questionnaire was comprised of three 5-point Likert scale items that assessed resident satisfaction with each patient encounter. Resident satisfaction was defined as the mean of the 3 items. The resident satisfaction items had a Cronbach's alpha of 0.88. Other items assessed by the questionnaire included contextual aspects of the clinic, diagnoses of the patients, continuity, and demographic data. RESULTS: Overall, the majority of clinic visits were satisfying to residents. The mean satisfaction score of all resident clinic visits was 4.02 (5-point scale) with a standard deviation of 0.91. However, patient diagnosis had a significant impact on resident satisfaction. The mean resident satisfaction score for patients diagnosed with general medical problems was 4.30. The mean resident satisfaction score for patients diagnosed with pain complaints was 3.58 (p < 0.0001) and for psychiatric disorders it was 3.27 (p < 0.0001). Clinic visits with patients diagnosed with pain complaints or psychiatric disorders were more satisfying to residents if the patients were male rather than female (p < 0.05). 1st year residents were less satisfied with patients diagnosed with pain complaints or psychiatric disorders than 2nd and 3rd year residents (p < 0.05). On the other hand, 1st year residents found visits with patients diagnosed with general medical problems more satisfying than 2nd and 3rd year residents (p < 0.05). A number of factors were not associated with resident satisfaction including resident gender, rotation, and call status. CONCLUSION: Although resident satisfaction with patient encounters in the outpatient clinic is high, resident satisfaction is influenced by the patient diagnosis. Seeing patients with pain or psychiatric diagnoses is associated with decreased resident satisfaction in their continuity clinic. However, this appears to be influenced by the experience level of the resident. Future research can explore whether early resident training in the treatment of pain and psychiatric conditions may increase resident satisfaction with such patients. PURPOSE: Approximately 25% of Americans are functionally illiterate and low literacy has been associated with poorer physical health, psychological health, and higher health care cost. This study was conducted to see if residents could identify patients with potential literacy problems based solely on interactions with patients during a clinic visit. We hypothesized residents will overestimate patients' literacy abilities and not identify many patients at risk for potential literacy problems. METHODS: Residents completed a questionnaire measuring a number of constructs regarding continuity clinic in general, and their perception of their continuity patients' literacy. The residents were asked,``Do you feel this patient has a literacy problem?'' Patients in continuity clinic were given the Rapid Estimate of Adult Literacy in Medicine-Revised (REALM-R), a new instrument to quickly screen for potential literacy problems. The REALM-R has been previously correlated with the Wide Range Achievement Test-Revised (WRAT-R) and the Rapid Estimate of Adult Literacy in Medicine (REALM), two well-validated instruments but impractical for large scale screening in a busy clinic. The REALM-R asks patients to read a series of eight medical words and a correct response is given for each correct pronunciation. A score of six or less was used to identify patients with a potential literacy problem. Data were available from 182 patients who completed the REALM-R and whose residents completed the literacy question. RESULTS: Patients ranged in age from 17 to 93, and 85% were Caucasian. Scores on the REALM-R ranged from 0 to 8 with a mean of 6.8 and S.D. of 2.1. Twenty-three percent of patients read at the eighth grade level or less according to the WRAT-R. Despite these baseline characteristics, residents believed only 10% (18) of patients had a literacy problem based on their clinical interactions. Residents perceived 90% (164) of patients to have no literacy problem, but 36% (59) of these patients scored 6 or less on the REALM-R. Conversely, residents identified three patients as having a literacy problem whom scored higher than six on the REALM-R, while the other 15 all scored six or less. CONCLUSION: Residents perceived a significant number of patients to have no literacy problem, although these patients scored poorly on the REALM-R. Residents incorrectly identified only a few patients. Residents' overestimation of patients' literacy is concerning given that many patients will hide literacy problems from physicians and the poor health outcomes associated with poor literacy. A brief screening instrument like the REALM-R may help clinicians identify patients in whom literacy may complicate their health care. METHODS: This data is derived from the intake questionnaire for participants in the PSPA study. We obtained names and addresses from the AAMC roster for faculty that entered academic medicine as Assistant Professor June through December of 1995. In 2000, 183 faculty members from 35 different states volunteered to participate. Participants indicated they were clinician-educators (CE) or clinician-investigators (CI). If participants indicated``other'' or`c linician'', we assigned them a priori to one of the two former categories based upon their percent effort allotted to various work activities. RESULTS: Participants had a mean age of 40 years, and had been working at their current position for 4.7 years. 63% of the faculty were clinician-educators (CE) and 37% were clinician-investigators (CI). More CI's than CE's were minorities (30% vs. 14%, p < .05), but there was no significant difference in gender (35% female). Whereas 98% of CI's reported a publication expectation to be promoted, 25% of the CE's reported no such expectation (p < .001). Significantly more CI's had career mentors available than CE's (68% vs. 32%, p < .001), and 92% of CE's for whom they are not available indicated they would like one. 79% of CI's indicated >10% protected scholarly work time, compared to only 35% of CE's (p < .001). 53% of CI's vs 32% of CE's (p < .01) meet more often than yearly with their chief/ chair for performance review, and more CI's have seen written promotion guidelines (72% vs. 51%, p < .01). CE's and CI's assigned similar importance to clinical research, written scholarship, and reputation (top 3 out of 11 items in importance for both groups) as activities leading to their promotion. The top 3 important activities for CE's according to promotion committee chairs in our previous study (teaching skills, clinical skills, and mentoring), were ranked 4th, 10th, and 8th by CE's in this study. Both CE's and CI's agreed that CI's more likely get promoted (82% vs. 79%). CONCLUSION: CE's indicated fewer performance reviews with their chair/chief, fewer mentors, and less knowledge of written promotion guidelines than their CI colleagues. Although university promotion chairs have indicated that teaching and clinical skills are most important in the promotion of CE's, CE's believe research and written scholarship are most critical to their promotion. PURPOSE: The purpose of this study was to identify residents' perceptions of the socioculturally and linguistically based barriers they face in caring for a diverse patient population. We also attempted to determine whether, and in what ways, a required crosscultural curriculum helped them deal with these barriers. METHODS: We conducted structured interviews with the entire intern class of an urban academic internal medicine residency program (n=40). These were carried out with the stated goal of identifying general barriers to quality care for their patient population. Interviews were performed within six months of the completion of an eight-hour, case-based, cross-cultural curriculum. Residents were not aware of any connection between the research assistant who conducted the interview and the primary researchers who taught the curriculum. Probes for the interview focused on: general barriers to care, sociocultural and linguistic barriers to care, if and how they had been prepared to deal with these barriers and the type of training they had received, and whether the cross-cultural curriculum had helped them and affected their attitude towards these issues. Interviews were recorded, transcribed, and coded by two independent outside researchers experienced in qualitative analysis. RESULTS: Several barriers to delivering care were identified:\n1. Language differences between patient and resident 2. Limited time to address important cross-cultural issues in medical encounter 3. Understanding socioculturally-based patient expectations and perceptions medical care (including mistrust) 4. Differences between patient beliefs and physician beliefs regarding disease and illness Effect of cross-cultural curriculum on attitudes and practice:\n1. Greater acknowledgement of the role of sociocultural issues 2. More probing into patients' beliefs and practices 3. Overall very helpful in addressing barriers CONCLUSION: Residents cite several barriers to caring for their socioculturally and linguistically diverse patient populations. While they identified several benefits of a crosscultural curriculum, they also highlighted the need for effective interpreter services and increased time to care these patients.\nD.E. Bonds 1 , R. Watkins 1 , J.C. Mychaleckyj 1 ; 1 Wake Forest University, Winston-Salem, NC PURPOSE: Previous studies have found that Internal Medicine faculty and residents rate their ability in ambulatory care procedures as poor. Our objective was to assess faculty and residents ability in those skills related to Women's Health, and whether that ability varies by gender or status (resident vs faculty). METHODS: A descriptive survey was administered to all Internal Medicine residents and teaching faculty at one academic medical center. Respondants were asked to rate their ability to perform 5 ambulatory skills (breast exam, pelvic exam, wet mount/KOH, pap smear, endometrial biopsy) and comfort in obtaining 4 areas of history/counseling (sexual activity history, domestic violence history, preconception counseling, contraceptive counseling). A 4point Likart scale was used. RESULTS: 88 Internists completed the survey: 91% of residents (72/79) and 80% of faculty (16/20). Women comprised 32% (32% residents, 31% faculty). Greater than 70% felt able to perform all physical exam and procedural skills except endometrial biopsy (0% able). Conversely, less than 40% rated their comfort level as high for domestic violence history, and areas of counseling. Women rated their ability higher than men, and faculty higher than residents. See table 1 for full results (* indicates p < =0.05). CONCLUSION: Faculty and residents rated their ability high for exam and procedural skills, but most felt uncomfortable in obtaining history or performing counseling. Women rated their skill level higher, as did faculty. Both faculty and residents need additional training in ambulatory skills related to women's health. To assess the relative degrees to which a variety of factors are perceived to contribute positively and negatively towards motivating internal medicine residents to improve their physical examination skills. METHODS: In May 2000, forty G1 internal medicine residents at the Mayo Clinic completed an objective, structured clinical examination (OSCE). Immediately following this exercise, they were anonymously surveyed regarding the degree to which certain factors tended to either increase or diminish their motivation for improving their skills in physical examination. Twelve potential motivators and thirteen potential barriers were ranked on a Likert scale from one to seven. RESULTS: Those factors ranked as the strongest motivators for improvement had to do with the role of the physical examination (PE) in patient care decisions (mean score = 5.9), the regret of missed findings in the past (5.9), and the perceived contribution of strong PE skills to one's overall excellence as a clinician (6.0) or teacher (5.3). The need to enhance one's performance on formal measures such as the OSCE (3.6) or to fulfill the expectations of faculty (4.6) or peers (4.0) on rounds were rated as weaker motivators. Perceived barriers to improvement included lack of time to study (5.2) or apply (5.1) the methods of PE and a lack of faculty emphasis on the importance of PE (4.8). Although the sense of exam findings being eclipsed by test results in making clinical decisions was rated as a moderately strong demotivator (5.1), other factors designed to look for overall``PE nihilism'' were ranked as only weak hindrances to improvement (2.7). Likewise, a sense of discouragement over one's past performance and future prospects for improvement seemed to be a minor barrier (3.4) , as did reliance on a perceived supervisorỳ`s afety net'' (2.0). CONCLUSION: Internal medicine residents in their first year of training seem to be most strongly motivated to improve their PE skills by factors which relate to their desire to improve the quality of patient care, to avoid regret, and to build personal confidence as an excellent clinician and teacher. The most significant demotivators are lack of time to study and apply the methods, and lack of faculty emphasis on the importance of these skills. The perception of test results eclipsing the contribution of the physical exam to clinical decisions also seems to demotivate at some level, and educational strategies which emphasize those situations wherein the exam makes an indispensable contribution to patient care may help to overcome this potential barrier. Few studies, however, explore fellows' training expectations or their satisfaction with the program they select. We sought to evaluate these factors and identify areas of training that lead to a productive and satisfying fellowship experience. METHODS: We developed a 43-item questionnaire that addressed fellows' initial training expectations, satisfaction with the quantity and perceived quality of training (teaching and research), availability of mentors, scholarly productivity (publications, grants) and anticipated career track. Question format included best answer and Likert scales. Directors of the 37 active fellowship programs listed in the 2000 SGIM Fellowship directory were contacted by e-mail and asked to forward our web-based survey to current fellows. To calculate an accurate response rate while maintaining respondent anonymity, each fellow used a program-specific code to initiate the survey. RESULTS: Forty-three percent (65/152) of fellows completed the survey, with at least 1 respondent in 65% (24/37) of the programs. Nearly three-quarters (74%) anticipated careers as clinician-investigators. Eighty-two percent had high or very high initial expectations for research training, 26% for teaching and 20% for learning leadership skills. The availability of mentors (95%), program flexibility (94%) and protected time for research (91%) were rated as the most important factors in fellowship training. Most fellows were satisfied or very satisfied with the actual availability of mentors (80%), program flexibility (85%) and protected research time (88%). Over 80% agreed that their expectations had been met for both the quantity and quality of research opportunities, while 63% reported this for the quantity and 40% for quality of teaching opportunities. The availability of mentors and the perceived quality of research opportunities were significantly correlated with fellowship scholarly productivity, 0.507 and 0.539 respectively (p < .001). CONCLUSION: This study suggests that the majority of GIM fellows enter training with the expectation for advanced research skills and are satisfied with most aspects of their experience. These data highlight the important correlation between mentoring, high quality research opportunities and scholarly productivity during fellowship. That relatively few fellows are satisfied with the quality of available teaching opportunities is concerning and deserves further exploration. METHODS: We designed a 10-page survey to determine the extent of faculty development (FD) activities that focused on``improving the teaching/educational skills'' of faculty. For those hospitals with ongoing activities we asked about the subjects covered, the methods of teaching, the formats offered, and the evaluation of the programs. In April 2000, we mailed the survey to Department of Medicine chairmen at the 390 teaching hospitals with internal medicine residency training programs in the U.S. We classified teaching hospitals with membership in the Association of Professors of Medicine as university hospitals (N=114), and those without membership as non-university. We performed descriptive analyses of hospitals with and without ongoing FD, and used chi2 to detect differences in categorical variables and t-tests for differences in continuous variables. RESULTS: Three hospitals responded that their training programs had closed. Of the remaining 387 hospitals, 279 responded for a response rate of 72%. Only 39% of teaching hospitals have ongoing FD, including 48% of university and 34% of non-university hospitals (p=.007). An additional 34% have occasional FD; 27% have no FD activities. At the hospitals with ongoing FD activities (N=109), an average of 10 content areas are covered, with university hospitals including more than non-university hospitals (11 vs. 9, p=.036). The most common content areas are general teaching principles (90%), giving feedback (88%), outpatient precepting (72%) and evaluation of learners (72%). FD is taught most commonly in small group sessions, with 65% of hospitals using them frequently/always, and in lectures, with 50% using them frequently/always. The most common format for FD is a 1/2 day workshop (78%), but 21% offer courses lasting more than a month. Compared to university hospitals, non-university hospitals are more likely to offer 1/2 day workshops only (p=.008), or courses < 7 days (p=.013), and less likely to offer longer courses (p=.006). Evaluation of FD programs is limited to forms filled out by participants at the end of the program. Most programs had trained: < 50% of hospital-based and < 25% of community-based faculty; < 50% of general internal medicine faculty and < 25% of subspecialist faculty. 2/3 of hospitals with FD, but only 37% of hospitals with no FD, arrange for faculty to go off-site for FD.\nCONCLUSION: A minority of teaching hospitals have ongoing faculty development programs for improving their faculty's teaching skills and evaluations are primitive. University hospitals are more likely to have ongoing FD and to have programs that are more developed. Overall, only a limited percentage of faculty have been trained, especially community-based and subspecialist faculty. These gaps should be considered in future funding decisions to provide learning opportunities for all faculty. PURPOSE: This study was done to determine the relationship between students' ratings of faculty on a clinical teaching evaluation form and a medical lecture evaluation form. METHODS: Twenty-three faculty received ratings between July 1999 -December 2000 from medical students immediately after a didactic lecture on an 11-item lecture evaluation form that used a scale from 1 (not satisfied) to 7 (very satisfied) to assess lecture skills (Chronbach's alpha=.96) and at the end of a clinical rotation on a 15-item clinical teaching form that used a scale from 1 (never/poor) to 5 (always/superb) to assess attending/teaching behaviors in the patient care setting (Chronbach's alpha=.97). An average of 6 and 16 students per faculty completed the lecture and the clinical teaching forms respectively. Average students ratings on all items for each faculty member were calculated. Pearson correlation coefficients were computed between the average scores for each item on one form and the overall average scores on the other evaluation form. RESULTS: Overall there is a correlation of .69 (p < .01) between the average scores on the two forms. Only correlation coefficients above .65 (to explain >45% of the varaince) were considered meaningful. The following five items from the lecture form significantly correlated (p < .01) with the average clinical teaching scores: how well the course was organized (r=.69), how effectively time was used in class activities (r=.67), the clarity of the instructor's explanations (r=.70), the instructor's use of examples or illustrations to help clarify the material (r=.67), and having learned something which I consider valuable (r=.67). The following three items from the clinical teaching form significantly correlated (p < .01) with the average medical lecture scores: teaches diagnostic skills (r=.73), teaches effective patient/family communication skills (r=.72), and teaches principals of cost-appropriate care (r=.70). Interestingly, these three items were the only items that included the word``teaches'' suggesting direct didactic instruction as opposed to the teaching strategies suggested in the other items (i.e., adjusts teaching, coaches, asks). Items from the lecture form that did not correlate with the average clinical teaching scores (e.g., specifying objectives, summarizing material, encouraging class discussion) are often considered fundamental in classroom teaching and may not easily translate to clinical teaching. Yet, the larger number of related items from the lecture form suggests that lecture techniques do crossover to clinical teaching, especially with providing explanations and using examples. CONCLUSION: There appear to be some differences in the skills involved in clinical teaching and lecturing; only 47% of the variance is explained between them. Although requiring more work by students, using two forms to evaluate teaching may better enable instructors to identify specific strategies where they are strong or need improvement for the various teaching activities they perform.\nAVAILABILITY OF SAMPLE MEDICATIONS AND RESIDENTS PRESCRIBING PATTERNS. J. Diaz 1 , M.J. Fagan 1 , A. Etienne 1 ; 1 Brown University, Providence, RI PURPOSE: The use of sample medications in clinical teaching settings is controversial. One concern is that the presence of sample medications may affect physician compliance with treatment guidelines. This project examined the availability of anti-hypertension sample medications and the selection of specific medications by internal medicine residents to treat hypertension. METHODS: From 12/4/00 to 12/20/00, charts of patients presenting to the residents' clinic at Rhode Island Hospital were reviewed if hypertension was listed as one of their diagnoses and the patients were receiving medications. All anti-hypertension medications prescribed, the date of visit and insurance status of each patient were recorded. During the same time period, inventory of the clinic's medication sample closet was taken for each clinic session and names of all antihypertension medications available were noted. RESULTS: During the 3 week period, 288 patient charts met criteria for review. 56% (161/ 288) of patients had no insurance coverage for medications. A total of 41 different blood pressure medications were documented as being prescribed and 18 (44%) of these were available as samples. The 5 most commonly prescribed medications were hydrochlorothiazide (86/288), Norvasc (amlodipine-73/288), Accupril (quinapril-66/288), Vasotec (enalapril-42/288) and Toprol XL (metoprolol-32/288). Three of these, Norvasc (amlodipine), Accupril (quinapril), and Toprol XL (metoprolol) were available in the sample closet during the study period and one, Vasotec (enalapril), had recently been available. Except for diuretics, for each class of antihypertension medication, the most commonly prescribed agent was also available as a sample. Of 146 notations of ACE-Is, 65 (44.5%) were Accupril (quinapril). Of 118 calcium-channel blockers (CCBs) prescribed, 70 (59%) were Norvasc (amlodipine). Of 65 notations of betablocker use, 31 (48%) were Toprol XL (metoprolol). No diuretics were available as samples, but the diuretic most commonly prescribed was a generic agent, hydrochlorothiazide (70%, 84/120). Stratifying by medication coverage, a sample closet CCB and a sample closet ACE-I continued to be the medications most commonly prescribed in their drug classes. Norvasc (amlodopine) accounted for 54% (38/70) of CCBs prescribed to patients without medication coverage and 67% (32/48) of patients with coverage. Accupril (quinapril) accounted for 42% (36/85) of ACE-I prescribed to patients without coverage and 47% (29/61) of patients with coverage. CONCLUSION: This pilot study suggests that residents selecting medications to treat hypertension may be influenced by the availability of sample medications. With the exception of hydrochlorothiazide, for each class of anti-hypertension medication, a drug available in the sample closet was the one most commonly prescribed. Although this reflects patterns at only one institution, residency programs should be aware of how sample medications may influence the prescribing patterns of physicians in training.\nPURPOSE: Several studies over the past two decades have examined the issue of depression in house staff, but none have attempted to relate it to house staff performance. The ideal study to examine this question would be prospective and longitudinal, using validated psychiatric rating instruments and quality assurance data. Since IRB concerns have thus far precluded such a study, a pilot retrospective survey was developed to obtain preliminary data. METHODS: After IRB approval was obtained for the pilot, the survey instrument was distributed to interns, junior residents, and senior residents in the internal medicine residency program at our institution. Participants were asked whether they had worked with someone they thought was depressed, if they had worked with a colleague whose performance had been affected by depression, and if they had ever been depressed themselves during the residency. RESULTS: 45 members of the house staff (33%) replied to the survey. 34 respondents (76%) believed that they had worked with a depressed colleague during the residency, and 23 respondents (51%) believed that a coworker's performance had been affected by depression. Fifty-one percent of the respondents also reported having experienced depression themselves during their residency. CONCLUSION: Although the sample size is small and these data are only preliminary, depression appears to be prevalent in some internal medicine residency programs. Furthermore, house staff depression may affect patient care. The current pilot study indicates the need for further research in this area. format, the other 2/3rds was interactive. Each workshop included a case with a clinical question, the search strategy, and an article selected to answer the question. While in the small groups participants assessed the validity of the study, interpreted the results and applied the results of the study to the clinical question. We assessed the usefulness of the workshop, materials and effectiveness of the facilitators by asking participants to fill out an evaluation form using 5-point Likert scales to rate the individual items. The participants were asked to rate the workbook and its content, the facilitators, the format of the workshop, the didactic sessions, the interactive sessions, and if they would recommend the workshop. RESULTS: Sixty-one participants completed the evaluation sheets. The median scores for all items were 4 or 5 for all items in both years. The workshop``practice session'' was slightly higher rated in 2000, compared to 1999 (p = 0.047). There were no other significant differences between years. CONCLUSION: A 90 minute workshop can effectively teach the principles and methods for the development of a successful EBM-CDM journal club to faculty members. PURPOSE: Evidence based medicine (EBM) is part of the curriculum of most medical schools and residency training programs in the US. We hypothesized that the concepts and skills may not be integrated into clinical practice because there is little support outside the classroom setting. We surveyed academic internal medicine generalists and subspecialists to assess whether they have different attitudes about evidence based medicine and whether they rate the importance of specific EBM skills differently. METHODS: We surveyed a stratified random sample of academic internists and subspecialists from 11 medical centers in the Chicago area (n=330) in the Spring of 2000. Respondents completed a 39-item self-administered questionnaire, which was piloted and revised for internal consistency prior to use. The three survey questions required respondents to: 1) rate the importance of 9 core skills and 5 EBM skills on a 5-point Likert scale, 2) choose the five most important skills in residency training, and 3) indicate the level to which they agreed or disagreed with 15 attitudinal statements about EBM, also on a 5-point Likert scale. We also collected information on year of graduation from medical school, teaching responsibility, and exposure to a formal course on clinical epidemiology or evidence based medicine. RESULTS: The response rate was 63%. Core medicine skills (clinical reasoning skills, differential diagnosis skills, physical exam skills, interviewing skills, and doctor-patient interaction skills) were scored highest, with all EBM skills rating at least 1 point lower: average core skill score 4.4 vs. average EBM skill score 3.5 (p < 0.001). None of the EBM skills were listed in the five most important skills. EBM skills were rated higher among younger faculty (p = 0.01) and those who had previously taken an EBM course (p = 0.03), but generalists and subspecialists rated the importance of EBM skills similarly (p = 0.56). However, generalists had slightly more positive attitudes about EBM (p = 0.014). Neither year of graduation nor exposure to an EBM course predicted faculty attitudes toward EBM. CONCLUSION: Contrary to our initial hypothesis, generalists and subspecialists differ little in their attitudes about EBM and in their ratings of the importance of EBM skills in residency training. Although both groups rated EBM skills positively, the skills were prioritized much lower than all core skills. Perhaps EBM skills are not routinely reinforced outside formal teaching settings because they are not a high priority among academic faculty-generalists and subspecialists alike. PURPOSE: Few studies have examined the effectiveness of Internet-based medical educational programs for physicians. We wanted to determine whether a skin cancer triage intervention, developed and proved successful in a face-to-face, on-site application, could be effectively delivered over the Internet with the same positive results. METHODS: Physicians were randomly assigned to an Intervention or Control Group. Intervention Group physicians completed modules which included a Pretest, Pretest Scores with Individualized Feedback, Instruction (text and hyperlinks to digitized images of skin lesions), and two Posttests. Control Group physicians completed the Pretest and Posttest I. The program could be completed in 3-4 hours, at participants' convenience. There were 14 outcome measures including diagnosis and evaluation planning for malignant melanoma, basal cell carcinoma, and squamous cell carcinoma. RESULTS: 71 physicians completed the program through Posttest 1, and 46 (27 of 39 in the Intervention Group) completed the entire program. The Intervention Group showed significantly greater improvement than the Control Group in 9 of 14 outcome measures. By Posttest II, Intervention Group improvement was maintained in 5 of the 9 outcomes. CONCLUSION: Our brief, Internet-based, educational intervention with individualized feedback was successful in improving the skin cancer diagnosis and evaluation planning test performance of primary care physicians. The IOM's report on medical errors focused attention on the importance of measuring quality and outcomes. As part of the UME-21 project funded by HRSA, the UCSOM developed a Quality Improvement Curriculum. This abstract examines the impact of student CQI projects on the quality of care delivered at community practices. METHODS: Eighty second year students working in groups of 3-4, initiated a CQI study on Diabetes Mellitus at 23 community-based primary care practices collecting baseline data, implementing a results-specific intervention and re-measuring quality indicators 8 months later. Students attend community-based primary care continuity clinics one-half day/week, year I-III. Students were evaluated using Likert-scale rated attitudinal items as well as open ended questions. RESULTS: 512 charts were abstracted for the baseline sample with 383 charts abstracted postintervention. The number of documented foot and eye exams increased significantly from baseline to remeasurement (51% to 67.8%; p < .001 and 26.4% to 35.5%; p= 0.003, respectively). The mean value for glycohemoglobin dropped from 7.71% at baseline to 7.2% at remeasurment (p=.0001). Analysis of student attitudes revealed acknowledgement of the benefit of outcomes measurement in clinical practice despite frustration with the tedium of the chart abstraction process. Feedback was used to modify curriculum design for future classes. CONCLUSION: Medical student-driven CQI projects can have significant impact on improving the quality of care at the practices in which they participate while introducing them to the process of quality measurement and improvement. Formative input from students should be used to optimize CQI experiences. The use of medical students to lead CQI efforts in private practices may represent an underutilized resource in our efforts to improve the quality of care provided to the public. launched the first program to financially support community-based teaching by enhancing the capitation level of primary care preceptors. We studied the effects of these enhanced capitation payments on preceptors' attitudes towards student training and towards managed care's support of teaching medical students. METHODS: Participants were community-based preceptors who completed a questionnaire consisting of sections on attitudes towards teaching and attitudes towards managed care, given prior to the distribution of enhanced capitation payments (Time 1) and after receiving payments (Time 2). The instrument was given to three groups: AUSHC preceptors receiving enhanced capitation (86/93, 92.5% response rate) and control groups consisting of AUSHC preceptors who chose not to participate in the enhanced capitation program (57/66, 86.4% response rate) and non-AUSHC preceptors (62/68, 91.2% response rate). Responses were scored using a sixpoint Likert scale ranging from Strongly Disagree (1) to Strongly Agree (6). Statistical tests employed included chi-square for differences between the respondent groups, and principal factor analysis and t-tests for differences between Time 1 and Time 2. RESULTS: Factor analysis identified three underlying factors: 1.) Attitudes towards managed care support of physicians, which became less positive, decreased from 3.58 to 3.43 (p = 0.28); 2.) Attitudes regarding the impact of teaching on their practices, remained positive, although slightly decreasing from 4.70 to 4.57 (p=.043); 3.) Attitudes towards their roles as trainers, which was very positive, remained unchanged (5.06 to 5.06, p = .956). No significant difference was found in attitudes when comparing those who received enhanced capitation to the control groups. CONCLUSION: While the enhanced capitation program has provided community-based preceptors with additional financial support, it did not improve preceptors'attitudes concerning managed care's support of their teaching activities. We believe this program is an important first step but more is required. Just as attitudes towards managed care are the result of many factors, initiatives to change those attitudes should be multifactorial.\nMODALITIES. E.H. Green 1 , R. Granieri 1 ; 1 University of Pittsburgh School of Medicine, Pittsburgh, PA PURPOSE: Internal medicine residency training involves exposure to different learning modalities including rounds, lectures, small group discussion, and independent study. Little is known about learners' views regarding different educational interventions. We administered a questionnaire to assess current medical residents' attitudes towards different learning modalities. METHODS: Second and third year medical residents from an academic residency program and closely affiliated community residency program were surveyed (n = 91). Respondents were asked to rate, using a Likert scale and list ranking, the contribution of various educational activities to their understanding of clinical medicine, preparation for standardized tests, and overall education. Subgroup analysis was done according to level of training, career goals (primary care or subspecialty medicine; academic or community), gender, college major, and additional education. RESULTS: A total of 50 residents (55%) responded.``Noon conference'' (core lecture series) and morning report were highly valued, with 82 and 84% of residents rating these endeavors as moderately or very important to their overall education. Attending rounds were less important, with 68% rating them as moderately or very important. Further, 44% of residents felt``too much'' time was devoted to attending rounds. Medical grand rounds had the least contribution to residency education with 33% rating it as moderately or very important. In contrast, 92% of residents rated independent learning with as moderately or very important. Independent study was the only educational activity that residents thought prepared them equally well for clinical medicine and standardized exams. Residents actively pursued independent study with a mean of 12.69 topics researched in the 3 months prior to the study. However, 52% of respondents felt they had``too little'' time for this endeavor. Independent study and morning report were independently identified as the two most important educational activities during residency. The only significant finding from subgroup analysis was that residents with advanced degrees valued independent study more than their colleagues (p = .038). CONCLUSION: In this study, internal medicine residents valued lectures and morning report over attending rounds or medical grand rounds. However, independent study is the most highly valued educational method, and house officers are very involved in this despite a perceived lack of time. Medical residents seem to be mastering the principles of adult, independent learning: future curricular reform should consider incorporating more time and opportunities for independent study. used; whether PDA's replaced medical texts; and subjective responses regarding the impact of PDA's on patient care and medical education. 114 of the 133 surveys were returned (86% response rate). RESULTS: 62% of medical residents owned PDA's (100%-based on the Palm operating system). The mean age of the cohort was 28 years, and 55% of PDA owners were male. Ownership was inversely correlated with both age and level of training. The most commonlyused medical applications were drug formulary references, medical formula calculators, medical note-taking, and reference texts. Interestingly, only 15% of PDA owners replaced their paperbased drug references with the PDA version. Relatively few residents used their PDA's for patient tracking, appointment scheduling, or to log procedures. The most-commonly used nonmedical applications included the built-in address book, memo pad, to-do list, calculator, local city guides, and games. 83% of owners felt that PDA's improve efficiency, and a majority felt that they were useful in medical education. 69% responded favorably when asked if PDA's improve patient care, but only 48% believed that PDA's decrease medical errors. 37% of nonowners were committed to purchasing a PDA within 12 months. CONCLUSION: The majority of internal medicine house officers at New York-Presbyterian Hospital already own PDA's. Most residents use drug references and medical calculators, perhaps because they provide point-of-care answers to direct questions. The majority of residents believe that PDA's are useful for improving efficiency, medical education, and patient care. However, more research is required to correlate these positive perceptions with objective improvements in clinical outcomes, as well as broadening the applicability of the results to other specialties. Furthermore, as PDA's and the availability of medical applications continue to proliferate, issues of medical privacy and quality-assurance of medical content will become increasingly important. PURPOSE: Despite calls to incorporate active learning strategies into medical education, many teachers believe that efficient delivery of content can only be achieved through didactic lectures. The goal of this study was to examine the effects of reducing didactic lecturing and adding small group work, a commonly employed active learning strategy, on learner participation, attitudes, and knowledge acquisition. METHODS: We recruited residents in internal medicine, pediatrics, and family medicine to attend a session on effective use of diagnostic tests, and assigned them to one of two groups. The control group received a standard 'slide and lecture' session where the lecturer spent a full hour delivering content. The intervention group received a session where residents were assigned to groups of 4-5 and completed a series of 3 small-group tasks with large-group lecturer-facilitated discussion between tasks; in this session, the lecturer delivered content only during large group discussions (approx. 30 minutes). Both sessions used the same lecturer and covered the same content. We measured resident demographics before and self-perception of active learning after both sessions. We measured attitudes toward the session content and knowledge before and after both sessions. We also employed trained observers, blinded to the study purpose, who used a standardized instrument to observe resident patterns of behavior during each session. RESULTS: 27 residents completed the control session and 36 completed the intervention session. The study groups did not differ significantly in demographic variables, baseline attitudes about, or baseline knowledge of the session content. As measured by the trained observers, during the intervention session more students were communicating with other students (p < .001) than during the control session. Students in the intervention group also selfrated their participation in the session higher (p=.001). While both groups improved in both knowledge and attitude scores from pre-to post-session (p < .001 for all comparisons), there were no significant differences in the amount of improvement between groups. CONCLUSION: In these sessions conducted in a controlled environment, we reduced the time spent 'lecturing' by the teacher by 50 percent and covered the same amount of content with no detrimental effects on knowledge acquisition or attitude enhancement. In addition, our incorporation of active learning strategies produced residents who both self-rated and were observed to be more actively engaged with each other and with the learning process. Our future work will measure the effects of this active engagement on long-term retention of content and attitudes. PURPOSE: Existing data suggests that physicians who demonstrate patient-centered behaviors enjoy high patient satisfaction. However, little data exists to link physician attitudes about medical care with patient-centered outcomes. In this study, we explored associations between student physicians' attitudes toward the physician-patient relationship and patients' perceptions of students' humanism during the medical interaction. METHODS: At the beginning of a primary care (PC) clerkship, we surveyed 293 third-year students using the Patient-Practitioner Orientation Scale (PPOS), a validated instrument that measures attitudes toward the physician-patient relationship. PPOS scores range from patientcentered (egalitarian, whole person-oriented) to physician-centered (paternalistic, less attuned to psychosocial issues). During the PC clerkship, all students performed a medical interview with a series of 5 standardized patients (SPs) who were trained to portray varying biopsychosocial issues. All SPs were unaware of the students' PPOS scores. After each interaction, each SP completed a 7-item validated instrument that measured their perceptions of the student physician's humanism. We calculated a total humanism score for each student and compared these to students' attitudes as measured by PPOS.\nRESULTS: Mean humanism scores were lowest for students who scored in the most physiciancentered quartile (79.6 9.4) and highest for students who scored in the most patient-centered quartile (83.4 7.0) on PPOS. In linear regression analysis controlling for month of the thirdyear on the primary care clerkship, patient-centered PPOS scores (p=.01) and female gender (p=.05) were found to be associated with higher total humanism scores. CONCLUSION: Student physicians with more patient-centered attitudes demonstrated higher degrees of humanism as perceived by standardized patients. Patients' perceptions of humanism were also influenced by student gender. Further research is needed to explore the nature of associations between physicians' attitudes and patients' perceptions of care, as well as the impact of medical education on students' attitudes toward the physician-patient relationship. PURPOSE: Although training in the skills required to perform pelvic examinations is ubiquitous in medical schools, few residency programs assess or reinforce these skills, and only limited evidence justifies the substantial cost of post-graduate training. We report results of a controlled study of training for Internal Medicine interns utilizing professional instructors who also serve as models for the exam. METHODS: 48 interns from two university Internal Medicine residencies completed questionnaires about their experiences with pelvic examinations. 23 participated in a two-hour training session after which their skills were assessed. At follow-up four months later, the skills of 13 of the trained interns were compared with that of 24 interns who had not been trained. Assessments were based on the observation of verifiable behaviors. Chi-square tests were used to assess the statistical significance of comparisons. Inter-rater reliability was assessed with the kappa statistic. RESULTS: Mean age of the 37 interns in the controlled study was 28.4 (range 24-40), with 57% women and 89% graduates of U.S. medical schools. 70% of the interns described having received specific pelvic exam skills training in medical school, 78% felt comfortable performing the exam, and 91% felt they give patients clear instructions. Interns who participated in the current training displayed 10 of 12 key behaviors more frequently when compared with interns who had not been trained. Overall, trained interns demonstrated 87% of key behaviors, compared with 63% for untrained interns. Specific items for which the largest improvements occurred were in providing an outline of the exam (100% for trained interns vs. 38% for those not trained) and explicitly giving permission to ask questions (69% vs.17%; p < 0.01 for all three comparisons). Independent video and in-person observers agreed well with ratings of instructors (kappas > 0.7). CONCLUSION: Most interns described exposure to medical school pelvic examination training, but many overestimated their skills. A program employing professional trainers can reliably evaluate and improve key skills and improvements are demonstrable months after training. Further work is needed to demonstrate consistent benefits in larger groups of interns and residents, to ascertain whether training efficiency could be improved, and to measure the impact of training on subsequent encounters with actual patients. The mini-CEX can facilitate interactive feedback (FB) between faculty and residents to help residents correct deficiencies and reinforce good clinical skills. However, little is known about the nature of the FB that results after a mini-CEX is completed, and whether the FB is interactive. PURPOSE: To assess how frequently interactive FB is given after completion of a mini-CEX with PGY-1s in an outpatient setting. METHODS: Prospective cohort study of mini-CEXs performed with PGY-1s in the outpatient setting at three internal medicine residency programs: Yale University Primary Care, Washington Hospital Center, and the National Naval Medical Center. Feedback sessions were audiotaped after completion of the mini-CEX. All tapes were transcribed with all identifying information deleted. Transcripts were independently coded by two investigators (ESH, SH). Disagreements were resolved by consensus. Interactive FB was defined as: a) any recommendation(s) given to the PGY-1s; b) PGY-1s were asked to self-assess their mini-CEX performance (self-assessment); c) PGY-1s were given an opportunity to react to the FB (learner reaction); d) and faculty stated a specific plan to change PGY-1's future performance (action plan). RESULTS: For this preliminary analysis, 30 attendings provided FB sessions to 41 different PGY-1s at the 3 residency programs. Fifty sessions have been transcribed and analyzed. Forty-five (95%) FB sessions included at least one recommendation. The median number of recommendations per session was 2 (range 0-9). Recommendations were given most commonly for communication skills, notably patient interviewing (31 sessions, 62%) and counseling (21 sessions, 42%). Feedback on components of the physical exam was given in 13 sessions (26%). Attendings asked PGY-1s to self-assess performance in 19 sessions (38%) and to specifically respond to the FB (learner reaction) in 32 sessions (64%). Despite the high frequency of recommendations, only 3 sessions (6%) included an attending's discussion for an action plan to help the PGY-1 correct noted deficiencies. CONCLUSION: For this cohort, the mini-CEX effectively generated recommendations to improve clinical performance, especially in communication skills, and the majority of attendings asked PGY-1s to react to the FB. However, programs planning to use the mini-CEX should also consider strategies to encourage their faculty to provide more FB on other observed skills, promote more self-assessment, and establish definitive action plans to correct deficiencies.\nA.L. Hull 1 , H. Copeland 1 , M.G. Hewson 1 ; 1 Cleveland Clinic Foundation, Cleveland, OH PURPOSE: Clinicians' self-perceptions of teaching effectiveness will affect their interest and committment to improving teaching ability. This IRB-approved study investigates the relationships among self-perception and characteristics of clinical teachers including demographics, interest and satisfaction with teaching, and perceived institutional support for teaching. METHODS: Perceptions, attitudes, and characteristics of clinical teachers was obtained in a survey instrument sent to 164 Division of Medicine faculty who had taught and had teaching evaluation instrutments completed Internal Medicine residents in 1998 and 1999. 94 (57%) of the surveys were completed after 2 reminders. The responses were coded and matched to the respondents' average of a 15-item clinical teaching evaluation (which has been judged to be reliable and valid) completed by residents for any rotation during the 2-year period. The dependent measure was a 5-point Likert-scale survey item evaluating the self-perceived teaching effectiveness (TE). The independent measures included faculty characteristics (age, gender, years in practice), satisfaction with teaching (enjoy teaching, career satisfaction, perceived institutional support for teaching), percent time spent teaching, involvement in teaching improvement activities (workshops, reading), and average of self-ratings of the 15-item clinical teaching evaluation. SPSS/PC was used to compute descriptive statistics, correlations between the dependent and independent measures, and a step-wise regression. Self-ratings of the 15-item teaching evaluation were not included in the regression analysis in order to maximize the effects of the other independent measures. RESULTS: The average TE score was 3.85 (+À .63). Statistically significant positive correlations (p < .05) were found between TE and average resident evaluation score (r=.213), percent time teaching (r=.251), average self-rating of the 15-item teaching evaluation (r=.457), and satisfaction from teaching residents (r=.336). All other characteristics were not significantly correlated with TE.\nStep-wise regression only resulted in reported enjoyment of teaching residents predicting TE (R=.414 [difference in N causes difference between r and R]). CONCLUSION: Internal Medicine faculty believe that they effectively teach residents, and self-perceptions of teaching effectiveness are correlated with actual resident evaluations of teaching (suggesting accuracy of the faculty member's perception), and time spent and enjoyment from teaching (faculty self-select activities that they enjoy and are good at). Faculty self-preceptions in general are not correlated with demographic characteristics, effort at improving educational competence, and perceived support for teaching, suggesting that faculty development efforts may need to be more individualized and focused on faculty who elect to participate rather than institution or department-wide programs.\nRESIDENTS AS TEACHERS. R.W. Janicik 1 , M.D. Schwartz 1 , A. Kalet 1 , S. Zabar 1 ; 1 New York University School of Medicine, New York, New York PURPOSE: We sought to measure residents' attitudes, preparation, and confidence about teaching and how these relate to their plans to teach. METHODS: We conducted an anonymous, cross-sectional survey of Medicine and Psychiatry residents at NYU. The questionnaire assessed attitudes and preparedness to teach, selfperceived teaching knowledge and skills, and plans to teach. RESULTS: Sixty-one residents (42 Psychiatry, 19 Internal Medicine) completed the questionnaire (79% response). Factor analysis confirmed scales for measuring residents' attitudes (Teaching Enthusiasm, Important Role in Department), preparedness to teach (Teaching Exposure, Teaching Capability) and self-perceived teaching knowledge and skills (Clinical Reasoning, Patient Management, and Procedural Skills) . Scales were dichotomized to present the % of residents with high scores: Teaching Enthusiasm -67% agree; Important Role in Department -34% agree; Teaching Exposure -only 7% reported high amount; Teaching Capability -23% high confidence; Knowledge of Clinical Reasoning -30% confident; Patient Management -49% confident; and Procedural Skills -21% confident. When asked the frequency with which they plan to teach, 75% planned to directly observe learners, 71% planned to give feedback, and 74% planned to teach with the patient present, all at least once per week. Plans to teach was not associated with resident age, gender, year of training, or department. Nearly significant trends suggested that residents with high teaching enthusiasm were more likely to plan to observe their learners and those with high prior teaching exposure were more likely to plan to give feedback. Teaching capability and all 3 knowledge variables were correlated with plans to teach with the patient present (p < .05). CONCLUSION: Residents value and look forward to their teaching roles despite having little preparation for teaching, and only modest confidence in their ability and knowledge to teach. Resident attitudes and self-perceived capabilities influenced their plans to teach. Programs to prepare residents as teachers that address attitudes, skills, and knowledge in these areas may lead to more teaching by residents. were general internists, 24% family practitioners and 5%``other medical specialties.'' Forty percent of respondents received some form of spirituality training. Twenty-nine percent received this training while medical students compared to 18% as residents or as fellows and 23% as attending physicians. The training received in medical school was largely in the form of small group lecture series, but during residency and fellowship, the training consisted of informal clinic discussions. Attendings primarily received training through self-education (books and journals). Forty-two percent of respondents agreed that they would desire further spirituality training while 20% disagreed, and 38% were neutral. The physicians scoring highest (greater than 4 out of a possible 6 on the Spiritual Well-Being Scale) were more likely to desire further training. In addition, those physicians who attended worship service (48%) and those who agree to the statement``faith alone could cure disease'' (60%) were more likely to desire further training (all p-values < .05). Also family physicians had a greater tendency to desire further training compared to general internists. CONCLUSION: Formal spirituality training related to medical care is sparse in medical school and almost non-existent thereafter. Although physicians with stronger religious predilections desire formal training, nearly half of all primary care physicians who returned the survey felt that further training in spirituality and medicine was desirable. A curriculum concerning patient spirituality and medicine at the medical school and residency level appears to be warranted. PURPOSE: Chart audits are used to assess physician performance and quality of care. Typically, records are reviewed for practicing physicians. Only recently have resident report cards been described. The purpose of this study was to compare performances of interns, senior residents, and faculty on adherence to national guidelines regarding prevention and management of common outpatient medical conditions. METHODS: The study was conducted at the outpatient practices of a university based internal medicine training program. For interns (n = 50), rosters of all new general medicine patients with 2 or more visits between July 1999 and April 2000 were generated. For senior residents (n = 39) and faculty (n = 30), the time frame extended from July 1998 to April 2000. A random sample of 9 patients per physician was identified. All visits with the designated provider through June 2000 were abstracted by nurse abstractors using a 115 item form. 91% (n= 965) of targeted charts were abstracted: 366 for interns, 349 for senior residents and 246 for faculty. Performance scores (the percent of indicated actions taken) were determined for each chart in the areas of cancer screening, immunizations, counseling, management of diabetes (DM), hypertension (HTN), asthma, coronary disease (CAD), and depression. Differences in intern, resident and faculty performance were assessed with ANCOVA and correlations. RESULTS: The mean patient age was 52.9 (sd=17.6); 68% were women. Patients had a mean of 0.77 comorbidities and 4.1 visits with the provider. These factors were covariates in the ANCOVA due to group differences. Faculty performed better than interns and senior residents overall (p = .03) and in the areas of counseling (p = .0007), immunizations (p = .003) and HTN (p = .002). Unadjusted mean performance scores for interns, senior residents and faculty were as follows: overall (46%,49%,52%), counseling (26%,27%,37%), immunizations (36%,37%,43%), and HTN (44%,50%,57%). There were no differences between intern, resident and faculty performance in the other areas. Mean performances were: cancer screening (66%), management of DM (48%), asthma (56%), CAD (67%), and depression (41%). Higher performance scores were associated with more visits (r = .19, p=.0001), fewer comorbidities (r = À.08, p = .02) and younger age (r = À.12, p = .0002). CONCLUSION: On only a few domains did performance improve with experience. In those domains, there was steady improvement through training. Average performance rates fell short of national guidelines for all groups, though some actions were likely not documented. Future efforts should explore how to improve adherence in training programs, including among the teaching faculty.\nIMPACT OF A NIGHT FLOAT SYSTEM ON HOUSESTAFF SATISFACTION. S. Kripalani 1 , M.V. Williams 1 ; 1 Emory University, Atlanta, GA PURPOSE: In an effort to reduce workload, a night float (NF) rotation was established in July 1999. Three of twelve ward teams were on call each day and stopped admitting patients at 2300. They remained overnight to complete work-ups and cover their own service. From 2300 until 0700, NF teams performed all medical admissions and covered the other 9 teams. Two or three NF teams (consisting of an intern and resident) worked each night. In the morning, they handed-off the new patients to ward teams and attended a teaching conference before going home. We conducted this study to evaluate the impact of the NF system on satisfaction and quality of care. METHODS: Ward residents (WR) and night float staff (NFS) completed a 2-page questionnaire at the end of each month from July 1999 to April 2000. Survey questions focused on the following domains: workload, educational activities, morale, and quality and continuity of care. RESULTS: The overall response rate was 81% (n=92 WR and 51 NFS). WR got an average of 4.5 hours of sleep on call, compared with 2.4 hours the previous year, p < .0001. Nearly all (89%) felt more rested post-call, and 71% thought they could take better care of patients during the day because of the NF system. According to 87% of WR, the workload was significantly better, and 63% had more time for educational activities. Approximately 90% agreed that NF had improved housestaff morale and the overall experience of the ward rotation. Regarding the NF admissions, most WR appeared satisfied with the quality of history and physicals (63%), choice of diagnostic testing (72%), and initial treatment (71%). However, many WR disagreed with the assessment and changed the treatment plan about 25% of the time. One-third expressed some concern that continuity may suffer when patients are handed-off in the morning, but only 2% felt that overall patient care suffered, and 94% were glad that the NF system was in place. Each NF team received a mean of 3.9 admissions per night (range 2-11) and a median of 4.8 crosscover calls, which required about 45 minutes to answer. Nearly 95% of NFS agreed that the overall workload and number of admissions were reasonable. However, only 66% were satisfied with the amount of learning/teaching on the NF rotation. Working nights for a month was difficult for 67% of NFS, and 80% felt it affected their social life. Daytime sleep was often unrestful, afternoon continuity clinics disrupted sleeping schedules, and NFS had limited time with friends and family. Nevertheless, 90% agreed that the month was a positive experience overall, and 96% were glad that the system was in place. CONCLUSION: Residents on the ward teams reported improved morale, increased sleep, more time to read and attend conferences, and better quality of care delivery. In spite of complaints about the nocturnal lifestyle and educational content of the rotation, housestaff on the NF teams favored the system as well. We implemented a clinic-based coaching model using rating forms and observed interviews. The purpose of this study is to describe the use and modification of the rating scales for coaching purposes, and to discuss the variations among specific communication skills by resident year and gender. METHODS: The Calgary-Cambridge Referenced Observation Guides were selected as a teaching tool because of their specificity and ease of use during observed interviews. To provide skill discrimination for each rating item, a 5 point Likert scale was created for each item ranging from poor to excellent performance. After obtaining patient consent, resident physicians were shadowed by the residency behavioral scientist (BRL) for one clinic afternoon or morning, and their skills were rated using the instrument. 25 resident physicians were observed for a total of 48 interviews. Likert scores were dichotomized into excellent (5) and less than excellent (1) (2) (3) (4) categories for each item; and mean scores generated for the following domains: Initiating the session (5 items); Exploration of problems (7 items); Understanding the patient's perspective (5 items); Structuring the consultation (4 items); Building the relationship (7 items); and Explaining and planning (5 items). General Linear Models procedures were used to compare mean differences in domain scores by gender and year of resident training (R1, R2, R3). RESULTS: No significant gender differences were found for any of the domains (p < .05).\nOverall, the resident physicians demonstrated the least proficiency with the domaiǹ`U nderstanding the patient's perspective'' (p = 0.01, mean domain score (MDS) = 1.07). When scores were analyzed by resident year, significant maturation effects were observed for:`E xploration of problems'' (p = 0.01, MDS: R1 = 2.6, R2 = 4.2, R3 = 4.8);``Understanding the patient's perspective'' (p = 0.01, MDS: R1 = 0.28, R2 = 1.23, R3 = 1.94); and``Explaining and planning'' (p = 0.02), MDS: R1 = 1.5, R2 = 2.1, R3 = 3.4). Resident evaluations of the coaching were uniformly positive. CONCLUSION: The development of physician-patient communication skills is an essential part of resident education. The modified Calgary-Cambridge guides provide data that identifies areas of general strengths and weaknesses in communication skills. Using this instrument, we saw maturation effects in domains that require facilitation of emotional expression and shared decision-making. These results can be used to focus educational efforts, and may be useful for evaluating the impact of interventions to improve physician-patient communication. METHODS: Using a 58-item clinical teaching evaluation instrument previously employed to determine the construct validity of the framework for inpatient clinical teachers, a split sample of 4702 evaluations completed by 550 students rating 450 teachers was evaluated using factor analytic and standard item-reduction techniques. Students completed evaluations for any university-or community-based outpatient teacher with whom a minimum of three half-day teaching encounters had occurred during a one-month internal medicine ambulatory block rotation. Results were replicated using the second half of the data. Generalizability analyses were used to quantitatively determine the number of evaluations needed to obtain a stable estimate of a teacher's performance using the reduced instrument. RESULTS: As in the construct validity testing with inpatient clinical teachers, thè`k nowledge'' items statistically combined with``self-directed learning'' items and``learning climate'' items held up as a discrete construct; however, items from``control of session'',`c ommunication of goals'',``understanding and retention'',``evaluation'', and``feedback'' clustered into one remaining``global'' teaching construct. These three factors explained 67 % of the variance. Following item reduction, the three constructs were compressed to contain three items each. The items remaining in the``global'' construct were all``communication of goals'' items. Overall internal consistency of the reduced instrument was .90 with internal consistency of constructs ranging from .90-.97. The overall inter-item correlation of the reduced instrument was .55 for sample one and .54 for sample two (optimal inter-item correlations are approx =.6). A minimum of 5 evaluations are needed per teacher to obtain a generalizability coefficient > .90 using conservative estimates from both samples. CONCLUSION: For students having limited contact with outpatient teachers, setting the climate, communicating goals, and stimulating students' future learning emerge as focal areas. Further study is needed to see if this three-category framework persists as viewed by trainees with more extensive contact with outpatient preceptors. PURPOSE: Professional isolation, poor financial remuneration, and insufficient training to meet patient needs decreases career satisfaction, making rural and inner city areas less desirable for primary care practice. Changes to Internal Medicine (IM) and Family Practice (FP) residency curriculum have attempted to address health care disparities by better preparing physicians for generalist careers. The effect of these curricular changes on career satisfaction has not been examined. METHODS: We surveyed all IM (n=128) and FP (n=74) physicians 1 to 10 years after graduating from a large Mid-west community teaching hospital. The survey assessed physician and practice demographics (i.e., practice type and location, community size, case mix), career satisfaction, resource availability, and adequacy of curricular content for practice. RESULTS: Survey response was 84% (105 IM, 65 FP, p=ns). Respondents were primarily male (66%), Caucasian (88%), and in solo (13%), group (42%), or multi-specialty group practice (20%). Thirty-one percent practiced in a community of < 50,000 residents (9% < 10,000) and 28% noted some or all subspecialties were unavailable in their community. Most respondents (88.5%) were somewhat to highly satisfied with their present career, and 81% would choose medicine again as a career (78% IM, 85% FP, p=ns); those practicing in emergency room settings were least satisfied with their present career (p = 0.04). Greater career satisfaction was related to meeting expected financial remuneration (p = 0.046), satisfaction with training in palliative care (p < 0.01), faculty role modeling of compassion (p < 0.05), and support/assistance provided by residency faculty (p = 0.001). Career satisfaction was not related to community size (p = 0.6), subspecialty availability (p = 0.7), computer/internet searching skills (p = 0.11), or time spent in ambulatory training (p = 0.3). Training better focused to practice demands in differential diagnosis, neurology, orthopedics, and hematology were associated with greater career satisfaction (all p < 0.05). Using logistic regression, controlling for physician demographics, greater career satisfaction was associated with meeting financial remuneration expectations (OR=3.9, p=0.01), and better training in differential diagnosis (OR=5.8, p=0.03), and literature appraisal (OR=3.0, p=0.04). CONCLUSION: Results suggest that at this institution, nationally mandated curricular reform in several areas is associated with greater career satisfaction. Satisfaction was also related to residency faculty role modeling and support. Whether additional curricular reform further improves career satisfaction warrants investigation. The AAMC has mandated the inclusion of end-of-life (EOL) and palliative care (PC) in undergraduate medical curricula. A significant number of medical schools teach about death and dying, yet focus predominantly on ethical aspects rather than the development of clinical skills in EOL and PC. We report here on the design and implementation of an EOL curriculum directed towards specific communication skills. This curriculum was developed and initiated by students at Stanford Medical School. METHODS: A needs assessment of advanced clinical students demonstrated little formal training in EOL communication skills, while a literature review surveyed current EOL curricula on communication. These efforts were used to design a case-based curriculum that underwent faculty review. Next, faculty with national or local experience with EOL and palliative care training contributed to case development. Twelve of the faculty attended an orientation/training session on teaching the curriculum and then taught it in pairs to six groups of 10-14 medical students per group. RESULTS: The four-hour curriculum described here was taught to students at the start of their clinical training. Topics entailed breaking bad news and discussing treatment options in the setting of life-limiting illness. In two successive sections, students (1) discussed and critiqued a physician modeling these areas [Weissman DE], (2) role-played a prepared case in dyads, (3) evaluated successful elements of communication skills, and (4) assessed their performance in the role-playing dyads. CONCLUSION: Medical students require skill-based EOL and PC curricula that emphasize clinical skills specific to their level of training. This module taught early clinical students communication skills for the setting of PC and EOL care. Further interventions will be necessary to advance these students' communication skills as they progress in their clinical training. We propose next to evaluate the effectiveness of this curriculum by comparing students' EOL communication skills with standardized patients before and after this curriculum. The longitudinal clinic experience at Creighton University consists of participating in the same clinic one-half day every other week of their 2nd year of medical school in a primary care setting (general internal medicine, family practice, or pediatrics). The 4th year medical students were surveyed regarding their 2nd year longitudinal clinic experience and their 1st choice for their future career. Nonparametric correlations (phi and point-biserial coefficients) were used to assess the associations between the student's actual choice of specialty to their self-reported effect of the longitudinal clinic on their specialty choice and the specific specialty (internal medicine, family practice, or pediatrics) of the longitudinal clinic. Hometown and MCAT scores were used as control variables. RESULTS: Of the 106 students participating in the longitudinal clinic experience, 42% reported it had a positive effect on the career choice in primary care (e.g. internal medicine, family practice, or pediatrics), while 53% reported it had no effect, and the remaining 5% stated it had a negative effect. There was no association between the student's self-reported effect of the clinical experience on their actual choice of specialty (phi=.248, p=.05). There was also no association between a student's choice of specialty and whether they were involved in an internal medicine, family practice, or pediatric longitudinal clinic (p > .05 for all three comparisons). CONCLUSION: The clinic setting had no effect on the students' career choice. Other factors (e.g. market factors such as surplus/shortage of primary care physicians or clinical experiences during their 3rd year) may also be influential in such decisions. To determine if self study with a CD-ROM auscultation tool would increase internal medicine residents' knowledge and skills in cardiac auscultation. METHODS: The interactive computer-based auscultation program was developed by two of the authors. It includes 20 multi-media cases that center on common cardiovascular conditions, including valvular abnormalities, congenital heart disease, coronary artery disease and congestive heart failure. At 2 institutions, a total of 127 residents participating in their 1-month primary care rotation were enrolled in this fixed-group controlled trial from April 1999 through June 2000. Residents were assigned to the intervention or control arm based on their clinic site. Those allocated to the intervention group (n=56) were provided two hours per week during their month to use the program. Control residents (n=71) were exposed only to the content of their rotation.\nAuscultation knowledge and skills were assessed by a computerized validated 40-item test administered before and after the rotation. Ten of these items tested the recognition and interpretation of heart sounds (skills component), and 30 tested facts about auscultation (knowledge component). Baseline and post-intervention scores were compared within groups using paired t-tests, and between groups with independent t-tests. When possible, residents who dropped out (n=10) were analyzed with their last outcome carried forward. RESULTS: Pretest and posttest scores are shown in Table 1 . At baseline, the two groups did not differ in demographic characteristics, pretest knowledge or total scores. The intervention arm had a lower mean pretest skills score than the control group. While both groups had improved scores after the rotation, residents in the intervention group had significantly higher improvements in skills, knowledge, and total scores than those not exposed to the CD-ROM based tool (* all p < 0.001). CONCLUSION: Protected time for internal medicine residents to pursue self-directed study with a multimedia auscultation tool during a primary care rotation enhanced both cardiac auscultation knowledge and skills. (1) self-reported use of information resources, (2) self-reported comfort with EBM skills, and (3) PURPOSE: Although thyroid examination is the least invasive and inexpensive method to detect thyroid pathology, most physicians lack the ability to detect thyroid abnormalities by examination. We developed a single and brief multimodality session to improve the thyroid examination skills of physicians-in-training and evaluated its efficacy in a controlled trial. METHODS: Thirty-nine first-year internal medicine residents were randomly allocated to participate in a multimodality learning session on thyroid physical examination or to receive no intervention (control group). The 50-minute learning session consisted of a slide presentation, a computer graphics animation, videotaped and hands-on demonstrations of the appropriate technique, participant examination of a patient with thyroid abnormalities and a normal volunteer under preceptor observation, and an evidence-based handout. All the participants were assessed in two stations of an objective structured clinical examination (OSCE) for appropriate thyroid examination technique (through a 2-way mirror) and identification of thyroid abnormalities (using a scored participant report of findings). The OSCE preceptors and results analysts were blinded to participant allocation. RESULTS: Of the 19 residents in the intervention group and 20 residents in the control group: 6 and 3 observed the neck for thyroid abnormalities (32% vs 15%; p = 0.3); 17 and 16 residents used proper hand position (90% vs 80%; p=0.7); 13 and 15 residents had the patient swallow while palpating the neck (68% vs 75%, p=0.7), respectively. There was a significant difference between the mean scores on thyroid physical findings between the intervention and control groups (100 vs 52.5, p=0.047) CONCLUSION: A single and brief multimodality learning session did not statistically improve the thyroid examination skills of first year Internal Medicine residents, but did positively affect their ability to detect abnormalities. Future research (with larger sample sizes) should test the effectiveness of iterative sessions or of increased emphasis on hands-on thyroid abnormality detection rather than appropriate technique. \nQuestions ( Table I ). CONCLUSION: Conclusions : Clinical data and physical exams are essential for ID. However complementary tests are important to determine FD. Among the tests, it's important to highlight the importance of CT scan , although we must re-evaluate others like Chest X-ray and Electrocardiograms which were taken to all patients but they were of poor value in patients without specific symptoms. We need to rationalize others like biochemistry tests and consults to others specialist. The biopsies and therapy tests were very useful. These results reflect what has happened with Medicine in the last two decades, in which the technological progress allowed a better diagnosis, although it was initially established by physicians at the patient's bedside. (sixth) year medical students while they were rotating through general medical outpatient clinic for 2 weeks. Faculty internists evaluated student's clinical competence observing student's ability to assemble clinical information gathered through medical history taking and physical examination and to formulate working diagnosis. Students were rated as follows; A, able to integrate pertinent information for initial assessment, B, able to gather pertinent information, C, information gathering is passive and superficial, and D, detrimental to patient. A, B and C were further divided into three subgroups according to individual performances to develop a 10 point Competence Score. Patient satisfaction ratings for students were also obtained at the same time by using a modified Japanese version of Patient Satisfaction Questionnaire developed by the American Board of Internal Medicine (m-PSQ). Multiple regression analysis was used for statistical comparison. RESULTS: A total of 77 medical students were evaluated by 9 staff physicians and by 229 patients. The m-PSQ consisted of single factor with Cronbach's a coefficient of 0.90. Multiple regression test revealed that competence scores of students were strongly associated with the m-PSQ ratings (p < 0.001), but not with student's age and gender, Objective Structured Clinical Examination (OSCE) scores, patients' demographic characteristics such as age, sex, occupation status. CONCLUSION: Patient satisfaction ratings of medical students in outpatient clinics were strongly associated with student's ability to gather and integrate clinical information obtained through history taking and physical examination in a university hospital outpatient clinic. The use of Palm Pilots is rapidly expanding among residents and some training programs are even providing them for their housestaff. However, little is known about how residents view their utility. The purpose of this study is to determine 1) residents' perception of the utility of Palm, 2) potential obstacles toward their use, and 3) the influence of providing Palms to medical residents. METHODS: We conducted a survey of all medicine residents at two hospitals. One of the hospitals heavily subsidized and encouraged residents' purchase of Palms. The survey included questions regarding perceptions toward the benefits of using Palms to help them record patient data (record) and improve the quality of their signouts (signout). We also surveyed for potential obstacles to their use: cost, difficulty in learning how to use (learning), fragility and effort required to enter data (enter). A Likert scale (1=strongly disagree to 5=strongly agree) was used. The survey was piloted for reliability at a third hospital. RESULTS: 65 of 81 residents (80 %) in the hospital that did not subsidize the purchase of Palms (unsubsidized group) and 25 of 29 (86%) in the hospital that subsidized their purchase (subsidized group) returned the survey. 21 residents in the unsubsidized group (32%) and 24 residents in the subsidized group (96%) owned a Palm. The unsubsidized group rated questions on the utility to record patient data and signout higher than the subsidized group (table 1) . Regarding potential obstacles the unsubsidized group felt that cost was a greater obstacle while the subsidized group felt that the fragility of the device and the effort to enter data was a greater obstacle than the control group. Analysis by ownership of a Palm independent of the site also failed to reveal any significant results with exception to cost and learning (less of an obstacle to Palm users). CONCLUSION: Our study demonstrates that in a training program that subsidized the purchase of Palms, residents perceived them to be less useful and felt that the fragility of the device and the effort to enter data were greater obstacles to their use. These results imply that simply providing housestaff with Palms may not be beneficial. Studies are needed to see if providing medical software and additional training will increase their utility and decrease perceived obstacles. inquired about the most common uses in patient care as well as the use of any medical programs. Information on training level and whether they owned a Palm Pilot was also obtained. The survey was piloted for test ± retest reliability at a third hospital. Mann ± Whitney U tests were performed to compare groups and logistic regression was performed to adjust for resident genre, level of training, and program site. RESULTS: The survey was sent to 81 residents in the hospital that did not subsidize the purchase of Palm (unsubsidized group) and 29 residents in the hospital that subsidized their purchase (subsidized group). 65 residents (80 %) in the unsubsidized group and 25 (86%) in the subsidized group responded to the survey. 21 residents in the unsubsidized group (32%) and 24 residents in the subsidized group (96%) owned a Palm device. Palm owners in both groups responded that they used these devices to organize patient records (23%) and phone numbers used in patient care (82%), keep patient``to do'' lists (39%), schedule events and appointments (75%), and as an aid for clinical calculations (86%). The most frequently used programs were pharmacopoeias (93%), medical reference (44%), and clinical calculators (18%). Ownership of Palm and training sites were not independent predictors of using clinical prediction rules more frequently on logistic regression. CONCLUSION: Our study demonstrates that medical residents use Palms for a variety of purposes relating to patient care. However, owning Palm Pilots did not increase the likelihood of using clinical prediction rules and very few residents used them to organize patient records.\nFrom an educational perspective, at this point the available evidence does not support providing Palms to medical residents. Further research is still needed to determine if the use of Palm Pilots will result in better patient outcomes or resident efficiency. , 4) Menopause, Osteoporosis and Hormone Replacement, 5) Psychosocial Issues, 6) Resident Presentations and Posttest. Also included in the curriculum is a three-hour visit to the local domestic violence prevention center. All necessary reading materials are handed out during the first session after the pretest. The didactic sessions start with a brief case presentation by the participating resident(s); this is followed by an in-depth discussion of the topics of the day with expected resident participation. Each session except the introductory and resident presentation session ends with role-plays where the instructor plays the role of the patient and the resident the doctor. This enables the resident(s) to put into immediate practice what they have just learned. Clinical practice of women's health is expected to occur when residents see female patients in their primary care practices. To expand the scope of this curriculum beyond these limited topics both residents and faculty have been encouraged to incorporate into everyday teaching and learning how various conditions may differ or are unique in women. Participating residents are expected to make a formal thirty to forty-five minute presentation on a topic in women's health not covered in this curriculum. Learners are evaluated by the completion of a standard evaluation form used in the residency program. Evaluations are based on the residents' performance on the posttest, participation during the didactic sessions and their presentations. Learners evaluate the curriculum by completing a survey. RESULTS: The curriculum was well received by all residents. All residents surveyed either agreed or strongly agreed that they would now be able to evaluate, manage or appropriately refer female patients as a result of knowledge gained during the month. The mean posttest score was 81%, an improvement over the mean pretest score of 66%. Residents stated particularly that role-plays helped improve their understanding of the topics. A survey of these residents in their third year regarding their perceptions on the women's health education they received in this program and how well they think it has prepared them for their future practices year is planned. CONCLUSION: The curriculum improved the knowledge of residents in selected women's health topics. Using the same curricular model, topics can be adapted to meet the learning needs of residents in other residency programs. (COPD)). Each physician subject saw a simple and a complex case for each disease. Fifteen physicians were randomly selected at each site, yielding a total of 120 visits. From these, we randomly selected 10 percent of these visits to audio record the SP physician interaction yielding 1 recording for each of the 12 SPs. To make the visits as realistic as possible, physicians consented not to be informed if a patient was an SP or if the visit was being recorded. SPs rated clinical care provided by physicians using closed-ended checklists that contained explicit quality criteria developed from national guidelines. Transcribed recordings were scored identically using the same checklists to determine if SP reports were valid. We calculated the percentage of criteria where SP checklists and recordings were in agreement and disagreement. McNemar's chi-square test for paired proportions was used to determine the statistical significance of differences between the two methods. RESULTS: The overall rate of agreement between SPs and recordings was 90.3 percent (p = 0.00). The rate of agreement ranged from 86.9 percent (p < 0.01) for the complex depression case to 95.3 percent (p = 0.375) for the simple COPD case. Eighty-five percent of the disagreements between SPs and recordings occurred when SPs recorded that physicians performed actions that were not verified by the recording. CONCLUSION: The high rate of agreement between SPs and recordings indicates that SPs are valid measures of the quality of outpatient care. The higher rate of agreement in the COPD cases as compared to depression may be related to two factors: 1) the larger number of items in the depression checklists, and 2) the subtlety of checklist items in the history section of these cases. The preponderance of overrecording vs. underrecording suggests that SPs may tend to give physicians the benefit of the doubt in the presence of ambiguity. Unannounced SPs are attractive quality measurement tools in the outpatient setting because they are inherently adjusted for case mix. These prospectively obtained results indicate that SP reports are valid measures of the quality of care, and may be useful for comparative evaluation of physician training across health care systems. Little is known about changes in end-of-life care attitudes among medical and pharmacy students following experiences working with patients at the end of life. METHODS: 8 medical or pre-medical students and 19 pharmacy students served as volunteer \"patient advocates\" (n = 27), providing social support in the form of weekly phone calls and monthly visits to patients with end-stage congestive heart failure, chronic obstructive pulmonary disease, and cancer. Students worked in pairs with an individual patient for a 4-month period. In addition, students participated in a 10-hour series of physician-led discussions about end-stage illness, the patient and family experiences of illness, and the emotional and spiritual dimensions of end-of-life care. Using a confidential, self-administered questionnaire with 10-point Likert responses (1 =very negative; 10 = very positive), student attitudes about the end of life were assessed before and following their work as advocates and participation in the class. The mean values among all respondents pre-and post-training were compared and analyzed using the two-tailed t-test. RESULTS: The response rate was 70% (n = 19). Students felt that the quality of their experiences with patients at the end of life was more positive after the course than before (mean = 7.0 post vs. 5.7 pre; p = 0.013). Students also felt more comfortable working with patients at the end of life (mean = 7.3 post vs. 6.4 pre; p = 0.045). Additionally, following their experiences, students estimated the average quality of life for patients at the end of life as higher (mean = 6.3 post vs. 4.7 pre; p = 0.028). Following their experiences, student belief in the possibility for growth at the end of life did not change significantly (mean = 8.7 post vs 8.9 pre; p = 0.48). Following their experience, students' rank of the professional importance of working with patients at the end-of-life remained high (mean = 9.0 post vs 9.6 pre; p = 0.077). CONCLUSION: Direct contact with patients at the end of life and formal physician-led discussions about end-of-life care led to improved quality in students' experiences with people at the end of life, as well as increased comfort working with patients at the end of life. While endof-life care experiences led students to estimate that patients' quality of life was higher than initially expected, following their work, students were no more likely to believe that growth at the end of life was possible. The influence of early end-of-life care experiences on preprofessional students is generally positive, but should be explored further as students receive increased training in end-of-life care. PURPOSE: Mentorship has been associated with greater career satisfaction among academic physicians, but has not been studied during residency training. While female physicians may benefit more from mentorship, little is known about mentorship among female residents. We compared the prevalence and characteristics of mentorship between male and female residents in medicine. METHODS: We developed a questionnaire to collect data on attitudes regarding mentorship, satisfaction with mentorship, and satisfaction with residency education. After pilot testing the questionnaire, we mailed it to residents in the five internal medicine residency programs affiliated with Harvard Medical School. We sent two additional mailings to nonrespondents. We compared responses between men and women, and then used logistic regression to identify factors significantly associated with having a mentor and being satisfied with mentorship or career guidance. RESULTS: Among 329 respondents (response rate 65%), 47% were women. Of the women, 57% were white, 29% Asian and 9% underrepresented minorities. Although 50% of both men and women had a current mentor, and both men and women were equally likely to report satisfaction with mentorship (46% vs 38%, p=0.16), women were more likely to report that having a mentor was important during residency training (96% vs 90%, p=0.04). While women were more likely than men to want a mentor of the same gender (35% vs 8%, p < 0.01) and were more likely to have a female mentor (52% vs 17%, p < 0.01), having a female mentor was not associated with satisfaction with mentorship. In addition, regardless of whether they had a preference for a female mentor, women were equally likely to have a mentor and to be satisfied with mentorship. In multivariable models adjusting for ethnicity, year of training, being assigned a mentor and history of mentorship, women and men were equally likely to have a mentor and to be satisfied with mentorship. However, after adjusting for ethnicity, year of training and having a mentor, women were more likely than men to report receiving inadequate guidance with career decisions [adjusted odds ratio 1.8 (95% confidence interval 1.1, 2.8)] and were less likely than men to think that their residency was doing the best possible job preparing them for a future career [0.6 (0.4, 0.9) ]. CONCLUSION: Although women in residency training developed mentoring relationships as often as men and were equally satisfied with mentorship, they were significantly more likely to judge guidance with career decisions as inadequate, and were less likely than men to believe that their residency was doing the best possible job preparing them for a future career. PURPOSE: Since mentorship has been associated with greater career satisfaction among academic physicians, some residency training programs assign mentors to all house officers. We compared characteristics of mentorship between residents who were assigned a mentor by their training program, and those who were not assigned a mentor. METHODS: We developed a questionnaire to collect data on attitudes regarding mentorship, satisfaction with mentorship, and satisfaction with residency education. After pilot testing the questionnaire, we mailed it to residents in the five internal medicine residency programs affiliated with Harvard Medical School. We sent two additional mailings to nonrespondents. Using logistic regression, we identified factors associated with three different outcomes: having a mentor, being satisfied with mentorship, and being satisfied with career preparation. RESULTS: Among the 329 respondents (response rate 65%), 34% were in residency programs that assigned mentors (assigned), and 66% were in programs that did not assign mentors (nonassigned). Most (87%) residents in programs that assigned mentors knew that they were assigned a mentor. Residents in assigned programs were significantly more likely to identify a mentor than those in nonassigned programs (82% vs 43%, p < 0.01). Residents with mentors in assigned programs were less likely than residents with mentors in nonassigned programs to report their mentor was helpful with professional development (64% vs 77%, p=0.05) or personal development (45% vs 69%, p < 0.01). However, residents with mentors in assigned programs were more likely to be satisfied with mentorship than residents with mentors in nonassigned programs (53% vs 36%, p < 0.01). In multivariable models adjusting for sex, ethnicity, year of training and history of mentorship, residents in assigned programs were more likely to identify a mentor [adjusted odds ratio 8.1 (95% confidence interval 4.3, 15.\n3)] and to be satisfied with mentorship [2.2 (1.4, 3. 6)] than residents in nonassigned programs. Regardless of whether residents were in a program that assigned mentors, multivariable analyses showed that residents who had a mentor during residency were more likely to think that their residency was doing the best job preparing them for their future career [2.0 (1.2, 3. 2)] compared with residents who did not have a mentor. CONCLUSION: Internal medicine residents who were mentored were significantly more likely to report excellent preparation for future careers. Residents who were assigned a mentor were more likely to identify a mentor and to be satisfied with the mentorship they received. These data suggest that residency training programs should assign mentors to house officers. PURPOSE: Identifying potential mentors is an important part of residency training. Morning report (MR) provides exposure to a variety of attending physicians. We studied resident and program characteristics that were associated with the ability to identify a potential mentor at MR. METHODS: We performed a cross-sectional survey of 356 internal medicine residents from a convenience sample of thirteen residency programs. The instrument included questions about demographic characteristics, subspecialty fellowship plans and ability to identify a potential mentor in MR during the previous six months. RESULTS: The response rate was 63% (38% female). Women were significantly more likely to have plans to go into general internal medicine (GIM) (43% vs.58%, p < 0.001). Overall, 73% of residents were able to identify a potential mentor at morning report within the previous six months. Women were significantly less likely than men to be able to identify a mentor (63% vs. 79%, p < 0.001). Stratification by fellowship plans revealed that female residents not planning on subspecialty training were significantly less likely than male to be able to identify a potential mentor ( Figure) . Residents from programs with > 50% generalists at MR were less likely than those from programs with more specialists at MR to identify a potential mentor (57% vs 81%, p < 0.001).\nCONCLUSION: Future generalists may face barriers to appropriate mentorship. Even though female residents were more likely to go into GIM, those who chose to do so were less likely to find a potential mentor at MR. Residents from programs with a majority of specialist attendings at MR were more likely to be able to identify a potential mentor, regardless of their career plans. completed a questionnaire at the end of each continuity clinic session which asked them to assess the number of patients they saw during that day's clinic using a 5 point Likert-type scale (1=too few, 3=just right, 5=too many). Chi-square analyses, Student's t-tests and linear regression modeling was used to evaluate the variables that contributed to optimal scheduling. RESULTS: Residents completed questionnaires for 73% of their clinic sessions (N=105). Overall, they saw an average of 4.5 (SD=1.6) patients per session. 75% of the time, patient volumes below 3 patients were considered too low and volumes above 6 patients too high. During sessions where they rated patient volume as \"just right\" interns saw an average of 3.7 (SD=1.2) patients, second residents saw 4.6 (SD=0.7) patients and third year residents saw 5.3(SD=1.2). Linear regression modeling adjusted for resident track, gender and study period, confirmed that second and third year residents saw more patients than interns, 0.7* and 1.5* patients, respectively; that contact with less than 1.2* patients per session below average constituted``too few'' patients; and that contact with more than 1.8* patients above average constituted``too many''. (*p-values < 0.0001) CONCLUSION: Residents at all levels of training were comfortable seeing the minimum volume of patients recommended by the Internal Medicine Residency Review Committee (1 new and 3 follow-up patients). Patient volumes below 3 were too low and volumes above 6 were too high. In between these apparent limits, ideal volume depended on year of training, increasing by about 1 patient per session per year. While information from the medical education literature suggests that some aspects of the clinic educational program may suffer when patient volumes exceed 4 per session, we found that residents at more advanced levels of training preferred patient volumes higher than this. While one explanation for this observation is the higher skill level of the third year residents, it may also be that more advanced trainees derive greater educational benefit from direct patient care than from supplemental educational activities such as reviewing patient care with attendings. Important next steps will be to improve our understanding of the relationships between volume, type and complexity of patient visits, and the acquisition of expertise in specified aspects of ambulatory care, as well as to further delineate the relative educational value of the different clinic activities residents engage in. year. The aims of our study are to evaluate whether this clinical experience: 1) serves as a bridge between the basic science years and the clinical years, maintaining clinical skills and confidence, and 2) improves students' knowledge of Ambulatory Medicine. METHODS: At the end of the clinic year, the students took a standardized knowledge test and completed a validated questionnaire assessing their skills. MD/Ph.D. students who chose not to attend clinic and MD non-Ph.D. students also took the test and answered the questionnaire, serving as reference groups. To compare the groups, ANOVA was performed, followed by Tukey testing.\nRESULTS: The MD group had proportionally more women and started medical school 2 years later than either MD/Ph.D. group(p < .05). The MD/Ph.D. clinic students (n=20) were significantly more confident than the MD students (n=49) about their skills in every category of the questionnaire (history and general competencies, readiness to function as a doctor, physical exam, diagnosis and treatment) (p < . 05). They were more confident than the MD/Ph.D. nonclinic students (n=13) First-year students' perceptions of necessary professional skills focused upon behaviors relative to their current roles in their educational experience. The patient care related issues identified by students such as patient confidentiality and knowledge of limitations appeared to be appropriate for their level of training. Applying the ABIM format to the survey results shows that the majority of responses fall into the categories of excellence (dedication to learning), honor and integrity (honesty), and respect for others (respect). Presenting first-year students with a professionalism curriculum that is primarily focused upon patient-care related issues may not be as relevant as professionalism issues more closely related to their current level of training. Addressing issues such as dedication to learning, honesty, and respect may increase the relevance of professionalism to pre-clinical students. We are using this work to develop identify students' perceptions of stage-appropriate professional skills and behaviors to devise stage-appropriate educational interventions to teach professionalism. METHODS: Faculty members participated in a pre ± post study of a faculty development program consisting of three 90-minute interactive seminars teaching evaluation, feedback, and One-Minute Preceptor microskills. Audiotapes were collected of ambulatory teaching encounters with 3rd year medical students before and after the intervention. The audiotapes were transcribed and coded by individuals blind to the identity of the teachers and learners. Transcripts were coded using the Teacher Learner Interactive Assessment System, a qualitative tool designed to comprehensively code all utterances into mutually exclusive categories. Ten percent of audiotapes were double coded to assess inter-rater agreement. Written surveys after each encounter assessed learner, teacher, and patient satisfaction. Learner and teacher perceptions of the amount and quality of several aspects of the encounter, including feedback, were also evaluated. RESULTS: Nine teachers and 64 third year medical students participated, providing 45 encounters before and 48 encounters after the seminars. 17,859 utterances were coded. Coders achieved a high degree of agreement (Spearman's rho > 0.8). In the baseline encounters, 17% of teacher utterances were some form of feedback, predominantly (92%) minimal feedback statements such as \"right\" or \"I agree\". Only 8% of feedback utterances were specific and none were interactive. Most (91%) of the feedback was positive. After the faculty development workshops, the amount and quality of feedback increased; teachers were more likely to provide feedback (OR 1.21; 95% CI 1.07 ± 1.36) and that feedback was nearly twice as likely (OR 2.08; 95% CI 1.45 ± 2.99) to be specific. Subjectively, teachers reported a higher likelihood of allowing students to present their own plans (p = 0.004) and of providing specific feedback to students (p = 0.004) after the intervention. Students also reported greater ability to present their own plans (p = 0.02) and rated the encounters as more successful (p = 0.003 Results are presented as univariate descriptive statistics and using Pearsonchi ± square tests to assess the statistical significance of differences in proportions between groups on categorical variables. The level of significance for all tests was 0.05. All analyses were performed using SAS statistical software, version 6.11 (SAS Institute, Cary, NC, 1995) . RESULTS: A total of 55 of the 61 graduating third year residents were surveyed. Over their three years of training, residents reported caring for a median of two patients who died in outpatient primary care, and 10 patients who died during inpatient rotations. In addition, they reported caring for a median of only 3 patients who they felt might have had a life expectancy of 6 ± 12 months. They reported median``little/poor'' EOL teaching from faculty, and``little/ poor'' support from resident colleagues in their EOL clinical care. Only 6% rated oncology rotations as``relevant or helpful in learning about EOL care;'' only 10% rated intensive care rotations as helpful; and only 27% rated geriatrics rotations as educationally beneficial. CONCLUSION: These results suggest that residents are meaningfully and intensively involved in the care of remarkably few terminally ill patients, and when they are, these teaching opportunities are too often being squandered. They suggest that we must improve EOL education in acute inpatient settings, capitilizing on the opportunities to teach residents around the dying patients that they currently care for. They further suggest that we must increase residents' experience in EOL patient care in community settings, targeting primary care clinics, home care, nursing home and hospice services. PURPOSE: Although the ability to work with and interpret numbers is vital to patient care, little is known about the numeracy skills of health care professionals. To facilitate mastery of numeracy in medicine, we sought to (1) assess the numeracy skills of first-year medical students, and (2) determine how different risk presentations affect student ability to compare and calculate treatment benefits. METHODS: We surveyed 62 first-year medical students who were attending a seminar on risk communication. Students were asked to (1) answer three questions assessing their ability to handle numbers in a non-health setting (non-health numeracy; i.e. Imagine that we flip a fair coin 1000 times-what is your best guess about how many times the coin would come up heads?), (2) state which of two drug treatments for a hypothetical disease Y provided more benefit, and (3) calculate the effect of drug treatment for a patient with a given baseline risk of disease. Risk information was presented to each student in one of four randomly allocated risk formats-relative risk reduction (RRR), absolute risk reduction (ARR), number needed to treat (NNT), or a combination of all three of these risk presentation formats (COMBO). Students' abilities to compare and calculate drug treatment benefits were stratified according the number of correct answers they gave to the non-health numeracy questions: 0 or 1 correct=minimal numeracy, 2 correct=moderate numeracy, 3 correct=advanced numeracy. RESULTS: Fourteen (23%) students incorrectly answered one or more non-health numeracy questions. Lower non-health numeracy skills correlated with lower health numeracy skills. While more than 90% of students with advanced (n=48) or moderate (n=11) non-health numeracy skills correctly stated which of two treatments provided more benefit, only 33% of students with minimal (n=3) non-health numeracy skills correctly did so (p = 0.03). Similarly, 71% of students with advanced non-health numeracy skills, but only 36% with moderate and 0% with minimal non-health numeracy skills correctly calculated the effect of drug treatment on a given baseline risk of disease (p < 0.01). Regardless of the risk presentation format, students were able to correctly state which of two treatments provided more benefit (range 81 ± 93%). When asked to calculate the effect of drug treatment on a given baseline risk of disease, however, students had significantly more difficulty when risk was presented as NNT (25% answered correctly, compared to 75% for all other presentations; p=0.01). CONCLUSION: First-year medical students with lower non-health numeracy skills demonstrated less skill in estimating both relative and exact treatment benefits. For all students, information presented as NNT was interpreted less successfully than information presented in other risk formats. If these findings persist through training, educators may need to de-emphasize NNT relative to other formats that communicate the same information more successfully. Step 1 scores were available for 355 students (91%). Average total OSCE score was 59 6. Average Step 1 score was 234 17. Correlation coefficient for the total OSCE score with USMLE Step 1 score was 0.41 (p < 0.0001). In the linear regression model (R square = 0.52), 5 of the 16 station scores were significant (P < 0.05) predictors of USMLE Step 1 score, as were MCAT biological sciences and physical sciences sub-scores. The 5 predictive stations (alcoholism and abdominal exam, arthritis, calf pain, lung exam, and skin exam) were weighted toward identification of abnormalities (28% of all items on the 5 predictive stations, as compared with 17% of all 16 OSCE stations) and differential diagnosis (32% versus 19%). CONCLUSION: An OSCE in physical diagnosis taken by second-year medical students is correlated with scores on the USMLE Step 1 exam, with OSCE skills that foreshadow the clinical clerkships (identification of abnormality and development of differential diagnoses) most predictive of USMLE scores. This correlation suggests predictive validity of the OSCE and supports its use in identifying students who need remedial education. This OSCE provides an additional measure to predict students' success on subsequent national board exams. PURPOSE: Community-based experiences can help prepare medical students for addressing disparities in health care. Clerkship management and instruction can be difficult with multiple community-based teaching sites. To help address this challenge we have been developing Webbased components for our internal medicine clerkship. To date these components include forms evaluating the clerkship and individual preceptors, an interactive patient logbook with real-time faculty access to completed logs, clerkship calendar and, on a pilot basis, the ACP/ASIM clinical problem solving cases. This study addressed 1) if students were able to conveniently access these components from all of our six community-based campuses, each with multiple hospitals and clinics, 2) students' acceptance of these web-based components, 3) the efficiency gains of Webbased data collection and clerkship management. METHODS: We appended questions concerning the ease of accessing the Web-based components and students' preference for this format on the clerkship feedback form. Students completing the ACP/ASIM cases filled out a Web-based survey. Efficiency gains were based on comparison with the former paper-based evaluation system. RESULTS: To date we received feedback from 87 students who completed the third year basic and fourth year advanced internal medicine clerkship. 91% of the students indicated they had convenient access to the web-based components. 77% of the students preferred Web-based to paper evaluation forms while 60% preferred Web-based to paper logbooks. All eight students completing the ACP/ASIM cases found them useful or extremely useful and only one encountered any technical difficulty. CONCLUSION: Our students appeared to have little difficulty accessing the web-based components of the clerkship even though they were in a variety of community-based settings with no special provisions for Internet access. Students clearly preferred the Web-based to paper evaluation forms. While on the whole they preferred Web-based logbooks, they were less enthusiastic. The concerns they expressed to a large extent reflected design problems with the new system. Many of these have been resolved. The Web-based feedback forms and logbook dramatically streamlined the process of collating the data and producing feedback reports. These now take less than an hour to prepare and avoid the time consuming and error prone task of transcribing data from paper forms. Pilot results evaluating the use of the ACP/ASIM cases in the clerkship curriculum are encouraging and suggest these cases could provide a valuable adjunct learning experience. For the next academic year we plan to develop two Web-based instructional modules and a more comprehensive clerkship management system that provides additional real-time feedback to students and preceptors.\nCOMPARING CARDIAC AUSCULTATORY SKILLS USING REAL PATIENTS AND CD-ROM HEART SOUNDS. J. Solomonides 1 , D.S. Hatem 1 , M. Belanger 2 ; 1 University of Massachusetts Memorial Medical Center, Worcester, MA; 2 University of Massachusetts Medical School PURPOSE: Recognition of cardiac auscultatory events has been shown to be deficient. Whether the assessment method influences documented skills acquisition has not been determined. Our purpose was to compare second year medical students' abilities to recognize cardiac physical examination findings in patients with known murmurs with recognition of abnormal heart sounds produced by a CD-ROM. METHODS: Ninety-one second year medical students were evaluated on cardiac auscultation skills at the end of a Physical Diagnosis course. Students examined 1 Real Patient (RP) with a known murmur and listened to a CD-ROM (CD), to 2 murmurs and 2 extra sounds, then recorded their findings. Findings assessed included Heart Sounds (HS;5 items for RP and CD)and Murmur (M;5 items for RP, 4 items for CD) scores. RP murmurs included a systolic ejection murmur (SEM; n=44 students) and aortic stenosis with aortic insufficiency (AS/AI; n=47). Gold standard for the RPs was a recent exam by their cardiologist. The Criley CD-ROM was used to assess HS with abnormalities including S3 and S4 sounds (5 items; n=91), and murmurs with abnormalities including mitral regurgitation (4items; n=91) and aortic stenosis (4 items; n=91). Mean scores were compared using paired sample T-tests. RESULTS: Students' recognition of cardiac findings differed significantly with the two methods of evaluation. Overall mean scores for the CD-ROM cardiac findings (HS and M scores)were 77.5 while those for the RPs were 59.3 (p < .001). Mean HS scores were similar between groups (CD 81.7,RP 78.7; p=0.1) while M scores accounted for the differences in student performance for the two evaluation methods (CD 72.0, RP 37.7; p < .001). These mean score differences were similar when comparing single murmurs on CD-ROM to the RP with a single murmur (CD 68.5, RP 37.7; p < .001)and to the RP with two murmurs (CD 68.5, RP 38.1; p < .001). CONCLUSION: Medical students scored significantly higher when cardiac auscultation skills were assessed using a commercially produced CD-ROM compared to their skills in examining real patients. Differences may be due to examination technique, patient factors (body habitus), competing sounds (breath sounds), or patient complexity (2 murmurs v 1 murmur). The lack of difference in HS scores may be due to the small numbers of abnormal findings resulting in lack of discriminatory capacity as opposed to a true skills difference. The use of a CD-ROM alone to assess cardiac findings may overestimate student skills. This reinforces the need to focus our cardiac auscultatory skills teaching to enhance student examination skills acquisition. (3) increase the number of independent patient contacts by students in community teaching sites. Our current study looks at the impact of this mission based program. METHODS: We developed a survey evaluating six key components of an Active Learning Environment (ALE) (table 1) .Third year students rated their preceptors on each ALE component at the end of the 4-week internal medicine ambulatory clerkship using Likert-type scales to generate an ALE score (min.=6, max.=30). Faculty development programs were designed to improve preceptors' ALE teaching skills. Follow-up letters were sent to each participant reviewing program highlights and providing them with the ALE ratings they received from students for 6 months prior to their workshop. These were compared to preceptors' ratings received for 6 months after their faculty development participation. Student's t-test was used to compare differences in pre-and post-workshop mean survey results. RESULTS: To date 14 community preceptors have participated in three faculty development programs. Table 2 results show a trend towards higher post-workshop ALE scores. Their was a significant increase in the post-workshop independent patient contacts reported by students. CONCLUSION: A mission-based faculty development program can have a significant impact on the learning environment of community teaching sites in an ambulatory clerkship.\nComponents of an Active Learning Environment ( We hypothesized that residents could develop and implement a successful practice guideline as a way of gaining quality improvement experience. We studied their guideline development process and evaluated the effect of their guideline on patient care. METHODS: We asked a group of nine resident volunteers to develop a clinical guideline on a topic of their choice. The hospital funded the effort, extended after-hours availability to guideline-related diagnostic services and provided a nurse coordinator. We observed the process of guideline development, tracked guideline utilization, and measured physician satisfaction with it after six months of use. We also compared admission rate, length of stay and resource costs for a cohort of patients before and after guideline implementation. RESULTS: The residents selected acute chest pain evaluation and developed an evidence-based guideline. They focused on risk stratification and rapid diagnostic testing. They streamlined documentation by developing evaluation templates that substituted for written notes and orders. When applied to 319 patients 30 and older presenting with acute chest pain, 49% were classified as``possibly cardiac'' of which 41% were considered``high suspicion'' and 59%``low suspicion.'' An acute cardiac diagnosis was made in 12% overall (24% of possibly cardiac, 46% of high suspicion and 9% of low suspicion). This cohort of 319 patients was compared to 150 chest pain patients evaluated before the guideline was implemented. Median length of stay and cost of care both decreased by 34%. Admission rate was unchanged. Physician satisfaction with the guideline was uniformly positive six months after its implementation. Physicians rated 24-hour availability of a nurse ± coordinator and after-hours functional testing particularly highly. Guideline utilization remains high more than two years after its implementation. CONCLUSION: Resident physicians were the key participants in developing and implementing a successful clinical guideline that enjoys ongoing use. By experiencing the success of their own efforts, residents learned important lessons about quality improvement. Such experiences are an important addition to resident education and to the quality of care at academic health care centers. We asked students if they owned a computer with Web access, how many of their clinical sites had Web access, their level of comfort finding and using Web medical sources before and after the clerkship and the overall value of the site to their learning. A forward stepwise linear regression model was built to predict comfort and satisfaction in the use of the Web site as a learning resource. RESULTS: We surveyed 147 students (98% response rate) of whom 64% owned their own computer. On a scale of I to 5 (5= extremely valuable), 88% of students rated the value of the Web site > 3. While 76% were extremely or somewhat comfortable finding and using Web resources before the clerkship, 90% (p < .0001) reported the same at the end. The site was accessed on a weekly or greater basis by 58% of the students. Although 13% of the students experienced no clinical sites with Web access,61% had 1 or 2 sites with access and 26% had 3 or 4 sites with Web access. In a stepwise linear regression model, comfort in using Web medical resources at the end of the rotation was best explained by comfort in using the Web before the rotation, owning a computer and the number of clinical sites that had Web access (R2 =.48).\nOverall satisfaction with the Web site was explained by the time of year the clerkship was taken, owning a computer and the number of sites with Web access (R2 = .067). CONCLUSION: Students found value in an ambulatory clerkship Web site as an information resource. It could be made more useful to them by assuring better access to a computer at their clinical sites and providing a personal computer. While comfort in using a Web site as a resource improved by the end of the clerkship, this can been enhanced by training prior to the beginning of the clerkship and again assuring better computer access. Given the relatively low frequency of use however, it cannot yet suffice as the sole curricular resource. The curriculum consisted of 10 one-hour case-based seminars, including two devoted to pain management. We reviewed consecutive billing and pharmacy records of patients of medical residents (n=733) and a comparison group of patients of neurology and rehabilitative medicine residents (n=273) who received an opioid during two 8-month periods before (1/1/ 97 ± 4/30/97) and after (1/1/99 ± 4/30/99) the implementation of the curriculum. The data abstraction protocol was validated by standard chart review of a random subsample of 50 charts (concordance = 89.9%). Three outcomes were measured:1) % opioid orders for meperidine in non-gastrointestinal patients (excluded because of a longstanding belief that meperidine should be used in biliary disease);2) % opioid orders accompanied by a bowel regimen (to prevent constipation); and 3) % opioid orders accompanied by adjuvant nonsteroidal anti-inflammatory drugs (to achieve the``additive effect''). RESULTS: We had a power of 80% to detect a 10% difference at a= .05, two-tailed. Percent change before/ after for each outcome for each group was assessed by t-testing. Logistic regression models were developed for each of the outcome measures in order to control for independent variables that could be confounders and to assess the magnitude of effects. The models used controlled for age and race. Significance is noted by * if p < .05. CONCLUSION: These data suggest that a palliative care curriculum can improve the opioid prescribing practices of medical residents, over and above a secular trend among other house officers. Larger samples and further research are needed to understand these findings and how palliative care education can improve patient care. Delayed and incorrect diagnosis can lead to needless suffering, be potentially life threatening, and also be costly to society as physicians spend significant health care resources to work-up presenting symptoms. Because of increased public awareness of DV, and reports of underdetection by practicing physicians, many have sought to improve DV education at the medical student and residency levels. Have these educational interventions improved residents'\nabilities? In the current study, we examined the abilities of internal medicine residents to identify and care for a victim of domestic abuse. METHODS: Seventy-one internal medicine residents in four programs participated in a tenstation standardized patient-based Clinical Skills Assessment. One standardized patient (SP) portrayed a woman, complaining of headaches, who was a victim of domestic abuse. The SP assessed residents' elicitation of information and counseling interventions on a fifteen-item checklist. After the encounter, residents documented the patient's problems, etiologies, work-up and treatment recommendations. RESULTS: Forty (56%) residents correctly diagnosed domestic violence and discussed the diagnosis with the patient. Eighteen residents (25%) asked about immediate safety concerns. Twenty-three (32%) asked about concomitant child abuse. Forty-one (58%) did not refer the patient for DV counseling. Forty-eight (68%) made one or more incorrect recommendations. Some of these recommendations could be dangerous: recommending marital therapy (n=19, 27%), prescribing potentially addictive medicines (n=9, 12%), or suggesting that the patient bring her abusive partner in to the doctor (n=23, 32%). To work-up the patient's headache, 36 (52%) ordered unnecessary tests which would have cost $32,500. CONCLUSION: In our sample there were many deficiencies in the residents' abilities to diagnose and manage DV. The majority of residents provided inappropriate care that in some cases would be dangerous. In addition, the residents' ordering of unnecessary and costly tests is a drain to already-limited health care resources. There appears to be a need for more intensive training in DV at the undergraduate and residency levels. METHODS: Groups of 4 to 5 residents participate in 10 to 12 weekly 1-hour sessions during the rotation. After 2 didactic sessions to review basic biostatistics, clinical epidemiology, and other core EBM topics, the sessions follow a journal club format. One resident searches for, selects, and distributes an article relevant to the clinical question chosen for that session. They present a summary and initial evaluation of the article, which is followed by participatory discussion facilitated by the instructor. The Users' Guides to the Medical Literature series from JAMA is used to guide topic selection and as supplementary reading. Group discussions focus on the Users' Guides core concepts and evaluative criteria. The residents also do a more intensive final presentation in a similar format. Changes in EBM knowledge and skills were evaluated with a 38-item test given before and after the course, with blinding of participants and the instructor to the results. Participants also self-evaluated changes in their knowledge, skills, and practice on an anonymous evaluation form. . Pairwise comparisons revealed that the three categories within each teaching setting were all significantly different from one another (p < .04). Regression analysis revealed that time spent teaching in the IP setting was a significant negative predictor (p < .001) in the LC and CG ratings for IP teaching, while total years of teaching experience was a significant positive predictor (p < .001) in the SDL ratings for IP teaching. For the OP teaching ratings, time spent teaching in the OP setting was a significant negative predictor (p < .002) for CG and SDL, while LC ratings were unaffected by any of these variables. CONCLUSION: Our results suggest the need for focused faculty development programs to enhance CG skills-particularly for teachers who spend more clinical time in either the IP or OP setting, SDL skills for less experienced IP teachers and teachers who spend more clinical time in the OP settings, and LC skills for faculty with more inpatient clinical time. Internal Medicine residents at one urban, multi-center residency program were asked to complete a 71-item questionnaire. The survey was self-administered at conferences, on wards, and by e-mail. It was piloted on faculty, fellows, and chief residents for clarity of the questions and content validity. We solicited residents' observations of the impact of crosscultural issues on clinical care. Attitudes and self-perceived knowledge and skills were assessed with 6-point Likert scales. RESULTS: 71 of 133 residents (53%) responded. There were 35 PGY-1, 16 PGY-2, and 20 PGY-3. The mean age was 28 years, 52% were women, 60% white, 39% were fluent in a language other than English, and 49% had non-U.S. born parents. Residents reported experiencing a situation where a patient's care was negatively impacted due to sexual orientation (28%), gender (29%), ethnicity (38%), race (44%), socioeconomic status (56%), or a language barrier (90%). 76% of residents responded that cultural issues are``very''/``extremely'' important to patient care, but only 50% thought training in cross-cultural medicine could substantially improve health delivery. Residents viewed the following as major barriers to integrating cross-cultural issues in residency training: time constraints (60%), lack of experts (30%), lack of interest among learners (19%), lack of interest among faculty (16%), perception that these issues are already adequately addressed (12%), belief that these issues might offend some people (12%). Most residents rated themselves as being overall``somewhat'' tò`m oderately'' knowledgeable (69%) and skilled (81%) about cultural issues and medicine. A significant association was found between PGY level and perception of overall knowledge (p < 0.04%). Interestingly, 16 ± 18% of PGY-1 and 2 residents felt very knowledgeable about cultural issues and medicine, while no PGY-3 did. CONCLUSION: Successful development of curricula in cross-cultural medicine requires an understanding of trainees' attitudes and observations. Residents regard cultural issues as important to patient care and many report observing a direct impact. Despite diverse backgrounds, they do not consider themselves to be highly knowledgeable or skilled in this area. Based on these observations, a need for cross-cultural curricula exists. PURPOSE: Mentors are regarded as important for the academic success of junior faculty, but the availability and impact of mentors on the careers of junior faculty has not been well studied. The purpose of this study was to evaluate junior faculty perceptions of senior faculty mentors at one large academic medical center. METHODS: We surveyed all 154 physicians in the clinician ± educator (CE) and clinician ± scientist (CS) tracks from the Departments of Medicine, Surgery and Obstetrics/Gynecology who held the rank of Instructor or Assistant Professor at the University of Washington. We collected demographic information and asked respondents to describe their academic productivity and rate the adequacy of their mentors. RESULTS: Among 122 (79%) junior faculty who responded the mean age was 38.7 years (SD=4.3); 56 (46%) were women; 52 (43%) were in the CE track; and the mean years in rank was 2.5 (SD= 1.5). CEs reported authoring 2.6 (SD=3.7) book chapters and 4.5 (SD=8.1) original manuscripts whereas the means for CSs were 3.5 (SD=12.1) and 10.9 (SD=12.6), respectively. CEs and CSs had a mean of 1.1 (SD=1.8) and 3.1 (SD=2.6) funded grants, respectively. When asked if they had access to a senior faculty member in their department whom they trusted and who supported them in achieving their career goals, similar percentages of CEs and CSs answered affirmatively (75% and 74%, respectively). On the other hand, when asked if they had adequate mentoring, 36% of all respondents``strongly agreed'' or``agreed,'' (mentored faculty) versus 64% whom``neither agreed nor disagreed'',``disagreed'' or``strongly disagreed'' (nonmentored faculty). In bivariate analyses, mentored faculty were more likely to be men (44% vs. 27%, p=0.049), have completed fellowship training (41% vs. 21%, p=0.048) and to be CSs (51% vs. 15.4%, p < 0.0001). 93% of mentored respondents``agreed'' or``strongly agreed'' that they would achieve their short-term career goals vs. 65% of the non-mentored faculty (p = 0.0006). Gender (OR=3.6, p=0.02) and academic track (OR=9.9, p=0.0002) were independently associated with junior faculty perception of adequate mentoring in logistic regression adjusting for age, years at current rank, department affiliation and fellowship training. Mentored faculty reported more publications than non-mentored faculty. This difference did not reach statistical significance in linear regression (p = 0.1) adjusting for all independent variables entered in the logistic multivariate analysis. CONCLUSION: Over half of the junior faculty in this study did not feel that they were being adequately mentored. Women and CEs were more likely to perceive not being adequately mentored. Strategies to improve mentoring need to be developed and evaluated. Little is known about the quality of Pap smears obtained by internists. The presence of endocervical cells increases the quality of pap smears and has been previously evaluated in family physicians and ob-gyn. We examined the influence of gender, status (faculty vs. resident) and practice site (university vs. community) on rates of satisfactory smears as defined by the presence of endocervical cells. METHODS: Retrospective review of Pap smears evaluated by one cytology department over a 2 year period that were performed by internal medicine faculty and residents from 1 academic department at three clinic sites: two university-based clinics(one staffed by faculty and one staffed by residents) and one community-based clinic(staffed by both faculty and residents). Vaginal smears were excluded. RESULTS: 1086 smears were evaluated. 56% of smears were obtained at the faculty university clinic (FC), 18% at the resident university clinic (RC), 26% at the community clinic (CC). FC patients were older than RC or CC (mean age 56,40,42). 86 Internists were included: 65% males, 17% faculty (60% FC), 83% residents (52% CC). 75% of smears were satisfactory, 19% satisfactory but limited by no endocervical cells, 5.6% limited for other reasons, 0.18% unsatisfactory. Males and female internists had similar satisfactory rates (78% vs. 71%). Physicians at the university clinics had higher satisfactory rates (83%) compared to the community clinic (53%). University faculty had higher satisfactory rates than community faculty (83% vs. 43%). University residents had satisfactory rates similar to university faculty (83%) but higher rates than community residents (56%). Community residents had higher satisfactory rates than community faculty (56% vs.43%). Using the generalized estimating equations approach to control for volume of smears done by each physician we found that younger patient age (p = 0.02), faculty status (p = 0.03), and community clinic site (p = 0.0001) were all associated with lower satisfactory rates. The odds of a satisfactory reading were 1.13 for each additional decade of age, 2.04 for residents vs. faculty, and 6.22 for FC vs. CC, 3.49 for RC vs. CC, and 1.78 for FC vs. RC. CONCLUSION: Satisfactory rates among this sample of Internists were 11 ± 18% lower than published reports for family physicians and ob-gyn. Rates of satisfactory smears did not differ by gender. We did find higher satisfactory rates in older patients, residents and at university-based sites. These results suggest that academic institutions should place more emphasis on teaching Pap smear sampling technique to internists. METHODS: Mailed and web-based surveys were conducted of all 395 internal medicine residency programs regarding 45 procedural skills. We asked directors which methods of procedural skills training they used, which procedures residency graduates should master, and the amount of training needed to attain and maintain competency in each procedure. This paper reports analysis of responses of 269 (68%) program directors or their designees. RESULTS: As in the 1987 survey, the proportion of program directors who said all residents should master these procedures differed from the proportion who said that all residents do master them. For 21 of the 45 procedures in the survey, at least 30% more respondents said residents should master the procedure than said all their residents do master the procedure. This gap is larger in the new survey. For the 35 procedures that appeared in both surveys the new survey found 12% fewer reporting that all residents mastered those procedures. For example, although more than 90% of respondents in both surveys thought thoracentesis should be mastered by all residents, 79% in the 1987 survey vs.58% in the new survey thought it was mastered by all. In addition, estimates of the amount of training needed to master the procedures were lower in 31 of 35 procedures and were 30% lower overall. Some procedures were less strongly recommended, as might be expected from changes in practice or new regulations (e.g. bone marrow aspiration, gram stain). For 4 of 6 procedures required by both the ABIM and RRC, fewer than 60% of directors said that all of their graduates master them. CONCLUSION: Compared with 1987, similar numbers of IM program directors consider procedural skills' training necessary, but fewer think that their residents are mastering the skills. Program directors' estimates of the amount of training needed are 30% lower. Although residents appear to be getting less training in procedural skills, these skills remain important for internists to learn. Given the implications for patient care, this apparent mismatch between expectations and actual practice requires careful review and intervention by medical educators and policy makers alike. PURPOSE: Managed care curricula are being developed and implemented in graduate health professions education programs throughout the country. The purpose of our study was to measure the attitudes of resident physicians in three departments at a large academic medical center during 1999 and 2000. Specifically, we wished to compare the attitudes of incoming interns and graduating seniors, since differences between these groups could indicate effects of graduate medical education on physicians' attitudes towards managed care. METHODS: A 35-item questionnaire was administered to all first-and third-year residents in Family Medicine, Internal Medicine, and Pediatrics at a large midwest academic medical center through two cross-sectional surveys in 1999 and 2000 (n=320). Topics included the effects of managed care on physician autonomy, costs, and quality of care (7 items); practice guidelines (3 items); capitation (3 items); Medicaid managed care (4 items); factors necessary for success under managed care (7 items); and expected practice payor mix (4 items), geographic setting (6 items), and practice type (1 item). All items were 4-or 5-point Likerttype scales. RESULTS: Over 60% of residents agreed that managed care has restrictive effects on physician autonomy, compensation, referrals, and test ordering. Residents were relatively neutral regarding managed care's effects on quality and costs of care. Residents recognized ( > 60% of residents rated as important) several``non-traditional'' areas as important for success under managed care, including patient outcomes, assessing the needs of populations, and team building. First-and third-year residents' attitudes towards managed care were largely similar, though first-year residents were more positive about managed care's effectiveness in controlling costs (64% vs. 46% agreed, p < .04), and more likely to expect to care for underserved (79% vs. 66%, p < .005) and Medicaid patients (98% vs. 88%, p < .04). CONCLUSION: Resident physicians are concerned about managed care's threat to professional autonomy, are relatively neutral regarding other aspects of managed care such as quality of care or cost control. Differences between first-and third-year residents were consistent with greater idealism among interns than seniors. Residency curricula should directly address issues of autonomy under managed care and provide accurate information on the effects of managed care on quality and costs. Recognition of the importance of several``new'' physician competencies suggests a positive effect of recent changes in medical school and residency curricula. Future research should be directed towards whether differences in idealism between interns and seniors are due to the effects of training, or secular trends in attitudes towards managed care and practice intentions, or both. This questionnaire was sent to all US medical schools in May and the results tabulated at the national CDIM office. Questions asked 1) whether formalized courses on student professionalism existed at the school, 2) whether white coat ceremonies were held, 3) what percentage of third year students had been counseled by the CD concerning professionalism issues, 4) what percentage of students received unsatisfactory clerkship grades because of these deficiencies in professionalism and 5) whether separate forms or mechanisms existed to document and deal specifically with issues of misconduct. RESULTS: 74% (92 of 124) of US medical schools completed the survey. 25% of respondents stated that a formalized course for professionalism existed at their institution. 84% of schools hold a white coat ceremony or similar event. During the past year CDs counseled almost 4% (range 0 ± 15%) of third year students and 1% of all students received an unsatisfactory clerkship grade because of these deficiencies. Only 32% of medical schools reported a separate form or mechanism to document breeches of professionalism. CONCLUSION: The results of this national survey of Internal Medicine CDs confirm the increasing interest in professionalism in medical education. The vast majority of institutions have established white coat ceremonies to foster the values of the profession. CDs are counseling students for unprofessional behavior and for a small minority this has significantly impacted the grade they received. However, formalized courses and effective policies to evaluate and document student misconduct still need broader implementation. This study sought to examine and understand how highly respected physician role models think about role modeling. METHODS: In-depth semi-structured 30-minute interviews were conducted by the primary investigator in the offices of 29 of the 30 most highly regarded role models, as judged by the house officers, at two large teaching hospitals in Baltimore. Interview transcripts were independently coded by two readers and compared for agreement. Content analysis identified several major categories of themes, which were examined and conceptually organized. RESULTS: The analyses revealed that role models have identifiable characteristics. Subcategories under the domain of personal qualities include a commitment to excellence and seeking continual improvement, integrity, a positive outlook, leadership, and being interpersonally skilled. Under the domain of teaching, the subcategories were establishing rapport with learners, being committed to their growth, and developing specific teaching philosophies and approaches. Subjects thought there was some overlap between teaching and role modeling, but role modeling was felt to be more implicit, more intimate, and more encompassing. In the clinical domain, a recurrent theme was that being a strong clinician was necessary but not sufficient for being considered a role model for medical learners. The informants identified barriers to effective role modeling and they included being over-extended, being passive, and being impatient, inflexible and overly opinionated. Although any given role model might embody a range of talents, subjects believed it was valuable for each medical learner to have multiple role models. CONCLUSION: Highly regarded role models shared their opinions about the critical components of role modeling in medicine and the barriers that can make it difficult. The identification of personal qualities and features of teaching and clinical work associated with effective role modeling by physicians advances our understanding in this area. PURPOSE: Due to the dwindling numbers of generalists available to teach interviewing and physical exam skills to small groups of medical students in the first and second year, all clinical departments at the University of Wisconsin Medical School agreed to supply faculty for small group teaching in the Patient, Doctor, and Society Course (PDS.) Much concern remained, however, as to whether specialist and generalists could teach pre-clinical students with equal effectiveness. METHODS: Specialists and generalists were randomly assigned to the third semester of PDS to teach groups of four students. All leaders were provided with the same verbal and written orientation materials. The specialists from nine clinical specialties were assigned to lead 18 groups of students and the generalists from general internal medicine and family medicine were assigned to lead 16 groups. To determine from the students' point of view, whether specialists and generalists teach with equal effectiveness, students were asked to evaluate their small group leaders on a seven point Likert scale on nine items that included: enthusiasm for teaching; fosters discussion; prepared/ knowledgeable; availability; constructive feedback; timely return of work; good role model; treats students with respect; and an overall rating of the group leader. Scores for leaders were compared using a chi square test. In addition, a questionnaire was distributed to the leaders to determine whether specialists and generalists were equally confident in teaching basic history and examination skills. The questionnaire asked leaders to evaluate their confidence in their ability to teach the abdomen, neurology, cardiovascular, pulmonary exams, and in providing feedback to students in the complete history and physical exam skills. Confidence ratings for leaders were compared using a chi square test.\nTo determine if there might be differences in specialist and generalists' teaching effectiveness, student scores on an objective, structured, clinical examination (OSCE) were compared. Finally, we compared small group grades assigned by specialist and generalist leaders to their students. RESULTS: Responses from 77% of the students indicated that specialists and generalists teach basic examination skills with equal effectiveness. Responses from 71% of the leaders indicated that they are equally confident in teaching basic examination skills. There were no differences in the generalists and specialists assignment of grades to their students. Most importantly, there were no differences in the scores on the OSCE between students taught by generalists and specialists. CONCLUSION: Medical schools can expand their pool of physicians teaching basic skills by utilizing specialists without decreasing teaching effectiveness. With changes in the organization, financing and delivery of health care, this strategy could help schools preserve their academic mission in the face of mounting pressure to increase clinical revenue. Novel point-of-care echo machines are becoming available and may provide a rapid and cost-effective method for accurately assessing LV function. It is unknown, however, whether physicians without prior echo experience can be trained to use echo to assess LV systolic function. METHODS: Physicians, without prior echo experience, completed a 3-hour training course including didactic and hands-on training in echocardiography. Patients scheduled for standard transthoracic echocardiography as part of their clinical care were eligible for enrollment. Enrolled patients had a point-of-care echocardiogram performed and interpreted for LV ejection fraction (EF) by a trained physician within 24 hours of their standard echo. LV EF was classified as 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or 55%. Point-of-care echos were performed using the new Agilent``OptiGo'' cardiac ultrasound device. Each standard echo was read twice. All echocardiographic interpretations were performed blinded to one another. Agreement was defined as a difference in LV EF of 15%. Agreement rates between point-of-care echo and standard echo were determined and compared to standard echo interobserver agreement rates. RESULTS: Twenty-five physicians enrolled 510 patients. The mean age of the patients was 59.4 years and 52.8% were male. The indications for echocardiographic assessment was evaluate ejection fraction in 70.4% and evaluate murmur in 11.1%. A total of 57.9% of patients had an LV EF of > 55%, 14.2% had an LV EF of 45 ± 50%, 9.2% had an LV EF of 35 ± 40%, 7.2% had an LV EF of 25 ± 30%, and 11.5% had an LV EF of < 15 ± 20%. Rates of agreement for evaluation of LV function are shown in the table. CONCLUSION: Physicians without prior echo experience can be trained in 3-hours to assess LV function with a novel point-of-care echo device. Agreement between point-of-care echo and standard echo is similar to standard echo interobserver agreement. Wider availability of pointof-care echocardiography could improve medical care. CONCLUSION: Although only a minority of low income women report low levels of trust in their personal health care provider, lack of trust is strongly associated with lack of adherence to recommended cancer screening. Mistrust of health care providers in general and beliefs about unethical medical research practices are more common, but have less effect on use of preventive health services. Increasing trust of primary care physicians among low income populations may offer a novel approach to addressing socioeconomic health disparities.\nuniversally useful markers of medical underservedness. The purpose of this study was to determine how well race/ethnicity and income identify women who do not receive screening mammography in the City of Philadelphia. METHODS: Telephone survey of adult residents of the City of Philadelphia. Households were sampled using random digit dialing stratified by service area. Interviews were conducted in English or Spanish between June and September 1998. Women ! 50 yo were asked``About how long has it been since you last had a mammogram?'' and women who had not had a mammogram in past year were asked the most important reason they had not had a mammogram. RESULTS: Of 6,329 eligible households, 4,114 individuals (65%) agreed to participate. 828 respondents were women between 50 and 75 years of age. Mean age was 61.9 yrs; 46% were Caucasian, 48% African-American and 4% Hispanic; 26% had not completed high school. Mean annual household income was $19,000 (range < $8,000 to > $100,000). 75% had undergone screening mammography in the prior year, 9% one to two years previously, 11% more than two years ago and 5% had never had a mammogram. In the overall sample, mammography in the prior year was associated with African-American ethnicity (RR 1.19, 95% 1.1 ± 1.3), but not with age, income or education. However, there were significant interactions between ethnicity and income and ethnicity and age. Income was associated with mammography among Caucasian women only (RR 1.28, 95% CI 1.0 ± 1.7 for above vs. below federal poverty level) and age was associated with mammography among African-American women only (RR 1.34, 95% CI 1.0 ± 1.8 for over 60 vs. 60 and under). Among women who had not had a mammogram in the prior year, African-American women were less likely to cite lack of motivation as their reason for not undergoing screening (RR 0.70, 95% CI 0.48 ± 0.99) and more likely to cite system barriers (e.g. insurance, transportation) than Caucasian women. CONCLUSION: In the city of Philadelphia, low income, minority women no longer represent the underserved for screening mammography. Furthermore, the characteristics of women underusing screening mammography differ between African-American and Caucasian women. These data suggest that the sociodemographic characteristics of the medically underserved may depend upon type of medical care, geographic location and even ethnic group. Generalist efforts to reduce medical underservedness should be based on accurate assessments of the communities they serve. Our purpose was to describe the relationship between both selfreported (SR) and electronically monitored (MEMS) adherence (ADH) and HIV viral load (VL), and to characterize drug resistance patterns in subjects who failed to sustain virologic suppression on potent HAART regimens. METHODS: Subjects on HAART were recruited from a longitudinal study of HIV-infected drug users. During a median of 166 days, VL and ADH with all antiretrovirals were assessed by SR and MEMS at each of 6 monthly visits. ADH (% doses taken/doses prescribed) was assessed for both 1 day and 1 week preceding each visit, and mean ADH was calculated for the entire study period. We also studied drug resistance in 11 patients with viral rebound (VL > 1000 copies/ml for > 2 consecutive mos. after previously maintaining VL < 400 copies/ml for > 2 consecutive mos.). RESULTS: 501 SR estimates and 417 MEMS estimates were obtained from 85 subjects. Mean 1 week SR ADH correlated with mean 1 day SR ADH (76% v. 77%; r=0.84, p=0.0001), as did mean 1 week and 1 day MEMS ADH (55% v. 57%; r=0.86, p=0.0001). Though SR ADH was greater than MEMS ADH, both were correlated with VL (r=0.6 for 1 week MEMS, r=0.5 for 1 day MEMS, r=0.5 for 1 week SR, r=0.4 for 1 day SR). The following factors were associated with poor ADH: active heroin use (64% for active v. 80% for former use, p=.02), active cocaine use (38% for active v. 60% for former use, p=.009), active alcohol use (60% for subjects who drank several days/week v. 80% for subjects who drank less, p=.002), side effects (73% for subjects with > 2 side effects v. 83% for subjects with < 2, p=.08), and female gender (44% for women v. 60% for men, p=.04). Among the 11 patients in whom we analyzed ADH, resistance and rebound, we found that median ADH during viral suppression was 54% (range 0 ± 83%), and median ADH after rebound was 8% (range 0 ± 36%). Only 2/11 developed high-level phenotypic resistance, and 4/11 developed significant mutations in either RT or protease; M184 in 3/11 on 3TC, K103N in 2/2 on NNRTIs, and various IDV mutations in 1/11 on PIs. In 2/4 patients, M184 mutations were present without corresponding phenotypic resistance. CONCLUSION: High correlation between 1 day and 1 week estimates suggest that ADH does not change in the day before a visit. While SR overestimates true ADH, repeated quantitative SR estimates correlate with VL. Our data further show that SR ADH > 90% is necessary to maintain virologic suppression, and that active drug/alcohol use and medication side effects lower adherence. Our analysis of drug resistance suggests that significant RT or protease mutations may be present as early indicators of phenotypic resistance. However, due to lack of sufficient selective pressure, high level phenotypic resistance is uncommon after viral rebound when ADH is poor. PURPOSE: Pulmonary infections, including bacterial pneumonia, cause significant morbidity and mortality for patients infected with HIV. The purpose of this project was to determine the predictors of inpatient mortality among HIV patients with community-acquired pneumonia admitted in the early highly active antiretroviral therapy era, between 1995 ± 1997. METHODS: Trained abstractors performed retrospective chart reviews of hospitalized HIV patients diagnosed or suspected of having community-acquired pneumonia, who were admitted between 1995 ± 1997 at 86 hospitals in 7 geographic areas. We used hierarchically optimal classification tree analysis to develop a pre-admission severity of illness staging system for predicting inpatient mortality. RESULTS: Among HIV patients with confirmed or suspected community-acquired pneumonia (n=1112), the inpatient mortality rate was 9.5%. Multivariate modeling indicated a 5-category staging based on four predictors (Table 1) . Bootstrap validity analysis (50% resample) supported the stability of this model. CONCLUSION: Our staging system identifies HIV patients with low, moderate, and high risk of inpatient mortality from community-acquired pneumonia and may be useful for case-mix adjustment while exploring variations in community-acquired pneumonia mortality rates among hospitals and across cities caring for HIV patients. The potential risks of herbal supplement use is of growing concern. Reports of serious side effects and drug interactions have prompted many physicians to incorporate questions of supplement use into their medical history. Despite these efforts, clinicians are often faced with inadequate information due to the recent boom of combination herbal products known only by their trade names. Supplements marketed to consumers as weight loss remedies are one such group of products that frequently contain a multitude of``natural'' ingredients. This study was designed to characterize the ingredients found in a number of popular weight loss products in order to assist physicians in preventing potential adverse effects. METHODS: A systematic search of the World Wide Web was conducted to identify twenty herbal weight loss products for sale on the internet. Three search engines were used (HotBot, Google and Excite) and the search term``herbal weight loss.'' A site abstraction form was developed to collect data on the ingredients contained within each product. Descriptive statistics were used to characterize the most common ingredients found among the twenty combination products. RESULTS: Nine ingredients were identified in six or more of the products. These included: MaHuang, Ginger, Guarana, Siberian Ginseng, White Willow Bark, Chromium, Garcinia Cambogia, Gotu Kola, and Zinc. The number and frequency of each of these ingredients among the herbal weight loss products characterized are included in the table below. CONCLUSION: Despite little evidence of efficacy and the potential for significant toxicity, ingredients such as Mahuang, Guarana, and Chromium are found in a majority of weight loss products. Physicians need to take this into consideration when faced with a patient who discloses the use of an unfamiliar over-the-counter weight loss supplement. HIV prevention education is a part of substance abuse treatment at many correctional facilities, but the effectiveness of these programs has not been evaluated. The goals of our study were to evaluate who receives HIV prevention services in correctional substance abuse treatment programs, and to determine the impact of these services on short term risk behaviors. METHODS: This study was a secondary analysis of data from the National Treatment Improvement Evaluation Study (NTIES), conducted during 1993 ± 1995. A total of 1,223 HIVfree adult inmates, enrolled in nine correctional substance abuse treatment programs were evaluated. A composite index modeled after the validated Risk Assessment Battery (RAB) measured HIV risk behavior at treatment entry and at twelve-month follow-up. We employed multivariable analysis of covariance to assess the influence of receiving HIV prevention services, controlling for potential confounders, on HIV risk behavior at twelve-month follow-up. Because of assumed differences between those who were in and those who were out of custody during the follow-up period, all analyses were stratified by this variable. RESULTS: Overall, 77.1% of the sample received HIV prevention services while in treatment.\nAmong the out of custody group, Hispanics, persons of younger age and those in outpatient substance abuse treatment programs were significantly less likely to received HIV prevention services. Among the continuously incarcerated group, women, persons of younger age and those in outpatient substance abuse treatment programs were least likely to receive these services. Both the in custody and out of custody groups exhibited HIV risk behavior reductions after twelve months of follow-up. However, controlling for baseline HIV risk behavior and other possible confounders, receiving HIV prevention services was associated with less risk behavior only among the out of custody group (=À.14, P=.04). No association was observed among the group that was continuously incarcerated throughout the follow-up period. CONCLUSION: This analysis suggests that HIV prevention services are beneficial in reducing risk behavior among inmates whose discharge is expected in the near future. The benefit of administering these services in individuals with longer sentences earlier in the incarceration period is unclear. Lifestyle changes, such as smoking cessation, can play a pivotal role in outcomes following the diagnosis and treatment of coronary heart disease (CHD). However, many patients with CHD are not motivated to make lifestyle changes even after undergoing coronary revascularization. Prior studies have shown that time orientation is a major motivational determinate of an individual' s behavior and more specifically as it relates to healthy lifestyle choices. Therefore, we sought to examine the association of future orientation with readiness to make healthy lifestyle changes in a diverse group of patients at high risk of CHD. METHODS: Forty patients scheduled for elective coronary angiography were identified in the fall of 2000. High risk patients were those with one or more of the following: diabetes, hypertension, or hyperlipidemia. Following informed consent, a baseline 20-minute structured bedside interview was conducted, which collected information on the following: demographics (e.g. age, race, and sex), high-risk behaviors (e.g. smoking) using the interview format of the Computerized Lifestyle Assessment (CLA), and future orientation using Carstensen and Lang' s Future Orientation Scale. This latter scale measures the extent that one perceives there is sufficient time to achieve his/her goals. A follow-up interview was conducted 3 months from baseline to assess readiness to change as measured by the CLA. RESULTS: Follow-up data were completed on 33 subjects (83%). Their mean age was 58 years (S.D.=10.4), 64% were women, 42% were Hispanic, 27% were White, 21% were Black, and 10% were Others. Most patients (79%) did not exhibit any movement in readiness to change their lifestyle or habits as measured by the CLA subscale. Overall, 15% of patients had movement toward adopting a healthier lifestyle following angiography, such as improving their eating habits and exercising; 6% regressed. Patients who were future oriented (n= 16) had the most progress in movement toward making healthy changes in exercise (25%), diet (10%), and smoking (10%), compared to 17%, 23%, and 6% respectively of patients who were present oriented. However, 13% of future oriented subjects had an overall decrease in their readiness to change score following angiography, compared to none (0%) of the 17 patients who were present oriented. CONCLUSION: These data suggest that patients at high-risk of CHD, as well as those who are future oriented, may be slow to make lifestyle changes following angiography. These findings also indicate that there is a need for more research to ascertain patients' perception of their personal coronary risks. Primary care and other health providers can use such novel information to implement individually relevant educational programs to address motivational factors related to health behavior. RESULTS: There were 1006 patients surveyed with a refusal rate of 20.3%. The study patients did not differ from the total population who accessed care during this time period in regards to age, insurance status, ethnicity, or gender. After controlling for several factors, including age, gender, and the presence of chronic medical conditions, Hispanic patients were more likely than white patients to report having a regular source of health care (p = 0.008) or a regular physician (p = 0.018). However, Spanish speaking patients were less likely to note a regular physician (p < 0.0001) than English speaking patients. Black patients were more likely than white patients to note a regular source of care (p = 0.04) but not a regular physician (p = 0.8108). Hispanic patients were 1.7 times more likely to have visited the ED or UCC in the past year than our white patients (p = 0.006). However, Spanish speaking patients had less use of these services (p = 0.01) than English speaking patients. CONCLUSION: Sociocultural issues such as race and English fluency likely contribute to utilization of resources such as EDs and UCCs. For example, in our population minorities tended to utilize EDs and UCCs more often than white patients, but were also more likely to report that they had a regular source of care. Given the racial disparities in health outcomes there is an imperative to address this problem with additional studies. Simply attempting to redirect patients who inappropriately utilize EDs and UCCs to their regular source of care is unlikely to succeed unless language and cultural issues are addressed. where an aggressive approach to care following acute myocardial infarction (AMI) is more commonly adopted in comparison to a conservative approach, the aggressive approach may be associated with small incremental (marginal) mortality benefits. We conducted this study to evaluate the effectiveness of aggressive care following AMI in Canada. We hypothesized that the marginal benefits should be larger in Canada, as the country is operating on a lower margin because the approach to care is more conservative overall. METHODS: We conducted a retrospective cohort study using administrative data of acute care hospital admissions and in-and out-patient services for all patients who sustained a first AMI in Quebec in 1988 (n=8674). We used differential distances to hospitals offering aggressive care as instrumental variables to control for unobserved case-mix variation when measuring mortality up to 4 years after first AMI. RESULTS: Of the 4422 subjects who were > 64 years old, 11% received cardiac catheterization within 90 days after admission. In a previous study that applied similar methodology to the 1987 United States (US) Medicare population of first AMI patients, 30% of subjects received catheterization within 90 days. As in the US study, we found that subjects living relatively close to hospitals offering aggressive care were more likely to receive aggressive care (26% of``close'' versus 19% of``far'' subjects received cardiac catheterization within 90 days; 95% CI: 5% to 9%). Unlike the US study, we found no differences in mortality across the``close'' versus``far'' differential distance groups (unadjusted differences at 1 year: 1%; 95% CI: À1% to 3%). This absence of association was found in elderly ( > 64 years) and younger age groups. Adjusted results also showed no differences between subjects receiving aggressive versus conservative care (at 1, 2 and 4 years: 4%, 2%, À4%; 95% CI: À11% to 20%, À15% to 18%, À26% to 8%, respectively). CONCLUSION: Contrary to our hypothesis but consistent with results from numerous randomized trials and observational studies, the aggressive approach to post-AMI care does not appear to be associated with marginal mortality benefits even in Canada, where the approach to post-AMI care is conservative overall. Although clinicians may prefer results that are reported as``normal'' or``abnormal,'' this usually requires the specification of an artificial diagnostic criterion. Diagnostic information is typically lost in this simplification process. One application of the mutual information function is the calculation of the amount of information lost as test results are dichotomized. Consider, for example, a population of individuals in which the probability of disease is 0.5. Assume that the``healthy'' and``diseased'' individuals in this population are normally and identically distributed with respect to some diagnostic marker, except that the means of the two populations are separated by one standard deviation. In this case, I(Dx;T) = 0.16 bits. If, however, the results are dichotomized using the optimal cutoff (the midpoint between the means of the two populations) then I(Dx;T) is reduced to 0.11 bits. CONCLUSION: Information theory provides a framework for understanding diagnostic testing. This perspective suggests that mutual information, rather than the AUC, provides the appropriate summary index of diagnostic test performance. PURPOSE: Racial disparities in procedure use for acute myocardial infarction (AMI) have been well documented in selected populations in the 1980s and early 90' s, but little is known about recent trends in disparities in the general population. METHODS: We conducted a series of cross-sectional analyses of data from the Nationwide Inpatient Sample (which includes race and demographic, diagnostic and procedure data on all discharges from over 900 representative hospitals in 18 states) for 1995 through 1997 to compare rates of catheterization performed prior to discharge for acute myocardial infarction (ICD9 code 410) in whites vs. blacks. RESULTS: We identified 298,496 discharges among whites and 23,496 discharges among blacks with AMI as the first listed diagnosis during 1995 ± 97. Age-adjusted catheterization rates by race are presented in the figure. From 1995 to 1997, age-adjusted peri-AMI catheterization rates increased more sharply in blacks (39% to46%) than in whites (48% to 52%; p < 0.01). After adjustment for age, gender, comorbidity, region, and rural locale using Poisson regression, the incidence rate ratio (IRR) for peri-AMI catheterization in blacks compared to whites in 1995 was 0.84 (95% confidence interval (CI) 0.78 ± 0.90). During the next two years, the gap progressively narrowed, such that in 1997 the adjusted IRR was 0.90 (95% CI 0.83 ± 0.96). Similar results were obtained when we restricted the analysis to discharges from hospitals that performed catheterizations. CONCLUSION: These recent nationwide data suggest that, while blacks remain less likely to undergo catheterization during a hospitalization for AMI than whites, this racial gap appears to be narrowing.\nPURPOSE: Women veterans, like their male counterparts, are at increased risk for heart disease because they are more likely to be obese and smoke cigarettes than the general population. In order to develop clinical interventions that will improve risk behaviors among women veterans, we assessed their awareness of heart disease. The purpose of this study was to determine whether women veterans are worried about heart disease and to specify whether women with risk factors for coronary artery disease (CAD) are more likely to worry about heart disease compared to women without risk factors. METHODS: Cross-sectional survey of women veterans receiving primary care at the Durham VAMC. We measured demographics, CAD risk factors, other medical conditions, and worry about heart disease. Statistical analysis was performed using the chi-square test and logistic regression analysis using worry about heart disease as the outcome variable. RESULTS: Overall, 409 women veterans completed the questionnaire (response rate 71%). We excluded women with heart disease (n=64) and incomplete data (n=17 PURPOSE: Blacks are less willing to donate cadaveric organs, the most common type of transplantation, but whether the same race differences exist for other donations is unknown. We compared patterns of race differences in willingness to donate cadaveric organs with those of becoming a living kidney donor and donating blood. METHODS: We conducted a cross-sectional study in Maryland via standardized telephone questionnaire using random digit dialing to identify households. Persons age 18 ± 75 years were randomly selected within households. Subjects were considered willing to become cadaveric donors if they had designated themselves as an organ donor on their driver's license, livingrelated donors if they were willing to donate a kidney to a sibling, and blood donors if they had previously donated blood. Using logistic regression we analyzed the independent effects of race on willingness to donate adjusting for demographic, clinical and attitudinal characteristics.\nRESULTS: Of 385 respondents (84% of randomized households), 19% were White men (WM), 30% White women (WW), 12% Black men (BM), and 31% Black women (BW). BM and BW were more likely than WM and WW to have < high school education (p < 0.01), to have annual income < $40,000 (p < 0.01), to have dependents (p < 0.01), to be unemployed (p = 0.01), to consider spirituality important (p < 0.01), to believe hospitals``want to know more about personal affairs or business than they really need to know'' (p < 0.01), and more likely to believè`h ospitals have done harmful experiments on patients without their knowledge'' (p < 0.01). BM were least willing to become cadaveric donors (17%) compared to BW (34%), WW (59%), and WM (65%) (p < 0.01). However, WM were least willing to become living related donors (60%) compared to BW(61%), BM(72%), and WW(75%) (p = 0.05). BW were least willing to donate blood (40%), compared to WW (58%), BM (65%), and WM (86%)(p < 0.01). Despite adjustment for differences in respondent characteristics, BM were still much less likely to become cadaveric organ donors [odds ratio (95% CI)]=[0.2 (0.1 ± 0.7)], equally as likely to donate blood [0.8(0.3 ± 2. 5)], and almost 3 times more likely to become living-related donors [2.8 (0.9 ± 8.7)] than WM. It is unclear whether BW were less likely to be cadaveric organ donors [0.6 (0.2 ± 1.5)], but they were much less likely to donate blood [0.1 (0.04 ± 0.3) ], and equally as likely to be willing to be a living related donor [1.3 (0. PURPOSE: Ethnic minorities with coronary artery disease have worse outcomes compared to Caucasians. Depression is known to be a major risk factor for poor outcomes among patients with coronary artery disease. However, among ethnic minorities with coronary artery disease, little is known about the prevalence of depression. The purpose of this study was to determine whether ethnic variations in pre-operative depression exist between Caucasians and ethnic minorities undergoing angioplasty. METHODS: Subjects in the study were patients who had recently undergone coronary angioplasty and were eligible for enrollment in a randomized trial to reduce cardiac risk factors. Baseline evaluation included an assessment of demographic characteristics, cardiac history, and severity of illness. Depression was assessed using the Center for Epidemiologic Studies Depression Scale(CES-D). A score of 16 or greater indicated the presence of depression. RESULTS: Of 542 patients enrolled, 33% had evidence of pre-operative depression. Their mean age was 61(+12), 34% were female, 14 % were African-American and 16% were Latino-American. Patients who were depressed were more likely to be younger in age (P=03). A greater proportion of women were depressed in comparison to men (P=.003). Mean CES-D scores were significantly higher among ethnic minorities compared to Caucasians( P=.002). The highest CES-D scores were observed among Latino-Americans. In general, African-Americans and Latino-Americans were 2 times more likely than Caucasians to have evidence of depression ( 95 % CI 1.3 ± 3.2). CONCLUSION: Among patients undergoing coronary angioplasty, minorities were 2 times more likely to have evidence of depression at baseline. This finding is clinically important since baseline depression among patients with coronary artery disease is a poor prognostic indicator. Future studies are needed to determine whether baseline depression among ethnic minority patients with coronary artery disease is associated with poor outcomes. PURPOSE: Patients often receive medical information from the media and internet, but little is known about how information sources affect risk perceptions. Our objective was to investigate whether the source of medical information affected perceived risks for common diseases. METHODS: Women treated for breast cancer at an urban hospital were recruited through flyers and an oncology social worker. Participants completed a health survey about their sources of health information (doctor, television (TV), or internet) and their perceived lifetime risk of heart disease, hip fracture, and recurrent breast cancer. Risk estimates among patients receiving vs not receiving information from each source were compared using Wilcoxon Rank tests. RESULTS: The mean age of the 51 participants was 52 years, 16% were non-Caucasian, and the median educational level was 2 years of college. Patients receiving medical information from either TV or the internet had higher perceived risks for both heart disease and hip fracture than those not receiving information from those sources. Patients receiving information from TV perceived breast cancer recurrence as being more likely even though fewer among them had late stage or node-positive disease. Patients who received information from their doctors did not have consistently higher or lower perceived risk estimations compared to patients not receiving information from their doctors. CONCLUSION: Patients' perception of risk may be affected by the source of health information, with those who rely on the TV or internet having higher risk perceptions than those who rely on other sources. PURPOSE: Alcohol abuse is a common but frequently unrecognized problem in surgical patients, with important implications for Medical consultants. Studies have shown that 10 ± 50 % of hospitalized patients on surgical services have alcohol problems, depending on the subspecialty service and the detection methods used. We screened a large cohort of VA surgical patients to determine the prevalence of problem drinking in a combined inpatient and outpatient population as the initial step in a study of alcohol consumption and surgical outcomes. METHODS: All patients at the VA Pittsburgh Healthcare System admitted to surgical services or scheduled for elective surgery requiring at least an overnight hospital stay were considered candidates for screening. Patients were asked to complete a confidential screen for alcohol problems using the Alcohol Use Disorders Identification Test (AUDIT) and quantity-frequency measures. Non-cirrhotic patients who met criteria for alcohol problems (AUDIT score 8 or weekly consumption > 15 drinks in men or > 11 drinks in women) and control patients who did not meet these criteria were invited to participate in a descriptive cohort study. Controls were matched by surgical service and admission status (inpatient versus outpatient at screening, planned overnight versus longer admissions for elective procedures) in a 2:1 control:study ratio. Participants received a baseline preoperative assessment including demographic information and the Alcohol Timeline Follow-Back to measure alcohol consumption. Descriptive statistics were reported as mean standard deviation. Current drinking and drinking over the last 6 months were analyzed using a non-parametric test for trend. RESULTS: Among 1624 patients presenting for surgical admission or preoperative assessment over one year, 1067 (65.7%) were screened for alcohol problems, of which 99 (9.3%) screened positive. The mean AUDIT score for screen-positive patients was 12.0 | 9.6 and the mean number of drinks per week was 33.2 | 37.2, compared with 1.1 | 2.1 and 2.0 | 8.5, respectively, for screen-negative patients. Forty-seven (47.5%) screen-positive patients agreed to further assessment and follow-up, as did 93 matched controls. Mean AUDIT and alcohol consumption scores were not statistically different between enrolled and non-enrolled study patients (p = 0.3). Among enrolled problem drinkers, there was a trend toward less alcohol consumption in a typical drinking day as surgery approached, from an average of 9.2 | 12.5 drinks/day over 6 months prior to their assessment to 5.4 | 4.4 drinks/day over the previous 1 month (p = 0.13 for trend). Binge drinking episodes ( > 6 drinks in one day) showed a similar reduction, with the average problem drinker reporting 5.6 | 8.7 episodes/month at 3 ± 6 months prior to assessment but only 3.4 | 6.7 episodes/month over the previous 1 month (p = 0.18 for trend). The problem drinkers were most commonly found on the Orthopedic (29.8%)and General surgery (25.5%) services. CONCLUSION: The prevalence of problem drinking in our large cohort of veterans undergoing surgery was lower than that found in prior studies of surgical inpatients but similar to that seen in published studies of medical outpatients. The inclusion of outpatients and patients undergoing elective procedures may shift the drinking profile of surgical patients towards that of the general population. Intensive screening of surgical patients for problem drinking by Medical consultants may be most effective on selected subspeciality services. Alcohol consumption levels among problem drinkers tended to decrease as the date of surgery approached, suggesting that the perioperative period may provide a good opportunity for brief interventions.\nALPHA BLOCKERS AND HYPERTENSION Ð HOW MANY ARE AT RISK FOR SUBOPTIMAL THERAPY? C.L. Bryson 1 ; 1 Puget Sound VA, Seattle, Washington PURPOSE: This study defines the number of people at risk for suboptimal therapy with hypertension who are only on alpha blockers. Results of randomized controlled trials have provided substantial evidence that antihypertensives differ in their ability to mitigate outcomes of CHF, CVA, MI and renal failure in the hypertensive population despite nearly equivalent blood pressure control. This is of particular interest because of the early termination of one arm of the ALLHAT study in which the alpha-1 blocker doxazosin was associated with a doubling of the rate of incident heart failure. Doxazosin is less effective for preventing the outcome of CHF than chlorthalidone in hypertensive patients. This result was unexpected, since doxazosin has favorable effects on other surrogate markers including serum lipids. Alpha-1 blockers are an effective treatment for prostatism, and fifty percent of men have histologic evidence of BPH by 60 years of age. It is difficult to quantitate the exposure of alpha-1 blockers for hypertension in elderly men on pharmacy data alone because information about the patient' s comorbid illnesses is necessary to interpret the indication for the agent. This is true not only for alpha-1 blockers but also for other antihypertensives. METHODS: The National Ambulatory Medical Care Survey (NAMCS) is a national survey of physician visits that records not only physician diagnoses but also patient therapy. It was a sampling study that was carefully performed and allows extrapolation to the national visits during the study times. The NAMCS data was analyzed with STATA. The provided weights were utilized to expand the results to projected national averages. Summary measures were computed for variables of interest while maintaining acceptable relative standard errors. RESULTS: This presentation or poster will present the results of the analysis of the 1998 NAMCS for antihypertensive use, with an accent on alpha blocker usage as a single drug. Approximately 3.5 to 4% of the hypertensive population appears to be treated with only an alpha blocker, which is between 840,000 to 1 million patients. Interestingly, about half of these are women. Only 15% of hypertensives were on diuretics. Time trends and usage by indication may also be presented from prior NAMCS data. A correlation with VA data will be available as well by the time of presentation as a result of an ongoing project. CONCLUSION: An estimation of the use of different antihypertensive agents correlated with indication is important, particularly in light of recent clinical trials. An estimate of the use of alpha-1 blockers as single agents prior to changes in recommendations will help enumerate the size of the population at risk for suboptimal pharmacotherapy with regard to cardiovascular endpoints. It will also provide context for the importance of findings from clinical trials of antihypertensive agents. (3) clinical cues found empirically to be used by clinicians to differentiate sinusitis from viral URIs. Agreement between diagnostic approaches was determined using the Kappa statistic. RESULTS: Of 91 eligible patients, 61 (67%) agreed to participate in the study. Mean age was 42 years (range 12 ± 78), 43 (70%) were women, and mean duration of symptoms was 4.7 days (range 1 ± 23). Forty-four patients (72%), 6 patients (10%), and 11 patients (18%) were diagnosed with sinusitis by each diagnostic approach respectively. Agreement between diagnostic approaches ranged from kappa=0.08 (approach 1 compared to approach 2) to kappa=0.39 (approach 2 compared to approach 3). CONCLUSION: Three published approaches to diagnosing sinusitis modified for telephone use exhibited poor to marginal agreement. More research is needed to determine a valid tool for telephone diagnosis of sinusitis before the diagnosis and treatment of acute sinusitis solely through telephone contact can be recommended. Increasingly, hospices are developing affiliations with academic medical centers. However, little is known about hospice patients referred from academic medical centers, and how their needs may differ from those of the general population of hospice patients. Therefore, the purpose of this study was to identify differences between patients referred to an urban, non-profit hospice from academic vs. non-academic medical centers. METHODS: In this retrospective cohort study, 1,691 patients admitted to an inpatient and outpatient hospice program were identified between January of 1997 and January of 1999, and data were gathered until discharge or death. RESULTS: Patients referred from academic medical centers were younger, had higher incomes, and were less likely to have Medicare or Medicaid. Patients referred from academic medical centers were less likely to have a Do Not Resuscitate order or a living will, and had more medical and nursing needs. Survival analysis revealed no difference in length of stay between patients referred from academic and non-academic medical centers. CONCLUSION: Patients referred to hospice from academic medical centers have greater needs for nursing and medical care than do patients referred from non-academic medical centers. These results have important implications for hospice planning and policy. If they are replicated in other hospice settings, they suggest that hospices that establish ties with academic medical centers may be caring for patients with more symptoms, and more needs for care. Such differences should be considered in drawing up plans for affiliations. They might also be considered on a more global scale in assessing reimbursements to hospices by Medicare and other payers. were younger than 65 years, and 8 had concomitant valvular disease leaving 145 patients for analysis. Data collection was complete. The mean age was 77 years, and women comprised 55% of the cohort. According to the AFI criteria, 38% of the population had an annual stroke risk of < 4.3% while 50% of the population had an annual stroke risk < 3.6% by SPAF (p < .05). There was agreement between the AFI and SPAF in estimating the patients with the highest stroke risk of 8% annually (43% and 50%, respectively). The OBRI estimated 91% of all patients to have a bleeding risk of 12% over two years while 9% of the cohort had the highest estimated bleeding risk of 53% over 48 months. In all but the highest stroke risk groups, the OBRI-estimated bleeding risk exceeded the expected benefits of anticoagulation. CONCLUSION: In this population of elderly patients with atrial fibrillation, the use of published risk stratification indices has limited clinical utility, mainly because of the difficulty in balancing the relative risks in the group with low and intemediate stroke risk. In particular, the validated OBRI does not seem applicable to the stable outpatient elderly population, and its use might actually deter the appropriate anticoagulation of patients. PURPOSE: Cold angina is explained with increased coronary``vascular resistance''. The temperature of the lower extremities in cold weather and the temperature of blood during hibernation is approximately 228C. It is suggested that when blood temperature decreases from 36.68C to 228C, blood viscosity increases 26.13%. The aim of this study is determination of the effect of the increased blood viscosity on circulatory system. METHODS: Changes in circulatory parameters due to increased viscosity can be calculated with Poiseuille' s hydrodynamics equation. In the equation, blood flow rate in m3/sec is Q=%a4/ 8 L(F1-F2) and peripheric resistance of a vessel is R=(F1-F2)/Q, where a is the radius, L is the length of vessel, is blood viscosity, F1 is the initial and F2 is the final pressure at the two ends of a vessel. RESULTS: According to these equations, flow rate decreases 20.72 % and peripheral resistance increases 20.72 % when viscosity increases 20.72 % due to cold environment. This state can be compensated by increased cardiac work as 20.72 % increase in blood pressure or 5.9 % increase in the vessel radius. CONCLUSION: The term``peripheral resistance'' is more correct for the explanation of cold angina than vascular resistance, because peripheral resistance can cover blood viscosity (resistance against blood flow) and vascular resistance (change in diameter, length and shape of vessel). These calculations show that decreased temperature and increased viscosity of the blood in the skin and lower extremities in cold weather results in increased peripheral resistance and circulatory work. Due to sclerotic vessel has no vasodilatation capacity, Y  schemia risk is high for the coronary atherosclerotic patient. Unfortunately, similar circulatory changes caused by the cooling of the pulmonary blood in cold weather are unknown. The information above shows that the mechanism of cold angina gains an additional explanation with increased blood viscosity and peripheral resistance due to decreased blood temperature, for the first time. This method is important for further evaluation of the dynamics of the circulatory system. RESULTS: CC occurred in 13 patients (14.4%) including 8 patients who experienced 11 major complications. There was a trend toward more CC with age > 60, anesthesia duration > 3 hours, creatinine > 2.4, and vascular and emergency surgery. The ASA scale and revised cardiac index(Lee) performed better than the original Goldman and Detsky indices. The ACC and ACP algorithms were best at identifying low risk patients, but some of this effect was due to the results of non-invasive tests. (Table 1 ) Exercise capacity, Eagle criteria, and Larsen's index were not predictive of CC. CONCLUSION: Although our group of patients had relatively few major complications, we were able to validate the predictive ability of various indices to stratify perioperative risk; however, statistical significance of the differences could not be achieved due to the small numbers. The goals of risk indices are to identify low risk patients requiring no further workup and to select other patients potentially benefiting from additional testing or interventions. Other management strategies, including perioperative beta-blockers, will be needed to reduce complications in the intermediate and high risk groups, and in emergency surgery patients. The ACC and ACP each published guidelines for perioperative cardiovascular evaluation for noncardiac surgery. Both were evidence-based, but the ACC also used expert consensus opinion in the absence of strong evidence. Because of this difference, the guidelines may present conflicting opinions. The purpose of this study was to compare their recommendations with respect to non-invasive testing (NIT) and evaluate risk stratified clinical outcomes. METHODS: We retrospectively reviewed records of 90 patients (pts) undergoing 104 major intra-abdominal or vascular procedures in a large, urban teaching hospital. Patients were classified into low, intermediate, or high (including emergency) risk groups based on the algorithms. We then compared the ACC and ACP recommendations for NIT and subsequent clinical outcomes for each group. Major complications were defined as cardiac death, MI, ischemia, CHF, and VT. RESULTS: Eleven major complications occurred in 8 pts, 3 of whom had MI' s (1 cardiac death). Of the 104 procedures, 30 were emergent (1 MI). Additionally, 9 and 5 pts respectively were classified in a high risk group by ACC and ACP criteria. The remaining pts were divided into test or no test subgroups ( Table 1 ). The ACC recommended NIT for 11 of these lowintermediate risk pts, 2 of whom had postop MI' s, and no test for the other 54 pts, none of whom had major complications. The ACP recommended NIT for 6 of 14 intermediate risk pts, 1 of whom had an MI, and no test for the 55 low risk pts, 1 of whom had an MI. ACP guidelines recommended no test in 7 of the 11 pts (1 had an MI) in whom the ACC suggested testing . The guidelines agreed otherwise. CONCLUSION: Both ACC and ACP algorithms separated low risk patients from intermediate-high risk ones. Because of differing concepts regarding exercise capacity and NIT in non-vascular surgery, the ACP classified more patients as low risk and recommended fewer NIT' s; however, the clinical outcomes appeared comparable (despite the small numbers). Physician judgement and personal preference for the guidelines will determine the approach regarding NIT for an individual patient undergoing elective surgery. Future studies will need to clarify the best approach for high risk/emergency patients. were independent risk factors for lower extremity amputation in patients with PAD. Although the burden of certain atherosclerotic risk factors is higher in minority patients, the impact of this burden does not account for the increased risk of the outcome of lower extremity amputation in these two populations. As we do not have information on the severity of disease, race/ethnicity may be a marker for the severity of PAD. Further research is needed to better understand the reason (s) why race/ethnicity is independently associated with poor outcomes in PAD. PURPOSE: Whether patients and their providers make comparable assessments of the severity of pain, the likelihood of improvement and optimal treatment strategies when patients present with an acute episode of low back pain is not known. We sought to evaluate the level of concordance on these dimensions between patients and providers at the time that individuals with acute low back pain (ALBP) enrolled in a randomized controlled trial (RCT) of usual care versus choice of complementary and alternative medical (CAM) therapy. METHODS: Baseline data obtained by face to face interview at RCT enrollment. Eligible subjects were adult members of a multi-specialty medical practice who reported experiencing uncomplicated ALBP for less than 3 weeks with no co-morbid explanation for symptoms. Enrollees were randomized to either usual care or to a choice of chiropractic, massage, acupuncture or usual care. Enrollees' medical providers completed a written survey at the time they referred a patient to the study. RESULTS: Of the 80 enrolled in the study to date, the mean age was 42, 51% were women, and 58% were white. Sixty-seven of the enrollees' providers (84%) responded to a questionnaire about the enrollee's ALBP. The mean patient-reported severity score for back pain on a scale of 0 ± 10, with 10 representing the worst pain ever, was 8.0 (s.d. 1.6, range 4 ± 10); providers reported a mean score of 6.3 (s.d. 2.1, range 1 ± 9). Providers substantially underestimated patients' pain (t=5.48, p < .001), with 24% of the enrollees describing their pain as 10/10 and none of the providers rating pain higher than 9/10. Providers and patients had similar expectations for recovery. On a 0 ± 10 scale, with 10 representing complete improvement in 6 weeks and 0 representing no improvement, patients reported a mean score of 8.4 (s.d. 1.8, range 4 ± 10) and providers a score of 8.1 (s.d. 2.5, range 1 ± 10), t=.84, p=.40. The majority of patients (55%) expressed a preference for massage, 31% for chiropractic, 12% for acupuncture, and 1% for usual care. Thirty-two percent of the providers accurately anticipated the type of therapy the patient said s/he would choose, 18% thought that the modality the patient preferred was the best choice for that patient. Only 7% believed the therapy preferred by the patient was the best treatment for ALBP in general. Patients presenting to a university-based clinic were screened for MD using the PRIME-MD. Eligible patients also had a 17-item Hamilton Rating Scale for Depression (HRS-D) !12. Patient satisfaction with their PCP was assessed at baseline using a 5-point Likert scale and later dichotomized to``very satisfied'' or``not very satisfied'' for our analysis. The HRS-D was repeated at 6 months. Recovery was defined as a HRS-D 7. Information about depression treatment, such as pharmacotherapy, number of visits, and referral to a mental health specialist was abstracted from the EMR. RESULTS: Between 4/96 and 12/98, 211 depressed patients met all protocol eligibility criteria and completed a baseline interview. Of the 193(91%)patients who completed 6 month interviews, their baseline mean HRS-D was 20, 71% were women, 24% were African American and 119 (62%) reported being``very satisfied'' with their PCP. No differences in HRS-D or satisfaction were noted by gender, race or medical comorbidity. Although``very satisfied'' patients had significantly lower baseline HRS-D, the difference of only one point was not considered clinically significant. At 6 months 43(22%)of the patients recovered.``Very satisfied'' patients were more likely to recover at 6 months than those who were``not very satisfied'' (31% vs 8%, OR 5.1, 95%CI=2.0 ± 12.8). This association persisted after controlling for baseline HRS-D scores, medical co-morbidity and process measures for depression care (adjusted OR 5.3, 95%CI=2.0 ± 13.8). A baseline report of``very satisfied'' was not significantly associated with subsequent number of PCP visits, medications prescribed, or mental health referrals. CONCLUSION: Patients who are``very satisfied'' with their PCP are more likely to recover from a MD episode. Patient recovery has been found to be related to physicians engendering confidence in their patient's recovery. It is tempting to speculate that PCPs who stimulate greater optimism in their patients, improve satisfaction and subsequent recovery from depression. Assessment of patient satisfaction early in the therapeutic relationship may identify patients at risk for poor recovery from MD. Smoking is a significant issue among the homeless, but little is known about their willingness to quit or their interest in assistance with quitting. We therefore sought to determine what proportion of homeless persons smoked, whether they were ready to stop smoking, and whether they had preferences for smoking cessation treatments. METHODS: We anonymously surveyed homeless adults at nine sites in Pittsburgh, including emergency shelters, drop-in centers, transitional housing, a community health center, and residential substance abuse treatment programs. All persons at each site were approached during 2-hour blocks; 97% of those present completed the survey. Measures included demographic characteristics, living situation, smoking behavior, readiness to change smoking behavior (based on a stage-of-change model), self-efficacy to stop smoking (rated from 1 to 10), nicotine dependence (Fagerstrom scale), and preferred method of smoking cessation. RESULTS: 273 persons completed the survey, of whom 87% met criteria for homelessness. The sample was middle aged (mean age 42 years), male (81%), and minority (63%). Of the 68% who were current smokers, 87% had interest in quitting, and 39% indicated readiness to quit smoking within the next six months. Compared to those not ready to quit, persons were significantly (p < 0.05) more likely to be ready to quit if they had high self-efficacy (47% vs. 21%), knew persons who would support their smoking cessation efforts (47% vs. 13%), or smoked within 30 minutes of awakening (40% vs. 31%). Readiness to quit was not associated with gender, ethnicity, substance abuse, nicotine dependence, or type of shelter. Participants self-efficacy to quit smoking was significantly higher with assistance, compared to their selfefficacy without assistance (6.3 vs. 4.8, p = 0.000). Treatments preferred by persons ready to quit included nicotine replacement (33%), counseling (15%), bupropion (Zyban) (7.5%), and nicotine in combination with other treatments (12%); one-third (33%) indicated``nothing'' or`q uitting cold-turkey'' would help. CONCLUSION: The majority of homeless persons smoke, many are ready to stop smoking, and most believe they will be more successful with treatments such as nicotine replacement. There is an urgent need to develop and implement smoking cessation programs for the homeless. METHODS: We used data from a national telephone survey that examined attitudes and beliefs of Americans toward participation in clinical research. A distrust index, scores ranging from 0most trusting to 7-most distrusting, was created using the sum of the responses to a seven-item measure of trust. The dependent variable in these analyses, high levels of distrust, was categorized as 5 on the distrust index. Of 1000 survey respondents, there were 527 Black and 382 White respondents eligible for this analysis (n = 909). RESULTS: Black respondents were more likely than White respondents not to trust that``their doctor would fully explain research participation'' (41.7% vs. 23.4%, p < 0.01) and less likely to believe that they could ask their doctor questions (15.2 vs. 7.6%, p < 0.01). Black respondents were also more likely to disagree that``their doctor would not ask them to participate in research if the doctor felt there was harm\" (37.2% vs. 19.7%, p < 0.01) and more likely to state that they felt their doctors sometimes exposed them to unnecessary risks (45.5% vs. 34.8%, p < 0.01). Blacks were more likely to believe that``someone like them'' would be used as a guinea pig without their consent (79.2 vs. 51.9, p < 0.01) and that their physician had given them treatment as part of an experiment without their permission (24.5% vs. 8.3%, p < .01). Black respondents has a significantly higher mean index score (3.1) than Whites (1.8) (p < 0.01). Nearly 30% of blacks compared to 9% of whites had scores 5 (p < 0.01) In unadjusted analyses, Blacks had more than 4 times the odds of whites of having a distrust index score 5. After controlling for other sociodemographic variables in the logistic regression model, race remained strongly associated with a higher distrust score (OR=3.6; 95% CI=2. 4,5.4) . CONCLUSION: We found important differences by race in distrust. Even after controlling for markers of social class, African Americans were less trusting. Investigators trying to engage African Americans in research must focus on developing interpersonal trust with community members by actively engaging the members in all aspects of research design development and dissemination of findings. We included clinical trials conducted in patients in the following areas: cardiovascular disease, diabetes mellitus, HIV/AIDS, and cancer. For each RCT we collected data on the number and percent of white and minorities (i.e., African Americans, Hispanic, Asian/Pacific Islander, and Native Americans) reported in the study samples. In addition to simple descriptive statistics, we used ANOVA to assess reporting of race before during and after 1993 ± 1995. RESULTS: A total of 253 RCTs met our inclusion criteria (29 diabetes, 84 cardiovascular and 50 cancer and 90 HIV/AIDS). Forty percent of RCTs did not report the race/ethnicity of the study sample. Of the RCTs that reported race (n=150), 29% of the study samples were minorities. There was no statistical difference in the percent of minorities in RCTs published before 1993 RCTs published before , between 1993 RCTs published before and 1995 RCTs published before , and after 1995 . In addition there was no difference in the percent minorities when stratified by disease. CONCLUSION: In diseases where there are significant disparities by race in health outcomes, a large number of RCTs did not report the race/ethnicity of the study sample. In addition, there has been no increase in the proportion of minorities in study samples before, during or after enactment of federal initiatives to increase the proportion of minorities in RCTs. For clinicians to have the highest level of evidence to effectively care for minority patients and reduce disparities in health outcomes, RCTs must consistently report race/ethnicity and the proportion of minorities included in study samples needs to increase. METHODS: 309 psychiatric outpatients underwent a personal interview assessing medical conditions and health status with the Medical Outcomes Study Short Form-36 (SF-36) survey. We assessed psychiatric severity with the Brief Psychiatric Rating Scale (BPRS) which rates psychiatric severity from 0 (none) to 6 (extremely severe) in 18 symptom constructs. Podiatric health was assessed using 9 items from the National Health Interview Survey (NHIS) and an additional item addressing foot pain. RESULTS: 57% of our sample were diagnosed with schizophrenia and the mean BPRS score was 22 reflecting moderate severity of psychiatric illness at the time of the interview. 70% of participants reported at least one of 8 medical problems. 82% of patients surveyed reported at least one podiatric problem and 40% reported at least 3 problems. The most common problems were foot pain (48%), nail disorders (35%), and corns/calluses (28%). The prevalence rates of podiatric disorders in our cohort were 4 ± 10 times higher than those reported by the general population in the 1990 NHIS. For example, 5.2% of the general population reported nail disorders, 5.0% reported foot infections, and 5.7% reported corns/calluses. In bivariate analysis, the total number of podiatric problems reported by the participants was inversely related to their scores in all eight domains and both summary scores of the SF-36 (all p < 0.001). After controlling for sociodemographic factors, psychiatric illness, and medical conditions, the total number of podiatric limitations remained significantly associated with lower patients ratings in 4 of the 8 SF-36 domains: bodily pain (p < 0.001), role limits-emotional (p = 0.002 ), social functioning (p = 0.03), and general health (p = 0.003) and both summary domains, physical component (p = 0.04) and mental component (p = 0.03). CONCLUSION: Persons with severe and persistent mental illness have markedly elevated rates of podiatric problems when compared to the general population. These problems are associated with worsened self-perceived health status. Addressing podiatric health may be a successful way to improve the overall health of this population. PURPOSE: HIV continues to be a destructive problem in the urban poor. The SRO hotel population, a unique subset of the urban poor, is inadequately described in the literature. Our purpose is to describe the level of comprehensive health care, perceptions of health care, and utilization of health care services in the SRO hotel population served by both the Department of Acquired Immunodeficiency Syndrome (AIDS) Services Income Support (DASIS), a division of New York City's Department of Welfare, and CitiWide Harm Reduction, a community-based organization in the South Bronx. METHODS: Following a pilot survey, we conducted an extensive community-based survey of SRO hotel residents in the South Bronx from 8/99 to 4/00. We went door-to-door in all 10 DASIS-and CitiWide-served SRO hotels administering anonymous surveys to residents. The 34-item questionnaire included demographic information, health care utilization patterns, status of HIV disease and treatment, perceptions of quality of and access to health care, and drug use. RESULTS: Of the 190 hotel residents approached, 97% completed the survey. Of these residents, 91% reported being HIV-positive, 63% male, 59% Black, 34% Latino, and 62% active drug users. The average age is 41 years (range 20 ± 74 years), and 56% are high school graduates. The median length of stay in a single hotel is 4 months (range 1 day ± 32 years), and 96% have Medicaid. Of the 167 HIV-positive residents, 67% report having primary care providers. Of these individuals, 71% report receiving care in an infectious disease (ID) or HIVspecialty clinic. However, only 22% of 18 eligible residents report taking Mycobacterium avium complex (MAC) prophylaxis, 65% of 57 eligible residents report taking Pneumocystis carinii pneumonia (PCP) prophylaxis, and 44% of 88 eligible residents report taking antiretroviral medications. In the previous 6 months HIV-positive residents report a mean of 1.5 Emergency Department visits, and in the previous year they report a mean of 1.7 hospitalizations. Quality of care and access to care are perceived to be less than``good'' by 44% and 36% of HIV-positive residents, respectively. CONCLUSION: Among South Bronx HIV-positive SRO hotel residents, a largely substanceusing population with Medicaid, two thirds report having primary care providers. Yet a significant proportion of these individuals are not receiving medically recommended HIV therapy. Approximately half are dissatisfied with their quality of care, and one third with their access to care. In light of these findings, steps are currently being undertaken to address this deficiency in health care among HIV-positive individuals living in SRO hotels. PURPOSE: Although use of electronic medical record (EMR) systems is increasing, the effects on actual communication between patients and physicians have not been studied extensively. We sought to assess physician-patient communication patterns associated with use of an EMR system in an outpatient setting and provide an empirical foundation for larger studies. METHODS: We conducted a cross-sectional, case-control study involving analysis of videotaped physician-patient encounters, surveys of physicians and patients, and medical record reviews. The setting was an academic general internal medicine practice. We studied three physicians who used an EMR system (EMR physicians) and three who used solely a paper record (control physicians). A total of 204 patient visits were included in the analysis (mean = 34 for each physician). Makoul's SEGUE instrument was utilized for content analysis of whether physicians accomplished communication tasks during encounters. A separate, qualitative analysis by the authors sought evidence of general patterns of EMR and paper chart use. RESULTS: Compared to the control group, EMR physicians adopted a more active role in clarifying information and providing patient education. Overall, there were no statistically significant differences between the physician groups in mean duration of encounters, number of treatment options offered, or number of lab tests ordered. The qualitative analysis showed that physicians in both groups tended to direct their attention to the patient record during the initial portion of the encounter. The physical orientation of the computer keyboard/monitor and physicians' facility in typing were important determinants of attentiveness to the patient throughout the EMR encounters. CONCLUSION: EMR physicians' communication behaviors may reflect styles established before they began using an EMR system. Coupling education on patient-centered communication when physicians are trained on the use of EMR systems could enhance the effectiveness of providers using this tool. that might deter women from recommended screening. Another possible explanation for avoidance of BCS might be that some women are not sufficiently concerned about the consequences of metastatic breast cancer to submit to screening. We, therefore, measured womens' perceptions of metastatic breast cancer and compared them to self-reported compliance with BCS. METHODS: A trained research assistant interviewed 106 women aged 50 to 75 years using a structured questionnaire that assessed patients' attitudes toward mammography and their compliance with BCS. Interviews were conducted in North Carolina and Florida. Patients preferences for the health state``life with metastatic breast cancer'' were measured using a standard gamble approach. Standard gamble results were converted to health utility scores(HUS) for which 1.0 = normal health and 0 = death. Descriptive statistics were compiled. Bivariate analysis was performed to identify possible associations with recent noncompliance with BCS. Demographic data, attitudes toward screening, and HUS were used as independent variables in this analysis. Due to the exploratory nature of this study and small sample size, a p-value of < 0.1 was used as the cutoff for statistical significance. RESULTS: Of the 106 women interviewed, 83% were high school graduates or above, 35% were African-American, and 89% had some form of health insurance. The mean age was 60 yrs. Two thirds of participants reported themselves to be at least in good health. The mean HUS for life with metastatic breast cancer was 0.39 with a median score of 0.25. Thirty-two of the one hundred-six women interviewed had not received BCS during the 2 years preceding this survey. Over 90% agreed that mammograms could save their life and that the actual x-ray would not lead to breast cancer or needless breast surgery. Bivariate associations with BCS non-compliance included worse self-reported health, education less that high school, African-American race, and high HUS (0.7 or above) for life with metastatic breast cancer. CONCLUSION: Poverty, African-American race, and lack of education have long been sited as sentinel characteristics for lower BCS rates. The above data suggest that optimistic perceptions of metastatic cancer might also contribute to these lower rates. If indeed, higher individual HUS, can be confirmed as an independent predictor of BCS non-compliance, then a standard gamble approach could be used to identify a group of patients that could benefit from a targeted educational intervention that aggressively portrays consequences of metastatic breast disease. PURPOSE: A recent analysis of secondary data revealed marked differences in lung surgery rates between African-Americans and whites who suffered from early stage, non-small cell, lung cancer. The disparity in treatment according to race led to an 8% reduction in 5 year survival for African-American patients. The authors were unable to sort out whether patient or physician factors explained the difference in surgical decision making. Because of this uncertainty, we decided to measure patient perceptions of progressive lung cancer and evaluate whether more optimistic perceptions of this disease could reasonably explain differences in surgical management. METHODS: We recruited a stratified, random sample of 160 individuals from church groups, health fairs, and medical clinics in urban areas of North Carolina and Florida. A trained research assistant administered an oral questionnaire to obtain demographic information and assess patient preferences using a standard gamble approach. Standard gamble results were converted to health utility scores (HUS) for which 1.0 = normal health and 0 = death. HUS for progressive lung cancer were analyzed according to race. We then constructed a decision model that evaluated lung cancer surgery vs. no surgery for stages I and II non-small cell cancer. The model accounted for racial differences in HUS for progressive disease. The model was intentionally biased against surgery by overestimating surgical risk and survival with progressive lung cancer. Sensitivity analysis was performed. RESULTS: The average age of the 160 patients interviewed was 59 yrs. Sixty-three percent were women, 42% were educated beyond highschool, 41% had incomes < $20,000, 15% had no health insurance, and 41% were African-American. The mean HUS for progressive lung cancer among African-Americans surveyed was 0.32 compared to 0.21 among whites. The decision model when applied to a 60 yr.old African-American male demonstrated a quality adjusted survival of 3.38 life years (QALY's) for the surgical therapy group compared to 0.48 QALY's for the``no surgery'' group. In the sensitivity analysis, if patient age was adjusted to 80 yrs., surgery still maintained a large advantage (1.58 QALY's for surgical treatment vs. 0.48 QALY's for``no surgery''). Even if surgical cure rate was reduced to 10% in the model, the advantage in QALY's remained for surgical patients(1.2 surgery vs. 0.48``no surgery''). CONCLUSION: Although African-Americans hold a much more optimistic view of progressive lung cancer than whites, the difference does not explain discrepancies in lung surgery decisions even when decision models are significantly biased against surgical intervention. Other issues, such as physicians' misinterpretation of patients' surgical risk and patients' mistrust or misunderstanding of physicians, need to be assessed in order to resolve discrepancies in lung cancer treatment and survival.\nINDIVIDUALIZED TREATMENT FOR ALCOHOL WITHDRAWAL. J. Daeppen 1 , P. Gache 1 , U. Landry 1 , E. Sekera 1 , V. Schweizer 1 , S. Gloor 1 , B. Yersin 1 ; 1 Alcohol Treatment Center, Lausanne PURPOSE: To assess the effect of an individualized treatment regimen on the quantity and duration of benzodiazepine prescribed for alcohol withdrawal. METHODS: A randomized double-blind controlled trial including 117 consecutive patients admitted in an alcohol treatment program was conducted. Patients were randomized into two groups: i) 56 patients were treated with oxazepam only in response to the development of signs of alcohol withdrawal (symptom-triggered), and ii) 61 were treated with oxazepam every 6 hours with additional doses as needed (fixed-schedule). The administration of additional oxazepam was determined using the CIWA-Ar, a validated measure of the severity of alcohol withdrawal. RESULTS: 40% of the patients in the symptom-triggered group were treated with oxazepam, compared to 100% in the fixed-schedule group (p < .001). Mean oxazepam administered in the symptom-triggered group was 40 mg compared to 230 mg in the fixed-schedule group (p < .001). Mean duration of oxazepam treatment was 20 hours in the symptom-triggered group compared to 63 hours in the fixed-schedule group (p < .001). There were no differences in the incidence of complications and in the measures of comfort between the two groups. CONCLUSION: A symptom-triggered pharmacological treatment decreases the quantity and duration of benzodiazepine prescribed in alcohol withdrawal, with similar safety and comfort. Since both HIV/AIDS and its treatments commonly cause adverse somatic effects, symptom assessment is necessary for delivering and evaluating care for people with HIV. This study compared three methods to assess patient reported symptoms (presence, frequency, and bother) and to validate report of individual symptoms using longer patient-reported scales and clinical parameters in people with HIV disease. METHODS: Prospective cohort study. A questionnaire completed by 160 individuals on two occasions, four months apart. The questionnaire included 39 HIV-related symptoms, the Medical Outcomes Study-HIV Health Survey (MOS-HIV), and Health Transition items. Correlation coefficients were calculated to assess relationships between summary scores for the three symptom methods with health-related quality of life domains, CD4 counts, and HIV disease stage. Analyses were stratified by HIV disease stage to determine if correlations differed with respect to clinical stage. RESULTS: Sixty-seven percent of subjects were male, 63.5% were African-American, and 26% had less than a high school education. The percentage of participants with AIDS, symptomatic HIV, and asymptomatic HIV were 46.2%, 28.5%, and 25.3%, respectively with mean CD4 counts of 194, 386, and 400, respectively. The average number of symptoms reported was 15.2 (SD=8.4). There were statistically significant correlations between CD4 count and symptom frequency, symptom bother, and symptom presence. As hypothesized, correlations with the MOS-HIV Quality of Life domain were moderate and statistically significant whereas there were no significant correlations with the Quality of Life Transition score. Examination of individual symptoms revealed that fatigue and decreased memory were significantly correlated with both the Health Transition and MOS-HIV scores. Presence, frequency, and bother scores for trouble falling asleep were correlated with Sleep scale scores. Presence and frequency of fever correlated significantly with CD4 count. Bodily pain had statistically significant correlations with the Pain domain of the MOS-HIV. Hot/cold spells were not significantly correlated with CD4 count. CONCLUSION: The three symptom methods and their scores were strongly intercorrelated. Participants with more symptoms reported greater frequency and bother from symptoms.\nSeveral individual symptoms correlated strongly and statistically significantly with longer measures of the same concept, like the MOS-HIV Health Survey. Overall, no one method is clearly superior; different individual symptoms were better measured using different methods. Studies are needed to develop a measure of symptoms commonly experienced by HIV-infected people. Medical College of Wisconsin, Milwaukee, WI PURPOSE: Women have been found to overestimate breast cancer risk. Perceptions are more accurate in higher educated and more numerate women. The objectives of this study are to evaluate the association between African-American (AA) race and breast cancer risk perceptions. METHODS: We assessed 5-year and Lifetime Risk Perceptions of AA and Caucasian women recruited from a primary care clinic. Actual risk was calculated using the Gail Model. Error in estimation (EE) was defined as perceived risk minus calculated risk. The following categories were defined. Under-estimators had an error of < À10%, Accurate Estimators had an error of > À10% but < 10%, Moderate Over-estimators had an error of > 10% and < 30%, High Overestimators had an error of > 30% and < 50%, Extreme Over-estimators had an error of > 50%. Nonparametric statistics were used to compare EE between groups. RESULTS: There were 254 subjects. Sixty-eight percent (68%) were Caucasian, 30% AA, and 2% other. AA were younger (mean of 55 yrs vs. 58 years, p = 0.03), of lower income (84% vs. 35% of Caucasians < $20,000/year, p < 0.0001), had lower Rapid Estimate of Adult Literacy in Medicine (REALM) scores (mean 53 vs. 65, p < 0.0001), and less likely to answer a set of three numeracy questions correctly (11% vs. 55%, p < 0.0001), compared to Caucasians. The calculated mean lifetime and five-year risk of AA women was lower than Caucasian women (lifetime risk: 5.5% vs. 9.7%, p < 0.0001, five-year risk: 0.89% vs. 1.8%, p < 0.0001). In univariate analyses, race, age, and reading level were not associated with accurate risk perception. A trend of increased accuracy of lifetime and five-year risk with higher numeracy was found (p = 0.08). The table shows error in estimation stratified by race (*p-values > 0.05 using a chi-square test). CONCLUSION: In a primary care population, AA women had similar levels of accurate breast cancer risk estimation as Caucasian women, despite lower education and numeracy levels. Both populations could benefit from education to improve the accuracy of risk estimation. PURPOSE: Deciding on anticoagulating the oldest-old (OO)with atrial fibrillation (AF)with warfarin is an increasingly common problem for general internists. Age is an important risk factor not only for AF, but also for stroke in patients with AF. In addition, other risk factors for stroke in AF, such as hypertension (HBP)and diabetes (DM), also increase with age. Though anticoagulation(AC)has been shown to be effective in preventing strokes in some patients with AF, these studies cover only short periods and include few OO. Also, contraindications and side effects of AC are greater in the OO, the fastest growing segment of the population. METHODS: A cost-effectiveness analysis was developed using 3 Markov states: 1ife without stroke, life with stroke residuals and death. Quality-adjusted life expectancy (QALE) was calculated for each state. Baseline probabilities and rates were obtained from systematic reviews and age-adjusted. Based on self-report, women were categorized into three subgroups Ð current, past, and never drinkers. A drinker was defined as someone who drank moderately ( < 2 drinks/day). Depression was evaluated by using the Geriatric Depression Scale (low score indicating less depression) and Social Networks were defined by using the Lubben Social Network Scale (higher score indicating worse social network). RESULTS: When compared to past drinkers, current drinkers are less likely to carry a previous diagnosis of depression, score low on the depression scale, and take antidepressants. Current users are more similar to never users for these measures. CONCLUSION: Women who currently drink a moderate amount of alcohol are less likely to be depressed and more likely to have a stronger social network than women who are past or never users. Questions regarding possible confounders and why past users quit using alcohol may be answered after analyzing a recent questionnaire describing this cohort's pattern of alcohol use. PURPOSE: Public health departments have traditionally provided episodic safety net services to the uninsured. Increasingly, these providers have taken up the role of providing primary care clinical preventive services as well, particularly to racial/ethnic minority populations. To assess the association of race/ethnicity and other patient and system characteristics with receipt of preventive health care services by low-income women, we studied patients cared for through the Los Angeles-County-Department of Health Services(LAC-DHS) Primary Care Network. METHODS: We sampled patients receiving primary care services at 50 facilities to represent all patients in care throughout the LAC-DHS Primary Care Network. 1,288adult females were interviewed for a response rate of 80%. The sample was racially and ethnically diverse-63% Latina/Hispanic, 22% white, 13% African American,1% Asian/Pacific Islander and 1% Mixed race/Other. We measured receipt of the following tests and services: Pap smear within 3 years, mammogram within 2 years among women age 50 years and older, flu shot among women at risk, and cardiovascular disease testing and counseling among women at risk. We used bivariate and multivariate logistic regression analyses to assess the association of patient characteristics (age, race/ethnicity, education, income, immigration status, health insurance status, health status) and system characteristics (type of facility-comprehensive health center, personal health center, hospital outpatient clinic and public/private partnership clinic) with receipt of preventive health care services by low-income women within a publicly funded health care system. RESULTS: Eighty-eight percent of women had undergone cervical cancer screening within 3 years and 82% of women age 50 years and older had undergone breast cancer screening within 2 years, with the highest rates of screening among Latina patients. Among women at increased risk for morbidity and mortality associated with influenza 41 % had received a flu shot within one year. For women at increased risk for cardiovascular disease 91 % had undergone cholesterol screening within 3 years, 82% had been counseled about the importance of exercise, 86% about nutrition and healthy eating and 46% about tobacco use. In multivariate regression analyses Latinas remained the most likely to have undergone cervical and breast cancer screening and among women at risk for influenza African Americans were the most likely to have received a flu shot. CONCLUSION: For low-income women who are able to gain access to a large urban publicly funded health care system for their primary care needs their level of receipt of preventive health care is comparable or better than that of women in other healthcare settings, although shortcomings remain. PURPOSE: Studies have demonstrated that people seek emergency department services for a variety of reasons, including not being able to otherwise access needed health care. To assess the use of emergency department services(emergent and non-emergent) by low-income patients with a regular source of care we interviewed a sample of patients who received their medical care through a large public health system. METHODS: We performed a stratified cross sectional study utilizing probability sampling techniques and survey methods. We sampled patients receiving primary care services through the Los Angeles County Department of Health Services Primary Care Network. 1819 adults were successfully interviewed for a response rate of 80%. The sample was racially, and ethnically diverse. We used bivariate and multivariate logistic regression analyses to assess the association of patient characteristics including insurance status and use of primary care services with visits ( 1 or more) to the emergency department within the preceding 12 months. RESULTS: Twenty-eight percent of the adults in this sample had made one or more visits to an Emergency Department for medical treatment within the preceding 12 months. Almost half of those who had used emergency department services reported that they felt their medical problem was an emergency (49%),but a substantial number of patients sought emergency care for financial reasons(10%) and ease of accessibility to needed medical care (12%). Multivariate analyses revealed that patient characteristics associated with a greater likelihood of having used emergency department services included being white, poor health status, having Medicaid rather than being uninsured, and having delayed seeking needed medical care. CONCLUSION: Patients within a publicly funded medical system have high rates of emergency department use. Type of health insurance and whether people had delayed receiving needed medical care during the preceding year were associated with use of emergency department services. Thus, access and barriers to needed medical care are related to use of emergency department services for emergent and non-emergent medical care. PURPOSE: Conflicting study results regarding the role of intranasal steroids in patients with rhinosinusitis create a dilemma for clinicians. Using a double-blind, randomized, placebocontrolled methodology, we examined whether the addition of an intranasal steroid to conventional antibiotic therapy speeds the recovery of patients with recurrent acute rhinosinusitis. METHODS: Ninety-five (95) patients, ages 18 or older, presenting with acute rhinosinusitis and a previous history of recurrent sinusitis or chronic rhinitis were enrolled from 22 primary care and otolaryngology practices nationwide. Either plain film sinus radiograph or nasal endoscopy supported the diagnosis of sinusitis for all patients. Patients with a history of chronic bacterial sinusitis, previous sinus surgery, and recent intranasal steroid or antibiotic use were excluded. Subjects were randomized to receive either 2 puffs of fluticasone proprionate (n = 47) or saline nasal spray (n = 48) in each nostril once daily for 21 days. All patients received 2 puffs of oxymetazoline hydrochloride in each nostril twice daily for 3 days initially and cefuroxime axetil 250 mg twice daily for 10 days. Patients recorded their daily symptom status in a diary during the 3-week treatment phase. Telephone follow-up was conducted at 10, 21, and 56 days after enrollment; 88 patients (92.6%) completed all telephone follow-up. The primary endpoint was time to clinical success over the 8-week follow-up period (defined a priori as cured or much improved). RESULTS: Patients using the regimen which included fluticasone propionate achieved higher rates of clinical success, 93% versus 74% in the placebo group (p = 0.009 by Chi-square test). The relative benefit increase was 26% with the inclusion of fluticasone to the regimen and the absolute benefit increase was 19%; the number needed to treat to achieve one additional treatment success was 6. Fluticasone-treated patients improved significantly more rapidly (median days to clinical success was 9.5 and 6.0 days, respectively; p = 0.012 by log rank test). Other covariates, including specialty vs. primary care study site, age, number of comorbidities, gender, race, recent upper respiratory infection, or history of allergy, were tested using a Cox proportional hazards model, but only the inclusion of fluticasone to antibiotic and decongestant therapy significantly influenced the clinical outcomes. CONCLUSION: The addition of a 3-week course of fluticasone propionate to oxymetazoline (3 days) and antimicrobial therapy with cefuroxime (10 days) improves clinical success rates and accelerates recovery of patients with a history of either chronic rhinitis or recurrent sinusitis who present for treatment of acute rhinosinusitis. PURPOSE: Disadvantaged populations are often the last to benefit from innovation, including quality improvement (QI) programs for depression. Public sector clinics may not have the resources to implement and maintain QI programs and QI programs designed for private patients may not address needs, treatment preferences, and resources available to depressed, indigent patients. Careful needs assessment can help formulate objectives of QI for such populations, but assessment may be limited by the fact that such populations have little prior exposure to appropriate treatments. We present the development of a unique approach that demonstrates respect for indigent patients and their providers by employing market research strategies to rigorously assess their preferences for and barriers to depression care, as the first step to developing a tailored QI program. METHODS: We conducted literature reviews of existing primary care depression interventions and reviewed methods for understanding patient treatment preferences, including the use of market research methods for assessing consumer preferences. Key informant interviews were conducted with 4 principal investigators of depression treatment interventions. RESULTS: The reviews indicated that few existing QI studies for depression explicitly incorporate assessment of and respect for patient treatment preferences in intervention design. Further, valid methods for assessing patient and provider preferences for and barriers to depression care have not been systematically applied to treatment interventions for the poor. Market research techniques including conjoint analysis surveys have been validated and used to design appealing and acceptable consumer products and services. Because market research methods, such as conjoint analysis, have not been commonly used in developing health services interventions, especially in populations with little treatment experience, we have developed an iterative process for assessing and validating patient and provider preferences and barriers to care. The method includes 4 phases: 1.) patient and provider focus groups, 2.) a market research survey (using conjoint analysis) 3.) a pilot depression intervention in which patient preferences, barriers to care, and opportunities for treatment are identified and results from step 2 are validated, and 4.) indepth qualitative interviews with patients and providers. For all phases of the design depressed patients from the general internal medicine clinic are identified using waiting room screening. Focus groups were held with depressed patients to inform the design of the conjoint analysis survey. Of 86 patients in the general internal medicine clinic screened for focus groups, 26 (36%) had current depressive disorders, 23 patients were eligible to participate in focus groups, and 15 (65% of those eligible) did so. The mean age of focus group participants was 52 (SD = 8.9); 14 of 15 were female; 73% had less than 6 years of education; and all were Spanish speaking. Only one participant reported prior treatment for depression. Patients preferred counseling as the first line treatment for depression, but would consider antidepressants if recommended by a caregiver. A majority preferred group over individual counseling. Patients preferred to receive treatment at the primary care clinic, but from a mental health specialist rather than a primary care provider. Patients reported that their lack of knowledge regarding depression and its treatment and their fear of treatment costs were the main reasons that they had not sought care. In the second phase, 175 depressed patients are recruited to complete a conjoint analysis survey. In this technique, key attributes of a proposed product and possible variations (called``levels'') of the attributes are defined. Hypothetical products with variations of the defined attributes are rated or ranked by patients according to their preferences. Conjoint analysis then determines the relative importance consumers attach to each of the attributes and the utilities they attach to each variation or level of the attributes. We assess the validity of conjoint analysis for determining preferences for and barriers to depression care by comparing survey results with actual treatment choices and barriers to care following a limited depression intervention. Finally, indepth qualitative interviews developed with a medical anthropologist are used to complement quantitative data gathered during the pilot intervention. CONCLUSION: Previous QI programs for depression in primary care have generally not included systematic assessment of patient and provider preferences for and barriers to care in their design. Rather than making assumptions about the needs of providers and consumers in disadvantaged settings, researchers should rigorously assess the preferences of those most affected by a planned QI intervention. A rational and respectful approach for assessing patient and provider preferences using market research methods is proposed for developing and adapting health care QI interventions for disadvantaged populations. PURPOSE: To use anti-Xa activity to monitor enoxaparin dosing in the following patient types: greater than 65 years old, with a creatnine clearance less than 30ml/min and in morbid obesity. To develop a weight adjusted dosing method for patients over 65 years old and for patients with a creatnine clearance less than 30mls/min. METHODS: All patients were dosed by their physician using 1mg/kg sub-cutaneous every 12 hours. Peak anti-Xa activity was obtained 3 ± 5 hours after the third dose, trough activity was obtained just prior to the dose. Anti-Xa activity was measured using Dioagnostica Strago instrumentation. aPTT was obtained simultaneously. RESULTS: Thirty-eight patients were involved in the evaluation. Thirty-six peak levels and 17 trough levels were obtained. Thirteen patients greater than 65 years old were able to be evaluated. All values are mean: age 77, Scr 1.29mg/dl, CrCl 42ml/min, peak anti-Xa 1.3IU/ml. aPTT was prolonged in the vast majority of patients who had anti-Xa activity > 1.0IU/ml. One patient experienced a major hemorrhage. CONCLUSION: Doses of 1mg/kg of enoxaparin in elderly patients, especially females, seems to produce higher than expected peak anti-Xa activity. This can result from a decrease in renal function and an altered volume of distribution. As a follow-up to this evaluation we developed a weight-adjusted dosing method for enoxaparin in our elderly patients. We realize anti-Xa acitvity is only one of several pharmacologic properties of enoxaprin. PURPOSE: Self-reports of chronic diseases and general measures of health status predict use of health services but it is not established how well they predict mortality or hospitalization. We developed the Seattle Index of Co-morbidity (SIC) using self-reported chronic illnesses and tested its ability to predict mortality and hospitalizations among general outpatients. METHODS: Using data from the Ambulatory Care Quality Improvement Project (ACQUIP), we conducted a prospective cohort study of patients from GIM clinics at 7 VA medical centers.\nOur primary outcomes were all-cause mortality and first hospitalization. Of the 34,103 subjects who were eligible for ACQUIP, 12,388 subjects returned both a health inventory and the SF-36 at entry to the study and were eligible for this analysis. The health inventory asked whether a provider had told patients if they had any of 25 common chronic illnesses. We used Coxmodeling to estimate the hazard ratio for mortality and for first hospitalization. Because patients below 50 years of age violated the proportional hazards assumptions, the analysis was restricted to the 10,947 patients over 50 years of age. These patients were followed for a mean of 722.5 (plus or minus 84.3) days. RESULTS: Using a derivation set of 5,469 patients, the SIC was constructed using age, smoking status and 7 of 25 self-reported medical conditions that were univariately associated with increased mortality. The SIC was predictive of both mortality and hospitalizations when tested in a validation set of 5,478 patients. Multiple imputation methods were compared with a strategy of enhancing the clinical database by merging it with diagnoses derived from administrative ICD-9 codes in hospital discharge data and day procedure data (Norris et al., 2000) . Logistic regression models predicting death at one year were based on the different missing data strategies applied to the 1995 data and were evaluated using measures of discrimination and goodness of fit. The strategies were further evaluated by examining how well the logistic regression models predicted outcomes in data collected from patients in 1996.\nThe different methods produced similar logistic regression coefficients. The Cstatistics for the logistic regressions were 0.825, 0.819, 0.815 and 0.802 for the MICE, transcan, norm and data enhancement methods respectively. Decreases in deviance from the null model were 469.4 (MICE), 432.0 (transcan), 412.3 (enhanced data) and 412.2 (norm). When the logistic regression models were applied to 1996 data, the C-statistics were 0.806, 0.803, 0.803, and 0.800 for the MICE, transcan, enhanced data, and norm models respectively. Decreases in deviance from the null model were 236. 0, 229.5, 226.5, and 225 .5 for the MICE, transcan, norm, and enhanced data models. CONCLUSION: The performance of the data enhancement and multiple imputation strategies, although not identical, was generally similar. Multiple imputation methods require considerable statistical expertise, while data enhancement requires availability of data resources.\nResearchers should therefore base their choice of methods on their team's expertise and data resources, rather than on performance considerations alone. Univariate and multiple-linear regression analysis showed that the level of fatigue after one month were significantly improved for the group who received iron in comparison with the placebo group (p = 0.0049). This improvement was independent of the depression or anxiety scores. Interestingly, among the women in the iron group, the best predictor of response (in a regression analysis) was the amount of iron consumed and not the serum ferritin at baseline (p= 0.007). After the first month , the patients who received iron because of a low level of ferritin were greatly improved at 3 months in comparison with no treatment at all (p = 0.0005). CONCLUSION: Iron supplementation even in the absence of anemia may be beneficial to women complaining of unexplained fatigue. PURPOSE: Some previous studies have found that depressive symptoms predict coronary artery disease. This association has been less strong and not as well studied in women, especially older women. Our aim was to analyze prospective data from a cohort of older women to determine if depressive symptoms predict myocardial ischemia or infarction. METHODS: The Fracture Intervention Trial (FIT) was a multicenter randomized control trial of alendronate vs. placebo among 6459 women with low bone mass 55-80 years of age. At baseline, each participant was examined and completed an extensive questionnaire that included the 16 item Geriatric Depression Scale (GDS). The 140 women with a previous myocardial infarction (MI) were excluded from this analysis. Over a mean follow-up of 4.25 years, 77 women had fatal or non-fatal MIs, and an additional 102 had ischemic events/unstable angina. Medical records, discharge summaries, and death certificates were collected from each event, and were blindly adjudicated by 3 cardiologists. Relative hazard models, adjusted for potential confounders, were used to model the association between GDS scores and documented cardiovascular events. Results are reported as relative risk and 95% confidence intervals. RESULTS: Mean GDS (SD) was 1.52.0, and 168 (5%) reported 5 or more depressive symptoms. In age-adjusted analyses, a GDS score of 5 or more was associated with a trend towards increased MI and acute myocardial ischemia. After adjustment for age, self-reported health, BMI, smoking, alcohol consumption, exercise, hypertension, stroke, diabetes, and use of estrogen, however, there was no longer an association between depressive symptoms and subsequent ischemia or MI (Table 1) . CONCLUSION: In this cohort of older women, there was no significant association between depressive symptoms and subsequent myocardial ischemia and/or infarction when the data was adjusted for potential confounders.\nRelative Risk (95% CI) of Cardiac Events in Women with GDS score of 5 or greater. Johns Hopkins University, Baltimore, MD PURPOSE: While poverty and minority status have been associated with poor health and health care outcomes, research suggests that social networks may improve health status. However, the relationship between the degree of social integration into a community and heath care seeking behaviors is unclear. The objective of the study was to evaluate the relationship between social integration and positive health care seeking behaviors in a low-income African-American community.\nMETHODS: This secondary cross-sectional analysis used data from a door-to-door survey of 2,196 residents in a low-income, urban, and African-American community. The measures of social integration included 1) frequency of church attendance and 2) duration of residence in that community. Health care seeking measures were 1) Pap smear, 2) mammogram and, 3) dental visit all within 2 years, and 4) blood pressure measurement within 1 year, 5) having a regular source of care, and 6) not seeking care when needed. In multivariate analyses, we controlled for socioeconomic factors (age, gender, education, employment, marital status, and health insurance) and number of chronic diseases. Interactions were examined between measures of social integration and other covariates. RESULTS: The study population had a mean age of 44 years, 37% male, 63% completed high school, 21% married, 74% had health insurance, and 25% had > =2 chronic diseases. Eight hundred and eighteen (37%) went to church at least once a month and 1116 (51%) resided in that community for 10 or more years. In multivariate analyses, regular church attendance increased dental visits (OR heath care seeking behaviors is unclear. The objective of the study was to evaluate the relationship between social integration and positive health care seeking behaviors in a lowincome African-American community. METHODS: This secondary cross-sectional analysis used data from a door-to-door survey of 2,196 residents in a low-income, urban, and African-American community. The measures of social integration included 1) frequency of church attendance and 2) duration of residence in that community. Health care seeking measures were 1) Pap smear, 2) mammogram and, 3) dental visit all within 2 years, and 4) blood pressure measurement within 1 year, 5) having a regular source of care, and 6) not seeking care when needed. In multivariate analyses, we controlled for socioeconomic factors (age, gender, education, employment, marital status, and health insurance) and number of chronic diseases. Interactions were examined between measures of social integration and other covariates. RESULTS: The study population had a mean age of 44 years, 37% male, 63% completed high school, 21% married, 74% had health insurance, and 25% had > = 2 chronic diseases. Eight hundred and eighteen (37%) went to church at least once a month and 1116 (51%) resided in that community for 10 or more years. In multivariate analyses, regular church attendance increased dental visits (OR PURPOSE: Airline passengers are particularly vulnerable to the effects of cardiac arrest due to a lack of access to emergency medical services. To offset this relative isolation, airlines are installing automated external defibrillators (AEDs) on commercial aircraft. Our objective was to measure the cost-effectiveness of airline AED programs and estimate their value to the flying public. METHODS: A decision analytic model was constructed to estimate the clinical and economic effects of airline AEDs. Inputs were obtained from published data and the FAA. Utility estimates were derived from cardiac arrest survivors. The medical event rate was .0004 per flight in the base case; 15% of medical events were cardiac arrest. Of these cardiac arrests, 48% were ventricular fibrillation or tachycardia. Cardiac arrest survival with an AED on-board was 40%, compared to 8% without AED access. The AED cost (including training) was $2.51 per flight. Sensitivity analyses evaluated changes in AED cost, probability of cardiac arrest (and arrest survival), and the likelihood of airplane diversion. Since AEDs may provide utility gains through`p eace of mind'' for passengers not experiencing a medical event, the impact of this added passenger confidence was also evaluated. RESULTS: AEDs on commercial aircraft cost an incremental $5.16 per flight (approximately $.05 per passenger). AED deployment resulted in an estimated additional $162,000 per QALY gained (16 quality-adjusted minutes per flight). Sensitivity analysis of event probabilities and cost inputs did not substantially change the results. However, the cost effectiveness of AEDs was significantly enhanced by the inclusion of utility gain experienced by passengers from increased peace of mind. While the magnitude of this benefit is unknown, an incremental increase of .003 in utility over the flight duration would reduce the incremental cost-effectiveness of AEDs to less than $50,000 per QALY gained. CONCLUSION: Our model estimated that when the benefits of on-board AEDs are limited to patients experiencing a medical event, the incremental cost-effectiveness is inferior to most recommended medical interventions. However, if passengers gain utility from knowing an AED is on the aircraft, then these incremental expenditures may be justified. Utility gains from``peace of mind'' may have significant implications in determining the value of health care interventions. Thus, further research should be conducted into this potentially important area.\nA CHF OUTPATIENT POPULATION. B. Fenster 1 ; 1 Stanford University, Stanford, CA PURPOSE: Angiotensin Coverting Enzyme (ACE) inhibitors have been well established as first-line therapy in a wide spectrum of congestive heart failure (CHF) patients (1, 2) . ACE inhibition has been proven to ameliorate symptoms, decrease hospitalizations, and decrease mortality. In patients in whom ACE inhibitors cannot be used due to contraindications, intolerances, or allergies, angiotensin receptor blockers (ARB's) or isosorbide dinitrate and hydralazine, particularly in combination, have been shown to provide similar therapeutic benefits (3, 4) . Although the use of ACE inhibitors in treatment of CHF has become standard of care, previous studies have demonstrated wide variations in utilization patterns. Furthermore, few studies have directly assessed ACE inhibitor utilization among CHF outpatients (5,6). Finally, little is known about the usage of ACE inhibitor-alternatives in the setting of ACE inhibitor intolerance. The purpose of this study is to determine the utilization patterns of ACE inhibitors relative to established ACE inhibitor-alternatives in an outpatient CHF population. METHODS: The Palo Alto Medical Foundation (PAMF) is a large multi-specialty clinic located in Palo Alto, California. The PAMF CHF Registry consists of a database listing demographic and clinical data for 229 PAMF outpatients who currently carry the diagnosis of CHF. A retrospective electronic medical record audit of the PAMF CHF Registry population was performed to assess current utilization and dose administration of ACE inhibitors. Patients not receiving ACE inhibitors were assessed for ACE inhibitor-alternative usage, specifically ARB's, hydralazine, or isosorbide dinitrate. Reasons for ACE intolerance, contraindications, or allergies were also recorded. RESULTS: Review of all 229 patients in the PAMF CHF registry revealed that 87% of patients were receiving with either ACE inhibitors or established ACE inhibitor-alternative therapy. Sixty seven percent of all patients were receiving ACE inhibitor therapy, and 68% of these patients were treated with doses considered to be within the target range. An additional 20% of the population was being treated with either ARB's or hydralazine and/or isosorbide dinitrate. Many patients (13%) who were treated with ACE inhibitors experienced allergies or intolerances. CONCLUSION: The overwhelming majority of PAMF CHF outpatients are receiving either ACE inhibitor or ACE inhibitor-alternative therapy. In addition, the majority of patients receiving ACE inhibitors are receiving doses within target range. Although current practice patterns are better than predicted, a small but significant population exists that may benefit from ACE inhibitor initiation and/or more aggressive titration of existing ACE inhibitors. However, such efforts may be limited by a significant number of ACE inhibitor intolerances, allergies, and contraindications. Patients with alcohol problems are common in primary care and there may be advantages to treating these patients in this setting. However, there are few systematic reviews that focus on the efficacy of treatment for alcohol problems in primary care. Our goal was to evaluate the literature on treatment of alcohol problems in primary care according to alcohol consumption outcomes and methodological standards. METHODS: We searched the published literature for years 1966 ± 1999 to identify studies on treatment of alcohol problems in primary care. We included studies from English language peer reviewed journals that were (1) performed in a primary care setting; (2) had as the main focus the treatment of alcohol problems; (3) had two or more treatment arms; and (4) reported alcohol consumption outcomes. Two reviewers appraised all articles for methodological content and results according to pre-specified coding criteria. RESULTS: We identified eleven studies on treatment of alcohol problems in primary care. Seven described treatments for at risk drinking, while four described treatment for alcohol abuse and dependence. The studies were performed in a variety of primary care settings and all reported on the efficacy of brief interventions (BI). There was marked variability in these brief interventions with 4 (36%) using a single session, 6 (55%) using a drinking diary and 5 (45%) discussing a treatment plan. BI were effective in reducing alcohol consumption compared to control treatments in only three of the eleven studies (27%). This finding was only demonstrated in studies of at risk drinking. The studies inconsistently adhered to methodological standards with only 3 (27%) using a single step recruitment procedure and reporting the participation rate, none (0%) providing a full description of patient demographics and medical/psychiatric comorbidity, 1 (9%) assessing prognostic susceptibility, 10 (91%) performing randomization, 1 (9%) assessing ancillary treatments, 0 (0%) assessing discrimination between the treatments, and 5 (45%) providing follow-up on all enrolled subjects. CONCLUSION: This systematic review, restricted to studies performed in primary care found that, in contrast to expert recommendations, there is contradictory evidence on the efficacy of brief interventions for at risk drinkers in primary care. Based upon evidence from the medical literature, patients in primary care with alcohol abuse or dependence are unlikely to decrease their alcohol consumption as a result of brief interventions alone. Finally, studies of brief interventions in primary care inconsistently adhere to methodological standards.\nREPLACEMENT AMONG AFRICAN-AMERICANS WITH OSTEOARTHRITIS? M.K. Figaro 1 , P. Williams-Russo 1 ; 1 Cornell University, New York, NY PURPOSE: Total Knee Replacement (TKR) improves mobility, decreases pain, and improves quality of life for patients with severe knee osteoarthritis (OA). Despite the higher prevalence and higher reported disability of knee OA among Blacks, whites are over 1.5 times more likely to have TKR than Blacks. This study examined beliefs of Harlem Blacks with OA regarding TKR, including its efficacy and safety. METHODS: Community-dwelling older adults were recruited from churches, senior centers or other social networks in Harlem. In-person interviews elicited familiarity with others in the community who had TKR, perceived knowledge of TKR, referral to specialists, concerns and beliefs regarding surgery. We also assessed demographic data and severity of OA using the Western Ontario and McMaster Osteoarthritis Index (WOMAC). RESULTS: Women made up 84% of the sample of 65 patients. The mean age was 73, with a mean duration of symptoms of 9 years; 61% had at least a high school education and 45% had an income < $10,000 in 1999; 99% of subjects had Medicare insurance. Subjects had a mean WOMAC score of 56 +18, indicating high levels of OA disability; 45% had been referred to specialist. Table 1 summarizes major factors and concerns.\nCONCLUSION: The most important of subjects' concern were regarding post-surgical pain, low expectations of the eventual efficacy of surgery, and concern over the cost of TKR. In addition, despite the high WOMAC disability score of the group, less than 50% of patients were referred to a specialist. Efforts to address the current disparity in use of TKR should attend to these potentially modifiable factors. We conducted a randomized effectiveness trial in the General Internal Medicine clinics at 7 Veteran Affairs facilities that had discrete firms. All patients who were assigned a primary provider and who had an appointment in the prior year were eligible. Randomization, intervention and analysis were by firm. Respondents to a baseline health inventory were regularly mailed the SF-36 and, as relevant, validated questionnaires about 6 chronic conditions: coronary disease, COPD, depression, diabetes, alcohol use and hypertension. From the SF-36, we computed physical and mental component scales (PCS and MCS). We also sent surveys about satisfaction with humanistic and organizational aspects of care and retrieved clinical data from computerized records. The information collected was presented to primary care providers of eligible patients at every visit for 2 years in graphical format (e.g., blood pressure, HbA1C, SF-36 scale scores and anginal frequency) plus interpretations (e.g.,``Patient unable to climb stairs due to shortness of breath'') and``tips'' derived from national guidelines. Clinicians also received summary reports comparing their patient panels with local and national norms and highlighting patients with potentially serious problems. ). There were also no significant differences in satisfaction in the humanistic or organizational domains between intervention and control firms. Similar results were found using several analytic strategies including restriction to patients who completed all forms. CONCLUSION: Routine collection and feedback of measures of general and conditionspecific health and satisfaction did not improve outcomes. It is likely that such data must be linked to specific management suggestions if they are to influence patients' outcomes. in several studies appears to be an independent risk factor for developing incident coronary artery disease (CAD). The biological mechanism explaining the increased risk is not certain. We wanted to determine if C-reactive protein (CRP), a marker of increased risk for CAD, is associated with major depression. METHODS: Our analysis is based on data from the third National Health and Nutrition Examination survey conducted between 1988-1994. The Diagnostic Interview Survey (DIS) was administered to 8451 individuals between 17 ± 39 years of age to assess current and past episodes of major depression. The analysis is based on 7091 individuals (3849 females and 3242 males) with complete data on CRP and other important covariates. Analysis was stratified by gender because of the influence of estrogens on CRP levels. Logistic models were used with detectable CRP as the dependent variable. Unadjusted and adjusted estimates for current (last year) and past major depression compared to never lifetime depression were calculated. Estimates are adjusted for sampling design and nonresponse. RESULTS: Men with current major depression (OR=3.29 95% CI 1.56, 6.92) and past major depression (OR=2.05 95% CI 0.84, 5.04) had elevated levels of detectable CRP after adjustment for age, African-American race, BMI, total cholesterol, log triglycerides, diabetes, systolic blood pressure, tobacco smoking (self-reported current or past or cotinine level above 10 ppm), and alcohol intake compared to never depression. A similar trend was found for single episode of depression (OR=1.13 95% CI 0.26, 4.90) and multiple episodes of depression (OR=3.87 95% 1.67, 8.96) compared to never depression after adjustment. There was a significant linear trend between number of lifetime depressive symptoms and elevated CRP (p < 0.001). The relationship between depression and elevated CRP was present for smokers, nonsmokers, and those with low or high total cholesterol. For women, there were no associations between any of the measures of depression and elevated CRP. Adjusting for current use of estrogens or ever use of oral contraceptive agents and excluding those who were pregnant did not substantially change the results. CONCLUSION: In young men, current major depression is associated with elevated CRP levels that appear to return toward normal when the depression remits. CRP may be one mechanism by which depression leads to increased coronary heart disease in men. The same associations were not present in women but we may not have been able to adequately account for their hormonal status. This cross-sectional data needs to be confirmed with prospective studies. PURPOSE: Understanding why people drink alcohol is important for the health and safety of individuals and the public. The aim of this study was to examine from a cognitive point of view the hypothesized link between drinking and stress. METHODS: 25 scenarios were constructed by combining two items, either two life-change events or a social situation and an emotional state. In the initial three experiments, 159 male and 43 female alcoholics and 157 male and 93 female non-alcoholics in France judged the degree to which these scenarios were stressful and subsequently the degree to which they stimulated an urge to drink. In the final experiment, 126 of the male alcoholics were studied at the beginning and end of an in-patient alcohol rehabilitation program. RESULTS: The alcoholics and non-alcoholics, regardless of gender, assigned similar stress values to the scenarios and used the same cognitive rules for combining the stress associated with two items (disjunctive rules for two life-change events and additive ones for a personal emotion combined with a social situation). They differed, however, in how they judged the urge to drink. The non-alcoholics reported little stimulus to drink from any combination of items, whereas the alcoholics not only perceived the individual items as stimulating an urge to drink, but also used the same cognitive rule in judging the combined urge to drink of two items as they used in judging the combined stress. After completing rehabilitation, the alcoholics judged the combinations of life-change events as stimulating less stress and less urge to drink; nevertheless, they continued to use a disjunctive combination rule. CONCLUSION: Stress and drinking are linked at a fundamental cognitive level among alcoholics, though not among non-alcoholics. Alcoholics should be helped to recognize this link, to reduce their feelings of stress, and to respond to stress in ways other than drinking. documented. Yet, widely used systems of monitoring quality such as the NCQA's HEDIS measures do not require plans to report results separately by patient sociodemographic group. This AHRQ sponsored project is investigating the need for and feasibility of plans reporting selected HEDIS measures stratified by key social groupings. Our initial analysis examines plans' performance on advising smokers to quit, stratified by social group. METHODS: We obtained HEDIS 2000 data for 3,663 commercially-insured and 2,824 Medicaid-insured members enrolled in 7 UnitedHealthcare plans located in 3 regions. As specified by the NCQA, the Consumer Assessment of Health Plans Survey (CAHPS 2.0) was used to collect information on whether the enrollee was a current or recent smoker (denominator) and advised to quit smoking (numerator). We obtained information on patients' sociodemographic characteristics, visits, and health from the survey. Response rates were 55% and 38% for commercial and Medicaid patients, respectively. We analyzed rates of advising smokers to quit separately for commercially insured and Medicaid patients as is customary with HEDIS measures. For comparisons between patients in different sociodemographic groups, we pooled data from the different plans. We used Chi-Square tests to compare the proportion of smokers advised to quit by gender (female vs. male), race/ethnicity [nonHispanic White vs. (nonWhites and Hispanics)], and education (high school or less vs. some college or more). Pending multivariate analyses will assess independent effects of these factors adjusting for age, number of visits, and self-reported health. RESULTS: Of the respondents, 693 commercially insured and 973 Medicaid patients were smokers. The average rate of advising smokers to quit was the same for commercial (66%) and Medicaid (66%) patients. Among commercially insured patients, men were less likely to be advised to quit than women (61% vs. 70%, P=.01) and a lower proportion of less educated patients were advised to quit than among more educated patients (70% vs. 64%, P=.10), however, the latter difference was not significant. In contrast, among Medicaid patients, nonHispanic Whites were more likely than other patients to be advised to quit (69% vs. 61%, P=.01). The proportion of commercially insured patients advised to quit was also higher for nonHispanic Whites than for others (67% vs. 60%), but the difference was not significant (P=.32 The study population consisted of 1394 diabetics with a mean age of 69.2 (+/À 7.2 SD), 98.4% male, and 1.6% female. The old-old diabetic population (n=360) comprised 13% of the Louisville VAMC patient population 75 years old and older in 1997 (equivalent to the national rate of diabetes in persons aged 75 and older (13.2%)). The mean cholesterol was 200 mg/dL (+/À 42.8 SD) and the mean LDL was 120 mg/dL (+/À34.3 SD). The oldest-old were least likely to have their cholesterol checked (p= < 0.01). They were also least likely to be placed on cholesterol-lowering medications when their LDL Cholesterol was greater than or equal to 160 mg/dL. However, in 1998 and 1999, the oldest-old were more likely to be treated with medication than the middle old. CONCLUSION: The oldest old are less likely to have their cholesterol checked, but once it is checked, they are more likely than the middle old to be treated. Since diabetes and cardiovascular disease are common, cardiovascular disease is more common in the elderly, and morbidity and mortality from cardiovascular disease is great, physicians need to attempt to reduce morbidity and mortality by adhering to the practice guidelines for cholesterol management Ð even in the oldest diabetics. Practice patterns demonstrated in this study suggest increasing use of cholesterol-reducing medications in older adults. . As indicated by a c-statistic significantly greater than 0.5, the two existing classification schemes predicted stroke better than chance: 0.78 for the scheme developed by the Atrial Fibrillation Investigators (AFI) and 0.82 for the Stroke Prevention in Atrial Fibrillation (SPAF) III scheme. However, the new CHADS2 index performed better with a c-statistic of 0.87. The stroke rate per 100 patient-years without antithrombotic therapy increased by a factor of 1.5 (95% CI, 1.3 ± 1.7) for each 1-point rise in the CHADS2 score (Table) . CONCLUSION: The two existing classification schemes and especially a new stroke-risk index, CHADS2, can quantify the risk of stroke for patients who have AF. Use of the CHADS2 index should aid in the selection of antithrombotic therapy for patients who have non-rheumatic AF. PURPOSE: Medication errors (MEs) and adverse drug events (ADEs) are common in the hospital setting. However, relatively little is known about the frequency of MEs and ADEs in outpatients. Therefore, we prospectively examined the frequency, type, severity, and preventability of MEs and ADEs in the ambulatory setting. METHODS: We studied 2 academic hospital practices and 2 community based clinics in the Boston area, and collected copies of prescriptions written by 24 primary care providers from Sept 1999 to March 2000 (6 weeks per clinic). Prescription copies were reviewed by a pharmacist to screen for MEs and potential ADEs. In addition, patients who received prescriptions were telephoned 2 weeks after their visit to ask about problems with their medications, with a response rate of 59%. Patient-reported problems were presented to 2 MD reviewers, who classified them as ADEs or not, and determined severity and preventability. Events were considered preventable if they persisted for an unnecessarily prolonged period (even if they could not have been completely prevented). RESULTS: Of 1173 prescriptions screened during this time period, 202 (17%) were rule violations (orders that violate strict standards but are generally understood and generate no additional work), 44 (4%) were MEs, 59 (5%) were potential ADEs. The most frequent rule violations were missing route, and the most frequent MEs were errors in route or dose. The most frequent potential ADEs were errors in frequency and dose. Of 661 patients surveyed, 178 (27%) reported a total of 206 ADEs. Of these, 29 (14%) were serious, 74 (36%) were preventable, and 13 (6%) were both. The most frequent types of ADEs and preventable ADEs were CNS-related (35%, 36%), gastrointestinal (21%, 9%), and cardiovascular (16%, 18%). The most frequent medications involved in preventable ADEs were antidepressants (22%), ACE-inhibitors (14%), beta-blockers (10%), and calcium-channel blockers (10%). Of preventable ADEs (n = 74), physicians were primarily responsible in 63% and patients in 33%. The main types of physician error were failure to act on results of monitoring or tests (50%) and inappropriate drug choice (24%). The main type of patient error was failure to inform their physician of problems (100% Unfortunately, educational brochures are often generalized, and do not address the different needs and barriers that impair the self-management required for care of this chronic disease. Few materials are available that cater to individuals of different ethnic backgrounds, especially those with low literacy or education backgrounds. This study evaluated a new multimedia software application for diabetic populations with these needs in mind. The target population of subjects included diabetic patients from an urban, minority community not accustomed to computer usage. METHODS: 22 diabetic African-American patients were consecutively interviewed and digitally videotaped in an urban general medicine clinic. These individuals provided testimonials regarding the barriers, fears, benefits, and myths surrounding the ophthalmologic eye examination including pupil dilation. Specific issues, such as faith, lack of insurance, and misconceptions about eye exams were addressed. The video clips were edited and integrated into a multimedia application that included simple activities to engage the user over 15 ± 20 minutes. The application was available near the waiting room of a clinic for patients to view when waiting for an appointment, or afterwards before leaving. The program uses a touch screen, so previous technological experience was not required. A separate sample of 26 patients viewed the multimedia program, and completed a brief questionnaire. RESULTS: 11 male and 15 female diabetic patients completed the multimedia program and survey, with 17 (65%) African-American, and 18 (69%) having elementary or high school education. 12 of 26 (46%) have never used a computer before, and 18 (69%) described the computer as``easy'' or``very easy'' to use. Preferences for medium of learning about diabetes were greatest for computers (11), followed by doctors and nurses (8), television (5), and brochures and articles (4) . CONCLUSION: A multimedia patient education program that addressed barriers related to literacy and culture was found to be acceptable by a convenience sample. Additional telephone surveys will provide data regarding knowledge and attitudes toward obtaining annual eye examinations, as well as actual compliance with visits. Given the growing interest in on-line consumer health information, the availability of similar applications for underserved populations may help to bridge the``digital divide'' (or, the gap between different communities in terms of access to technology). PURPOSE: While gender selection of physicians has been studied extensively, mostly by survey, selection of physicians by race has rarely been addressed. We used an experimental design and an actor-portrayed video doctor to investigate patients' preferences for physician gender and race. METHODS: Participants were asked which one of six video doctors (one male and female African American, Latino, and Caucasian) they would pick to be their physician after viewing each deliver a 45-second health prevention message. The videotapes were professionally produced and great care was taken to keep all variables constant, including the age, attractiveness, and clothing of the video doctors; the video setting; and the delivery of the prevention message (script, non-verbal behaviors, facial expressions, and interpersonal style Ð e.g., warm, empathic, collaborative). Each group of 20 ± 22 participants viewed one of 18 configurations of the presentation order of the six video doctors to control for any order effect. RESULTS: 400 participants were recruited in a shopping mall in the San Francisco Bay Area. The sample was diverse in gender (39% male, 61% female); race (29% Caucasian, 8% African American, 26% Latino, 30% Asian, and 7% Other); and age (18 years ± 87 years). When asked who they would choose to be their physician, both male and female participants were more likely to choose a female (82%) as their physician rather than a male (18%). Among female participants, 88% chose a physician of their own gender, while only 29% of males chose a male physician. When asked who they would choose to be their physician, 23% of participants chose an African-American physician, 32% chose a Latino physician, and 44% chose a Caucasian physician. Fifty-two percent of African-American participants chose an African-American physician; 44% of Latino participants chose a Latino physician; and 56% of Caucasian participants chose a Caucasian physician. Among Asian participants, 20% chose an African-American physician, 37% a Latino physician, and 43% a Caucasian physician. CONCLUSION: The vast majority of participants in our study chose a female video doctor as their physician, regardless of their own gender. Although approximately half of African-American, Latino, and Caucasian participants chose their own race, the other half were willing to choose a video doctor of a different race. Our data suggests that when selecting a physician, patients may strongly associate women, regardless of a standardized patient-centered delivery, with those qualities they want in their physician. Future studies may want to use video doctor technology to further investigate patients' preferences in selecting their physician, in particular the effect of physician race on patient choice. PURPOSE: Adolescent obesity continues as public health epidemic. Data from the National Health and Nutrition Examination Surveys (NHANES I, II, III) reveal that the percentage of youth above the 85th percentile for body mass index (BMI) increased to approximately 22% by the late 1980's. Further, since the beginning of these surveys, the prevalence of adolescent obesity has increased by 39%, with Hispanic, Native American, and African American children more affected than other populations. A 1997 survey conducted by the Allentown (PA) Health Bureau revealed a 31% obesity rate among adolescent students in the Allentown School District (ASD), where 57% of students are non-white and 62% are eligible for free or reduced lunches. Both decreased physical activity and poor eating behaviors contribute to obesity. General internists have a responsibility but limited opportunities to understand and influence teen nutrition, especially in minority populations. METHODS: To better understand students' attitudes toward nutrition and physical activity, as well as barriers to improving their choices, we conducted focus groups among representative students to determine their food preferences, and dietary and activity patterns. We developed a 57-question survey that we utilized in six focus group sessions with high school students in an alternative program within the ASD. Sessions were audiotaped, and responses to questions categorized into four areas: Food choices, Physical Activity, Self-Esteem/Identity, and Knowledge. RESULTS: In general, students expressed a willingness to eat healthier foods, if provided a variety including fresh fruits and vegetables, as well as culturally acceptable products. Adolescents desired daily gym class with a variety of activities, although admitted that much of their free time was spent engaged in sedentary activities. Groups also expressed indifference, although many had basic knowledge of the consequences of obesity.\nCONCLUSION: Providing greater choices and opportunities for food and physical activity can promote healthier behaviors among adolescents, impacting upon the prevalence of obesity in this population. In particular, minority students may have different needs than others. General internists can have a large impact on adolescent obesity and the subsequent development of related diseases by working together with community leaders and other health care professionals in advocating policies to provide adolescents with these opportunities. PURPOSE: While some studies have suggested that there may be sex differences in access to cardiac procedures, others have found no evidence of`gender bias' in cardiac care. Possible explanations for these inconsistent results include the focus, in some studies, on nonrepresentative patient populations, and the occasional use of data sources that lack clinical detail. We used a clinically-detailed cardiac database to study crude and adjusted rates of cardiac revascularization for males and females in a population-based cohort of patients undergoing cardiac catheterization. METHODS: The Alberta Provincial Project for Outcome Assessment in Coronary Heart Disease (APPROACH) is an inception cohort of all patients undergoing cardiac catheterization in the province of Alberta, Canada. We studied 21,816 patients undergoing catheterization in calendar years 1995 through 1998, and determined rates of percutaneous coronary intervention (PCI) and/or coronary artery bypass grafting (CABG) for males and females in the year following cardiac catheterization. Cox proportional hazards models were then used in a two-step analysis to determine adjusted relative risks for revascularization and adjusted procedure rates. An initial`partial adjustment'; controlled for variables such as age and comorbidities that are routinely available in most databases, including administrative databases. A subsequent`full adjustment' additionally controlled for extent of coronary artery disease and ejection fraction, variables that are only available in detailed clinical databases. RESULTS: For the endpoint of any revascularization procedure (PCI or CABG), the unadjusted relative risk for revascularization for females relative to males was 0.67 ( 1, 1995, and December 31, 1998 . Cases were linked via postal codes to Canadian census data to determine population rates of cardiac catheterization by census-defined geographic regions. Individuals were flagged as being of aboriginal ethnicity when they resided in regions where greater than 95% of 1996 census respondents indicated that they were of aboriginal ethnicity. Such regions generally represent native reservations. Using the Alberta-wide population as a reference, we employed indirect standardization to adjust rates of cardiac catheterization to sequentially control for 1) age and sex, 2) income quintile, and 3) urban vs. rural residence. We also evaluated survival and the occurrence of revascularization procedures after catheterization (i.e., coronary artery bypass grafting [CABG] The prevalence of type II diabetes is much higher among Mexican Americans than among non-Hispanic Whites. Although much of this difference is thought to be genetic, environmental factors such as diet and exercise may also play a role. One way to try to separate genetic from environmental factors is to use acculturation data in immigrant populations. Prior studies on the effects of acculturation on the prevalence of diabetes in Mexican Americans have reported conflicting results. A study of Mexican Americans in San Antonio, Texas showed a decrease in diabetes rate with increasing acculturation. This decrease was hypothesized to be secondary to improved diet and increased levels of exercise in the more acculturated group. The Hispanic Health and Nutrition Examination Survey (HHANES) studied Mexican Americans in the Southwestern United States and found no effect of acculturation on rate of diabetes in Mexican Americans. METHODS: All patients seen in the adult clinic of a large community health center in Denver, Colorado over a fifteen month period were included in our analysis. For each patient seen, diabetes was considered present if the ICD9 code for diabetes was billed as the primary diagnosis on at least one visit during the 15 months. Level of acculturation was assessed by using language preference. Hispanic patients who spoke primarily Spanish were considered less acculturated than those who spoke English. Demographic and language preference data were obtained from registration records. RESULTS: Approximately 5,300 patients were included in our analysis. Of these, 1,500 patients were non-Hispanic, 2,600 were English-speaking Hispanics, and 1,200 were Hispanic who spoke primarily Spanish. By our current diagnostic criteria, overall diabetes prevalence was 14%. Hispanics had a higher prevalence of diabetes than non-Hispanics at all age groups and this difference was more pronounced with increasing age. In the 55 ± 64 yo age group, 31% of Hispanics vs. 20% of non-Hispanics had diabetes. There was no significant difference in diabetes prevalence between primarily Spanish-speaking and English-speaking Hispanics. CONCLUSION: We found no significant effect of acculturation on diabetes prevalence in this clinic population of Mexican Americans. Our findings also support previous reports of increased diabetes prevalence among Mexican Americans when compared to non-Hispanics, and a magnification of this effect in the higher age groups.\nT. Gill 1 , C. Williams 1 ; 1 Yale University School of Medicine, New Haven, CT PURPOSE: When determining the incidence of disability, long assessment intervals maybe problematic because they do not account for the possibility of recovery not for losses to followup due to deaths. In this study, we tested two related hypotheses: (1) longitudinal studies with long assessment intervals underestimate the incidence of disability; and (2) these underestimates increase as the length of the assessment interval increases.\nProject, an ongoing longitudinal study of nondisabled, community-living persons, aged 70 years or older. After a comprehensive, home-based assessment, participants were categorized into three groups according to their risk for disability (low, intermediate, and high) and were subsequently followed for up to two years with monthly telephone interviews (98% completion rate) to ascertain the presence of disability in four key activities of daily living (ADLs).We compared the rate of ADL disability obtained from a single follow-up assessment with that obtained from monthly assessments for intervals of 6, 12, 18, and 24 months. RESULTS: For any ADL and for each of the four individual ADLs (walking, bathing, dressing, and transferring), the rates of disability at 12 months were at least twice as high for the monthly assessments as for the single follow-up assessment. For any ADL disability, for example, the corresponding rates were 0.22 and 0.10. Although the overall rates were lower, the results for persistent disability, defined as a new disability that was present for at least two consecutive months, were similar. As the length of the assessment interval increased, the``true'' rate of disability, as determined by the monthly assessments, was increasingly underestimated by a single follow-up assessment. For example, the rates of any ADL disability for the monthly and single follow-up assessments were 0.28 and 0.09 at 18 months and 0.37 and 0.11 at 24 months, respectively. While these underestimates of disability were due almost exclusively to high recovery rates for low-risk participants, they were due increasingly to losses to follow-up from deaths for high-risk participants. CONCLUSION: The incidence of disability is substantially underestimated by longitudinal studies with long assessment intervals. These underestimates could lead policy-makers to inaccurately estimate the health care needs of community-living older persons. While adolescents in general face many serious health risks, these youth face worse and more chronic health problems, psychiatric and behavioral problems, alcohol and substance abuse, sexually transmitted diseases (STD) and teen pregnancies. School-based clinics have been reported to improve health among adolescents through increased access. We hypothesized that increased access to medical care within the shelter environment would improve health outcomes in this under served population. METHODS: Homeless youth were surveyed in 1992 and 1998 at the only Denver-area shelter dedicated to youth under 21. The first survey, in 1992, occurred when medical services were provided in the shelter 8 hours per week. At the time of the second survey in 1998, medical services had increased to 36 hours per week. The surveys were of structured interview form based on the Adolescent Health Survey instrument. Outcomes assessed included access to health care, health satisfaction, STD's and pregnancies. RESULTS: The two study samples differed somewhat with regard to demographics. In 1998, there were more males (62%) than in 1992 (55%). There were also substantially more African-Americans (16% vs. 7%). The proportion of African Americans among shelter users was twice the proportion of African Americans in the community.`Good' or`Excellent'; self-reported health increased from 65% to74% between 1992 and 1998. Endorsement of frequent difficulty accessing care decreased from 3 1% to 14% between 1992 and 1998. Agreement with the statement,``the last time I needed healthcare I took care of it myself'' decreased from 24% in 1992 to 8% in 1998. The number of self-reported STD's decreased from 26% in 1992 to 17% in 1998, compared with the 2.4% reported in Colorado high school students in 1996. This decrease was seen at the same time a Centers for Disease Control-sponsored STD screening project was working to reach homeless youth, and detection rates may actually have increased. The number of teen pregnancies decreased from 14% in 1992 to 6% in 1998. CONCLUSION: Among homeless youth, increased access to medical care through increased availability of services in the shelter environment was associated with improvements in perceived health, access to health care, and reductions in some common health problems. Continued expansion of services within the shelter could have a profound impact on problems such as mental health and substance abuse.\nPURPOSE: Concussion has been associated with disturbance of physical well-being, thought process, and emotions, that may last 6 months or longer after the time of injury. Homeless adolescents are at higher risk for brain injury than adolescents from safer environments due to increased behaviors leading to injury. Impulsivity and affective liability are part of the DSM-IV definition of Postconcussional Disorder, These attributes have also been noted to be more common in homeless youth. We hypothesized an association between previous head injury and impulsivity and behavioral dyscontrol, as possible symptoms of postconcussional disorder. METHODS: Homeless adolescents accessing services at a shelter were surveyed about health histories, including the statement``Have you ever been hit on the head so hard you lost consciousness''. In the same survey a psychologic symptom battery, the SCL-90, was administered, The incidence of head injury was then compared with psychiatric symptom indices in the highest quartile. An odds ratio for the severity of psychologic symptoms in those with history of concussion was then calculated. RESULTS: Of the 97 youth interviewed, 38 endorsed a history of grade III concussion, generating a prevalence of brain injury which is 40% higher than that reported in one study of high school football players. Odds ratios for psychiatric symptom score in the 75h percentile or greater among those with history of grade III concussion were as follows:\nOdds Ration (95% Ci) Hostility 3.5(1.5 ± 8.4) Phobia 21(0.8 ± 5.2) Impulsivity 2.0(0.6 ± 7.2) Anxiety 1.5(0.6 ± 5.4) Obsessive/Compulsive 1.5(0.6 ± 3.7) Depression 1.3(0.5 ± 3.4)\nCONCLUSION: The symptoms of hostility, impulsivity, anxiety, and depression are included in the DSM-IV definition of Postconcussional Disorder. Impulsivity, anxiety, and depression were modestly, but not significantly, increased in those with history of grade III concussion. A significant association between hostility and concussion was found. As with other studies of brain injury in adolescents, the direction of the relationship between hostility and brain injury is unclear. However, the effect brain injury may have on the skills necessary to exit street life may be serious, and should be further investigated.\nPURPOSE: Echinacea is one of the most popular dietary supplements with over $10 million in annual sales in the U.S. However, since dietary supplements are not regulated by the FDA in the same manner as other over-the-counter products, there is concern as to whether contents are consistent with the labeling. Three species of Echinacea are sold commercially, augustifolia (EA), pallida (EPA), and purpurea(EPU). Each species has a different biochemical footprint, analogous to different contents. Clinical trials have reported decreases in cold symptoms using EPU, decreases in cold symptoms and duration using EPA, and no effect on cold symptoms using EA. It follows that labeling of species content on Echinacea supplements is important consumer information. We hypothesized that species content in Echinacea preparations may not be consistent with labeling. METHODS: Ten single-herb liquid-only preparations of Echinacea from 5 Denver area stores were purchased and sent to a blinded independent laboratory. The preparations were analyzed by thin layer chromatography (TLC) for fingerprints unique to each species of Echinacea. Of the 10 samples sent, 3 were not analyzed due to difficulties with glycerol in the preparation. RESULTS: The following RESULTS: Of the 182 persons interviewed, 120 (66%) eventually enrolled into the program. Enrollees in the work component were middle aged (mean age = 39), minority race (92%), and male sex (64%) and abusing primarily alcohol (31%), crack/cocaine (21%), heroin (5%) alone or poly-substance abuse (44%). They lived in emergency shelters (23%), doubled-up (49%), and bridge-housing (25%) arrangements. Self identified co-morbid medical and psychiatric illness were common and a third were on at least one prescribed medication. Fifty-seven (48% of enrolled) completed the work component of the program. Of these, 95% were subsequently employed at completion of the work component. During the work component, clients were abstinent (40%), improved their housing status (47%), attended medical appointments (87%), reported career skill improvement (69%), and arrest free (84%). Six months after the completion of the program, over 33% continued to be employed full-time, 39% had further improved their housing, and 36% remained abstinent of substance use. A vast majority reported having medical (90%) and psychiatric (93%) service needs met. CONCLUSION: A work stabilization program, supported by a collaborative network of community and academic partners, decreased substance use, criminal behavior, and health care utilization for a drug and alcohol abusing homeless population. Community and academic partnerships can be a powerful impetus to promote health and social improvement for this distressed population. The majority of depressed patients initially present in primary care with physical symptoms rather than psychological complaints. We examined the outcome of physical symptoms with antidepressant treatment of depression during a 9-month period. METHODS: This clinical trial enrolled 573 depressed patients cared for by 40 physicians in two primary care research networks. Patients were randomized to receive one of three selective serotonin reuptake inhibitor (SSRI) antidepressants: paroxetine, fluoxetine, or sertraline. Data was collected from participants via computer-administered telephone interviews both at baseline and after 1, 3, 6, and 9 months of treatment. This data included validated measures for depression, anxiety, and 14 common physical symptoms (SCL-20 and PRIME-MD), healthrelated quality of life (SF-36), work functioning (Work Limitation Questionnaire), and Medical Outcomes Study measures for social functioning, positive well-being, sleep, concentration, and sexual functioning. The prevalence of each physical symptom was assessed at baseline and all follow-up intervals and symptom severity graded from 0 (none)to 2 (bothered a lot). Stepwise linear regression models were used to determine the independent effects of physical symptoms and depression on HRQoL and other domains at baseline and over 9 months of treatment. All models were adjusted for demographics and anxiety scores. RESULTS: Most of the individual symptoms had a baseline prevalence in depressed patients of 50% or greater. Moreover, the prevalence of symptoms that were graded as``bothered a lot'' exceeded 10% for 11 of the 14 symptoms. Physical symptom prevalence and severity dropped substantially during the first 4 weeks of antidepressant therapy with minimal improvement thereafter. While also showing the greatest improvement during the first month, depression and all other outcomes continued to improve over the 9-month trial. Physical symptoms had the strongest association with physical functioning (11% ± 20% of the variance), pain (23%), and overall health perceptions (19%), whereas depression had the greatest association with mental (32% ± 49%), social (11% ± 36%), and work functioning (9% ± 37%). CONCLUSION: Many physical symptoms are present in at least half of depressed patients presenting in primary care. The predominant benefits of antidepressant therapy occur during the first 4 weeks of treatment for physical symptoms, while depression and other HRQoL domains continue to improve for some months thereafter. Physical symptoms and depression have diffential effects on HRQoL domains. METHODS: We surveyed a stratified random probability sample representative of managed care members aged 45 and older in 2 health plan locations (n=2168). Respondents reporting doctor-diagnosed arthritis or chronic joint symptoms (pain, stiffness or swelling of a joint within the past year that is present on at least half the days of a typical month) were considered to have arthritis. Weighted descriptive analyses were conducted to estimate the age-and gender-specific arthritis prevalence, and to describe the impact of arthritis (i.e., health status, activity limitations). We also describe treatments respondents reported using for arthritis and the type of physician who provided the arthritis diagnoses. RESULTS: The prevalence of arthritis ranged from 32.0% for the 45 ± 54 age group to 36.8% for the !75 age group. Women were more likely to report arthritis than men (38.3% vs. 29.9%). Compared to those without arthritis, respondents with arthritis were more than twice as likely to report fair or poor health (24.9% vs. 11.1%). Of those with arthritis, 39.9% indicated activity limitations related to their joint symptoms. Respondents reported using prescription medicines (43.9%), over-the-counter pain medications (57.2%), complementary therapies (i.e., herbs, glucosamine) (32.8%), physical or occupational therapy (20.2%), and exercise (i.e., walking, swimming) (70.5%) to treat their arthritis symptoms. Just over half of those reporting physician-diagnosed arthritis received their diagnosis from a primary care physician. CONCLUSION: One-third of these managed care members have self-reported arthritis and 40% report arthritis-related activity limitations. The prevalence and impact of arthritis on health-related quality of life suggest an opportunity to explore interventions, including education on self-help activities, that may complement care provided by clinicians. None have explored these issues in a multi-culturally diverse population of young women. The purpose of this exploratory study was to assess knowledge, beliefs and behaviors about osteoporosis prevention in this target population. METHODS: We performed a cross-sectional study of multi-culturally diverse college women attending classes over a two week period in 2000. In this convenient sample (N=50), we collected data using a survey instrument of 20 questions designed to measure knowledge, beliefs and behaviors about osteoporosis prevention. Incorporating the selfreported data, we characterized the respondents by demographic information, medical conditions and family history; calculated Body Mass Index, general consumption of calcium, their level of alcohol and nicotine use and degree of physical activity; and tabulated à`B elief-about-Disease'' Score to capture their perception of osteoporosis in relation to other medical conditions. RESULTS: Nearly a third of the respondents were non-White and 14% were of Asian origin. Respondents were able to identify the risk factors of low calcium intake(96%), lack of exercise(86%), and menstrual irregularities(68%). However, other risk factors such as White race(20%), Asian race(21%), and excessive exercise (42%)were less likely to be identified. Ten percent had a medical condition putting them at risk. There was no relationship between correctly identifying lack of exercise and low calcium intake as risk factors and getting adequate osteoprotective exercise ( > 360 minutes/month)(p = 0.53) and dietary calcium( > 1200mg/daily) (p = 0.85). Osteoporosis (3.31) (on Likert scale from 1 ± 5, 1 indicates not at all; 5, extremely)was believed to be significantly less serious than heart disease(3.60) * , breast cancer(3.66) * , AIDS(3.79) * , and Alzheimer's disease(3.54) * ( * =p < 0.05). There were no associations between ethnicity and respondents' responses, knowledge and beliefs. CONCLUSIONS: Many college women, regardless of race, lacked knowledge about the relationship between osteoporosis and low calcium intake, use of tobacco, alcohol, physical inactivity, and excessive exercise. Increasing levels of osteoporosis knowledge was not associated with young women's beliefs and behavior.Our data suggest a need to educate young women. Comprehensive osteoporosis educational programs during college should be evaluated as an intervention to improve osteoporosis prevention. Recently published studies suggest that automated external defibrillators (AEDs) on commercial aircraft may save the lives of passengers who have out-of-hospital cardiac arrest (OHCA). However, AED equipment and training costs are high, while cardiac arrests onboard aircraft remain rare. We conducted a cost-effectiveness analysis to explore the economic and health impact of AED deployment in the U.S. passenger air fleet. METHODS: We developed a decision analytic model using a societal perspective for costs and benefits. Estimates of the incidence of OHCA onboard aircraft, effectiveness of AEDs in resuscitation, subsequent hospital survival rate, and annual mortality after surviving OHCA were derived from the medical literature. Equipment, maintenance, and flight attendant training costs were obtained from AED manufacturers, the Federal Aviation Administration, and the Air Transportation Association. Hospitalization and downstream medical costs were abstracted from published cost analyses. Published estimates of healthrelated utility after cardiac arrest were used to express effectiveness in terms of qualityadjusted life years (QALYs) gained. We compared four strategies: 1) AEDs on all aircraft with basic life support (BLS) training for flight attendants; 2) AEDs on wide-body aircraft only; 3) AEDs on no aircraft±but attendants trained in BLS; and 4) no aircraft AEDs and no BLS training. RESULTS: Placing AEDs on all passenger aircraft with concurrent BLS training would cost $49,800 per QALY gained compared to no AEDs and no BLS training. BLS training without AEDs was dominated by a combination of strategies #1 and #4. Deploying AEDs exclusively on wide-body aircraft would be cost-effective only if total training costs could be limited to less than 49% of the cost of training all attendants. Sensitivity analyses indicated that the ability of AEDs to improve survival was the most important influence on cost-effectiveness. AEDs must demonstrate an incremental survival rate of at least 14% to have a cost-effectiveness ratio less than $50,000/QALY. A Monte Carlo analysis, which varied all assumptions simultaneously, indicated that the cost/QALY value for strategy #1 would be less than $50,000 at a probability of 34%, while the probability of the cost-effectiveness being greater than $100,000/QALY was only 0.1%. CONCLUSION: The use of AEDs on passenger aircraft is similar in cost-effectiveness to many widely accepted medical interventions and health policy regulations. Sensitivity analysis suggests this result is robust, even when underlying assumptions are varied widely. This study implies that requiring the placement of AEDs on U.S. commercial aircraft would be a cost-effective use of resources. PURPOSE: Pressures to shorten length of stay increase the chance that patients (Pts) may be discharged`sicker and quicker.' Pts hospitalized with hip fracture are very frail and maybe at particularly high risk. We sought to measure prevalence of active clinical issues (ACIs)on discharge (DC) in Pts hospitalized with hip fracture and their associated clinical and functional outcomes. METHODS: Information on vital signs, eating status, mentation, mobility, incontinence, and wound status, and other active problems on DC, and in the 24 and 48 hours prior to DC was collected on 559 Pts hospitalized with hip fracture as part of a 4 hospital prospective cohort study. Deaths, readmission, and functional mobility within 60 days of DC were ascertained by telephone interviews and querying the NY State hospitalization database. Mobility was measured with the validated Functional Independence Measure-Locomotion scale (FIM). Logistic and linear regression assessed associations between the number of ACIs on DC and post-DC outcomes. ACIs on DC were defined as any of the following in the 24 hrs prior to DC (Temp > 100, 100 > HR > 60, SBP < 90, DBP < 60, RR > 24, O2 sat < 90%, altered mentation, inability to eat, incontinence, and not mobilized beyond a chair (all different from baseline), and other active medical problems [dyspnea, chest pain, arrhythmias, CHF, or wound infection]). We used a validated risk adjustment model to control for covariates known to influence clinical and functional hip fracture outcomes. RESULTS: Pts mean age was 81 yrs, 82% were female, 22% had dementia, and 12% came from a nursing home. The mean length of stay was 8.56.2 days. Overall, 74% of Pts had!1 ACI on DC (29% had 1, 28% had 2, and 17% had!3 ACIs), most commonly not mobilized beyond a chair (37%), new urinary incontinence (34%), Temp > 100 (11%), abnormal mentation (5%), DBP < 60 (5%), and new bowel incontinence (3%). Within 60 days of DC, 3.8% of Pts died, 18.8% were readmitted, and 20.2% died or were readmitted (major events). The mean FIM locomotion score at Day 60 was 6.1 4.0 (scale from 2 ± 14; higher is better). The greater the number of ACIs on discharge, the higher the risk of post-DC death, readmission, and major events and worse functional mobility [FIM score] (p < .001 for all). The odds of major events increased 40% for every additional ACI on DC (OR=1.4; 95% CI 1.1 ± 1.8). The number of ACIs on DC remained a significant predictor of all post-DC outcomes even after controlling for age, sex, comorbidities, initial APACHE score, and pre-fracture nursing home residence, dementia, home health assistance, and FIM score. Similar results were obtained for outcomes at 30 and 90 days as well as for analyses that considered only the last set of vital signs along with the other ACIs or ACIs in the 48 hours prior to DC. CONCLUSION: The greater the number of ACIs on DC, the worse the post-DC clinical and functional outcomes even after controlling for other important prognostic factors. Clinicians should factor this information into deciding appropriateness for DC and the type and intensity of post-DC care and medical observation. PURPOSE: Elevated HDL is considered a negative risk factor for ischemic heart disease and HDL level > 75mg/dl has been found to be associated with the``longevity syndrome''. We wanted to study people with extreme HDL elevation as no studies have been done to evaluate them before. METHODS: We identified all persons presented to our hospital with HDL level > 95mg/dl in the last 2 years. All subjects were contacted by phone. RESULTS: 102 subjects were contacted (83 females and 19 males). The majority were African Americans (52 subjects) (13 males and 39 females). The mean age was 59.3 + 1.5 years. The mean cholesterol was 234 + 5 mg/dl with a mean HDL of 108 + 2 mg/dl, triglyceride of 85 + 4 mg/dl, and LDL of 112 + 5 mg/dl. The total cholesterol to HDL ratio was 2.4 + 0.2. Mean Body Mass Index (BMI) was 25.8 + 0.6 kg/m2. (table) Only 15% were younger than 50 years old. We found an unusually high prevalence of hypertension in our subjects (53%) especially in the African American population (71%). Seven patients have coronary artery disease (CAD) but all had other cardiac risk factors. CONCLUSION: Our study showed a predominance of African Americans and postmenopausal females. This population had normal TG, LDL, and BMI, and a high prevalence of hypertension and smoking. Half exercised or reported following a special diet. Although elevated HDL is associated with longevity syndrome, it may not be protective against CAD in people with multiple other risk factors. were advised not to have or were not offered AVR by a cardiologist or cardiothoracic surgeon, and 2 declined further evaluation for AVR. For the remaining patients a decision was made to proceed with AVR or no decision had been documented by 60 days. At the time of the chart review (2 ± 4 years after echocardiogram), 37 patients (39%) had AVR and 13 (14%) had palliative aortic valvuloplasty. Compared with patients who did not have AVR, patients who had AVR were younger (78 vs 84, p < .0001), more likely to be male (46% vs 26%, p=.05), had fewer comorbid illnesses (mean number comorbidities 1.1 vs 1.7, p=.04), and were less likely to need assistance with ADLs (mean number of ADL dependencies 1.1 vs 1.7, p=.04). In a multivariable logistic regression model including age, sex, number of comorbid illnesses, and ADL dependency, these differences persisted, but only the effect of age was statistically significant at p < 0.05. Of the 33 patients who had AVR at one of the study hospitals, 19 also had coronary artery bypass surgery, 2 also had mitral valve repair or replacement, 2 died postoperatively, and 1 suffered a stroke. CONCLUSION: In this cohort of elderly patients with severe, symptomatic AS, the majority did not have AVR surgery. Patients who were younger and who had better baseline health status were more likely to have AVR. Few patients who were selected for AVR died postoperatively or suffered a major surgical complication. PURPOSE: African American women experience higher breast cancer mortality than Caucasian women despite increasing rates of screening mammography. One postulated factor in these worse outcomes is a lower rate of follow-up for diagnostic testing once a breast abnormality is detected. We examined factors associated with inadequate follow-up in a referral group of predominantly low-income minority women. METHODS: The study population consisted of women referred to a Breast Center at an urban medical center from January to June 2000. Demographic information was collected via medical charts, registration files completed at the time of appointment scheduling, and consultation/referral forms. Adequate follow-up was defined as patient arrival to the scheduled Breast Center appointment and inadequate follow-up was defined as failure to arrive on the designated appointment day. We analyzed factors associated with inadequate follow-up such as patient age, race, median household income by zip code, referral source, and insurance type. Maintenance dose was defined as being greater than one month out from initiation of warfarin therapy with at least two consecutive measurements one week apart within the target range. Potential predictor variables included: age, sex, weight, race, indication for anticoagulation, individual medications and total number of medications. A combined approach using recursive partitioning and multivariate logistic regression was used to develop and evaluate potential prediction rules. Two different rules were tested: one looking at low maintenance requirements (less than or equal to 3 mg per day), the other looking at high maintenance requirements (greater than or equal to 7 mg per day). RESULTS: 131 patients meeting entry criteria were identified with an average age of 68 years (range 27 to 93 years), 52% male sex, 70% Caucasian race, 75% anticoagulated for either atrial fibrillation (38%) or deep venous thrombosis/pulmonary embolus (37%), average weight of 184 pounds (range 100 to 350 pounds) and average number of chronic medications (in addition to warfarin) of 6 (range 0 ± 29). The simultaneous presence of 3 of the following variables predicted low maintenance requirements with 80% specificity (95% CI 73 ± 87%), 67% sensitivity (95% CI 59 ± 75%) and a positive predicted value of 92% (95% CI 87 ± 97%): age greater than 50, weight less than 200 pounds and being on 5 or more chronic medications. Although there were factors (age less than 50 years, and weight greater than 200 pounds) that on univariate analysis were associated with high maintenance requirements, a statistically meaningful rule could not be developed. CONCLUSION: A prediction rule that identifies those with the lowest warfarin maintenance requirements has been developed which could potentially aid in determining how to initiate warfarin therapy. This rule needs to be validated and refined using different patient populations. PURPOSE: Our objectives were three-fold: To conduct a pilot study of risk factors and screening behaviors relevant to breast and cervical cancer in rural lesbian women, to assess women's willingness to participate in a study of these factors, and to assess the feasibility of contacting women through regional lesbian organizations. METHODS: Using a brief self-administered questionnaire, we obtained information concerning major risk factors and screening behaviors relevant to breast and cervical cancer. The questionnaire, targeted to rural lesbian who were at least 40 years of age, was distributed at lesbian community events in rural New Hampshire and Vermont between June and August 1997. A separate survey, assessing willingness to participate in a future study, was distributed (between October and December 1998) by regional lesbian organizations to their membership. RESULTS: The first questionnaire was completed by 105 women. Of these, 82% reported at least one physician visit during the previous year. With regard to breast cancer risk factors and screening, 66% were nulliparous, 66% had at least one clinical breast exam during the previous year, 43% had had a mammogram, and 40% conducted breast self-exams. With regard to cervical cancer risk factors and screening, 80% had a history of heterosexual activity, and 62% had undergone a Pap smear during the previous year. Over the past 3 years, 21% had an abnormal pap smear. The later survey assessing willingness to participate in breast and cervical cancer research was mailed to 250 women; 47 surveys were undeliverable due to unknown address. Of the remaining 203 (62%), 108 women (53%) expressed willingness to participate. CONCLUSION: This pilot study suggests that breast cancer risk may be higher among lesbian women than non-lesbian women, and that lesbian women are at risk of cervical cancer despite of their current sexual practices. Our preliminary data also suggest that rural lesbian women are medically underserved with regard to breast and cervical cancer screening. The mailed survey also supports the feasibility of recruiting large numbers of rural lesbian women for health research.\nPREVALENCE AND TREATMENT OF MENOPAUSAL SYMPTOMS AMONG BREAST CANCER SURVIVORS. P.F. Harris 1 , P.L. Remington 1 , A. Trentham-Dietz 1 , C. Allen 1 , P.A. Newcomb 1 ; 1 University of Wisconsin-Madison, Madison, WI BACKGROUND: Women diagnosed with breast cancer often experience early menopause secondary to treatment effects, yet physicians may be reluctant to prescribe hormone replacement therapy (HRT) in order to reduce their risk of cancer recurrence. The objective then is to assess the burden of menopausal symptoms, HRT use, and alternative treatments in recent breast cancer survivors compared to age-matched controls. METHODS: This is a population-based case-control study using breast cancer survivors and age-matched controls. Wisconsin women 18 ± 74 years old with a new diagnosis of breast cancer 8 ± 11 months prior to interview (n=110) and control subjects randomly selected from population lists (n=73). A standardized telephone questionnaire was administered to elicit information on menopausal symptoms, estrogen and alternative (prescription medications, vitamins, herbal preparations, soy products, acupuncture, chiropractic) therapies used to alleviate symptoms.\nMultivariate logistic regression was used to obtain odds ratios between cases and controls. Main outcomes were symptoms of menopause, use of estrogen, and use of alternative therapies. RESULTS: Breast cancer survivors were 5.4 (95% CI 2.9 ± 10.4) times more likely to experience symptoms, 25 (95% CI 8.3 ± 100) times less likely to use estrogen, and 7.4 (95% CI 2.5 ± 21.9) times more likely to use alternatives than controls. Soy, vitamin E, and herbal remedies were the most common alternative therapies reported by participants; use was greater in cases compared to controls. Among cases, tamoxifen users (n=62) reported a higher prevalence of symptoms and a higher prevalence of alternative treatments. Most soy users reported increasing soy products specifically to reduce the chances of a diagnosis of recurrent breast cancer. CONCLUSION: This is the first population-based survey of menopausal symptoms and treatments that compares breast cancer cases with disease-free controls. Prevalence of menopausal symptoms and use of alternative menopausal therapies were higher in breast cancer survivors than in controls; the rate of estrogen use was much lower. The increased use of soy products in this population has not been previously documented. (2) Test the effect of a personalized letter directed to women who have not had a recent mammogram Ð reticent beneficiaries. (3) Assess the cost-effectiveness of the intervention. METHODS: HCFA data for Medicare beneficiaries were used to identify women living in Michigan continuously from 1993 ± 98, age !65 in 1993, with no obvious comorbidity affecting screening, and no mammogram for !5 years (1993 ± 97). A randomized design included paired intervention and control women matched on residential zipcode and race. The study sample of 2,458 women had 1,229 pairs of either African American (AA) or non-AA women and either urban or rural women. The intervention used principles from the Health Belief Model in a personally addressed letter from the Medical Director of Michigan Medicare noting the individual's lack of use of the mammography screening benefit, with additional breast cancer related information enclosed. Letters were sent in Nov. 1997 with Medicare mammography claims followed through 1998. RESULTS: All women were age !70, with a mean of 79 in both control and intervention groups. Overall, 5.2% of controls and 8.1% of the intervention group subsequently had mammograms, + 2.9%, OR 1.6 (p < .005). The findings were similar by urban/rural and AA/ non-AA subgroups. Rates and the effect were higher among the women age 70 ± 79: 6.5% of control and 10.6% of intervention, + 4.1%, OR 1.6 (p < .02). Projecting to the Medicare population in Michigan, a total cost of $117,000 to $278,000 for a state wide intervention would produce 3,700 to 5,000 mammograms at $32 to $54 per additional mammogram in this reticent group. CONCLUSION: This approach for measurement and intervention is feasible, effective, and can be directly implemented in other states and nationally. Targeting a reticent group of older women, this intervention demonstrated a significant improvement in subsequent mammography. This targeted approach is likely to be more cost effective than``blanket'' community-based approaches with significantly higher overall cost but the same target group of reticent older women. Future research can address variations to increase the communication's effectiveness (e.g., multiple communications). The approach to measurement and intervention can be tried for other preventive services in the Medicare or other defined populations. However, few data are available about how often patients do not know or misunderstand the reasons for their medications. We studied a group of general medicine outpatients to assess patient understanding of their medications. METHODS: Patients seen in four Boston general medicine practices from Sept 1999 to March 2000 (6 weeks per clinic) and who received at least one prescription from a study physician (6 MDs per clinic) were eligible for the study. Approximately two weeks after the index visit, patients were telephoned (response rate 59%) and asked to retrieve all of their pill bottles, read the name of each medication, and state their reason for taking it. Answers were coded``know'' or`d on't know,'' and the reasons were recorded verbatim. The accuracy of``know'' responses was coded by a physician-reviewer on a 5-point scale.\nRESULTS: Of 598 patients, 58 (9.7%) did not know or gave definitely inaccurate indications for at least one medication. The drugs most often associated with inaccurate indications were blood pressure and lipid-lowering agents. An example of an inaccurate response was`a torvastatin is for my blood pressure.'' Education and younger age were directly related to knowledge (see Table) . There was a trend toward association of non-white race and male gender with poorer medication knowledge, but they were not statistically significant. Patients on more than one medication were far less likely to know the indication of all medications (p < .001). CONCLUSION: In this relatively well-educated population, knowledge about drug indications was high. However, older and less educated patients, as well as patients on multiple medications, were much less likely to know why they were taking all of their medications. Such patients may benefit from intensive education programs to enhance understanding of their medications, which could improve adherence. PURPOSE: Adherence to evidence-based practice guidelines is frequently suboptimal. We wished to determine whether an opinion leader quality improvement project would increase adherence to the Unstable Angina (UA) AHCPR guidelines compared to a traditional HCFA quality improvement model. METHODS: We designed a three-armed randomized controlled trial of 22 acute care hospitals in one state. The intervention arms were: 1) hospital-specific data-feedback of performance on quality indicators combined with a physician opinion leader-driven improvement intervention (OL); 2) hospital-specific data-driven feedback with traditional HCFA quality improvement model efforts (HCFA); and 3) no intervention (NI). We convened a national panel of experts to translate selected elements from the AHCPR UA guidelines into quality measures. We selected the following indicators: 1) ASA within 24 hours of admission, 2) ASA at discharge, 3) heparin during hospitalization, 4) beta-blockers at discharge, and 5) EKG within 20 minutes of presentation. For chart abstraction, we identified potential cases of UA using a stratified random sampling scheme based on ICD-9 codes from Medicare claims data. A computerized algorithm was developed to confirm UA cases from the abstracted data. Centrally trained abstractors reviewed the complete medical records. We used GLM to adjust for patients-nested within hospitals. RESULTS: Charts were abstracted for 2,516 patients; their average age was 72 11 years, of whom 45% were male and 85% were white. Average baseline performance for the indicators was 71.6 (ASA in 24hrs), 69. PURPOSE: Men with clinically localized prostate cancer have several treatment options including conservative management (C), radiation therapy (RT), or radical prostatectomy (RP). We used data from the population-based Prostate Cancer Outcomes Study (PCOS) to evaluate patient satisfaction with these treatments. METHODS: The PCOS evaluated 3,830 incident cases of prostate cancer reported in 6 regional Surveillance, Epidemiology, and End Results (SEER) programs between October 1, 1994 and October 31, 1995 . The current analysis was restricted to the 2,387 subjects with clinically localized prostate cancer who completed a 24-month post-diagnosis follow-up questionnaire. Weighted multivariate logistic regression analysis was used to determine factors associated with treatment satisfaction. Independent variables included demographics, socioeconomic status, comorbidities, tumor characteristics, additional cancer treatments, perception of being free of cancer, scales measuring self-reported bowel, urinary, and sexual function, and perception of problems with these three functions. RESULTS: Treatment selections were 431 C, 583 RT, and 1,373 RP. The cohort was 74% non-Hispanic white, 13% African American, and 13% Hispanic; 41% were 65 years and younger. Perception of being free of cancer was reported by 17% (C), 87% (RT), and 94% (RP), even though 16% (RT) and 14% (RP) required additional androgen deprivation therapy. Moderate/big sexual problems were reported by 33% (C), 44% (RT), 53% (RP); moderate/big urinary problems were reported by 4% (C), 3% (RT), 11% (RP); and moderate/big bowel problems were reported by 6% (C), 7% (RT), 3% (RP). Men undergoing radiation therapy were more likely to be delighted/pleased with their treatment choice (70%) than those undergoing either conservative management (55%) or surgery (59%), P < 0.0001. The following factors were independently associated with treatment satisfaction for conservative management: receiving androgen deprivation therapy (OR = 2.0; 95% CI 1. CONCLUSION: The majority of men with localized prostate cancer were very satisfied with their treatment decision. Sexual problems were common with all treatments but significantly decreased satisfaction for only RT and RP. RP most frequently led to urinary problems and this significantly decreased satisfaction. Androgen deprivation therapy increased satisfaction for C but decreased satisfaction for RT. For all treatments, the perception of being free from cancer was highly associated with satisfaction.\nPACIFIC ISLANDER CANCER CONTROL NETWORK. This study aims to assess frequency, content and impact of weight and diet counseling provided by primary care physicians to such patients. METHODS: We conducted an observational study on clinical prevention in 2 university-based primary care clinics staffed by 35 residents. We included 893 consecutive patients of whom 396 (44%) were overweight or obese (body mass index > 25 kg/m2). We interviewed patients after the index visit to assess counseling performed by residents during the three last visits. At 1 year, we mailed a questionnaire to assess patients' self-reported behavior change and weight loss (60% responders). We compared outcomes according to the quality of physician counseling (4 ± 6 vs. 1 ± 3 strategies used). RESULTS: Physicians infrequently advised overweight and obese patients about weight and diet: informing of health risks (40%); recommending to lose weight (40%); assessing motivation to lose weight (34%). They rarely used strategies facilitating behavior change: planning specific dietary changes (24%), setting a target weight (14%) and written material (14% 5) ). We calculated descriptive statistics using the Chi-square test and used generalized linear models to determine the independent effect of adverse computing habits on UEMD symptoms and functional impairment. RESULTS: Our sample included 46% women and 38% racial/ethnic minorities. 29% reported never having UE pain related to computer use, 30% had experienced pain in the past, 36% had pain in the past and current pain (in preceding 2 weeks), and 5% only had current pain. We defined``intensive computer users'' as students who typically computed for > 4h/day or spent !4h computing without getting out of the chair at least once a month. 66% were intensive computer users. Women were more intensive users (p = 0.05), reported more functional impairment due to UEMDs (p = 0.013), and had more upper arm (p = 0.07), shoulder (p = 0.001), and neck pain (p < 0.0001) than men. Worse mental health scores were associated with increased reported UE pain (p = 0.01) and functional impairment (p < 0.0001). Controlling for sex, mental health, and overall health, intensive computer use independently predicted higher UEMD symptom (Type III SS p=0.03) and pain severity scores (p = 0.01). CONCLUSION: Most college students in this sample engage in intensive computer use and have UE pain with computing. We found a significant independent association between intensive use and UEMD symptomatology. Our results suggest that programs to foster healthy computing skills may prevent long-term disability from UEMDs as students enter the work force. with attitudes (r = À0.15, P = 0.000 and r = À.0.12, P = 0.004, respectively) at the bivariate level. When included in the final model, the relationship between ethnicity and attitudes toward joint replacement was no longer significant; adjusted OR 0.66 (95% CI 0.38 ± 1.14).\nResponses on familiarity with JR did not mediate the relationship between ethnicity and attitudes toward JR. CONCLUSION: Differences in attitudes toward JR between AA and white patients is mediated by post-surgical concerns about pain and disability. If confirmed, our findings suggest an opportunity for generalists to address patient-level factors that contribute to racial disparity in the utilization of joint replacement. PURPOSE: Although racial and gender differences in mortality have been reported for systolic heart failure, little is known regarding diastolic heart failure prognosis; even though diastolic heart failure is the most common type of heart failure in the US. METHODS: Our sample consisted of 1058 patients 65 years of age or older who were admitted to 30 hospitals in Northeastern Ohio with a principal diagnosis of heart failure and had documented diastolic dysfunction by Echo. Diastolic heart failure in this cohort was defined as`h aving a principal diagnosis of heart failure and a LVEF of Ê 50% by Echo.'' Mortality data for all patients were obtained from Ohio MEDPRO files for Medicare beneficiaries. Logistic regression was used to compare 18-month mortality by ethnicity and by gender, adjusting for age, gender and comorbidities. RESULTS: Of the 1058 patients with documented diastolic heart failure (13% AA and 87% white), AAs and whites were comparable with respect to history of angina, stroke, being on dialysis, alcohol use, and the proportion of males. AAs were more likely to have hypertension (50% vs 36%; P = 0.001), diabetes (46% vs 29%; P = 0.000), history of tobacco use (27% vs 18%; P = 0.011), and high serum creatinine (1.99 2 vs 1.50 1; P = 0.003); they were also younger (76 7 vs 79 8; P = 0.000). Whites were more likely to have a history of ischemic heart disease (48% vs 32%; P = 0.000), metastatic cancer (3% vs 0%; P = 0.034), DNR status on record (14% vs 7%; P = 0.013), and atrial fibrillation (24% vs 14%; P = 0.002). The AA to white adjusted OR for 18-month mortality was 1.03 (0.66 ± 1.59). For men vs women (30% vs 70%), the above-mentioned comorbidities were comparable, except women were more likely to have DNR status (16% vs 7.3%; P = 0.000) and to be older (79.5 8 vs 77 7; P = 0.000). Males were more likely to have a history of tobacco use (30% vs 14%; P = 0.000), alcohol use (36% vs 15%; P = 0.000), and higher serum creatinine level (1.7 1.2 vs 1.4 1.1; P = 0.001). Men to women adjusted OR for 18-month mortality 1.06 (0.76 ± 1.46). CONCLUSION: In this cohort of elderly patients admitted with diastolic heart failure, there were no ethnic or gender differences in 18-month mortality.\nALTERNATIVE MEDICINE USE AMONG MEDICAL OUTPATIENTS. M. Herman 1 , L. Inouye 1 ; 1 Naval Medical Center Portsmouth, Portsmouth, VA PURPOSE: The use of complementary and/or alternative medicine (CAM) is increasing in popularity among patients in the United States. Hundreds of herbal products and homeopathic remedies are readily available to the consumer, but most of these have not been proven safe or effective. Potential side effects and interactions with traditional medications are often unknown. Studies suggest that between 30% and 50% of the adult population in industrialized nations use some form of CAM to prevent or treat a variety of health-related problems. The purpose of our study was to evaluate CAM use by Internal Medicine. The analysis would assess the proportion of patients in the clinic who are using alternative medicines, provide a description of the most common alternative medicines being used, and evaluate characteristics associated with alternative medicine use. METHODS: A convenience survey was completed by patients of the Internal Medicine clinic at Naval Medical Center, Portsmouth (NMCP) between 6DEC99 and 7JAN00. Information was gathered on the patients gender, age, race, medical problems and medications, educational background, military status, herbal and non-herbal usage to include confidence of efficacy and safety, and the source of information for herbal and non-herbal products. Chi-squared analysis was used to compare CAM use differences in gender and educational groups. RESULTS: There were 212 surveys completed. Of those responding, 15% were active duty or a dependent of an active duty member, and 60% were female. The mean age of respondents was 53 years. Complementary therapies had been used in 60% of the patients. The most common herbal products were garlic, ginseng, gingko biloba, and St. Johns wort. The most common non-herbal products were vitamins, chromium, glucosamine, and chondroitin sulfate. Sixty-five percent of females used CAM compared with 53% males, although this difference was not statistically significant (p = 0.125). The average age of CAM users was 53 years. Thirty percent of CAM users had a college degree, 15% were active duty, 35% were retired, and 50% were dependent. Sixty percent of CAM users had been using an alternative medicine for at least one year. Use of herbal CAM was associated with a higher educational background, although the difference was not statistically significant (p = 0.132). However, vitamin use was significantly correlated with a higher educational background. In those subjects with less education than a college degree, only 25% used vitamins. Among college graduates 54% used vitamins (p < 0.0001). Seventy-one percent of respondents felt that complementary therapy was safe and only 17% were very confident of its efficacy. CONCLUSION: CAM use among Internal Medicine patients at NMCP is higher than reported use in studies of civilian populations, which may reflect a difference in patient profile. Of interest is that a significant proportion of people using alternative medicines were not confident of its safety and only a few were very confident of its efficacy. This information may correlate with compliance issues pertaining to physician prescribed medications. While the social profile may reflect those most likely to use CAM, our data demonstrates widespread use of alternative medicine. This information has important implications for health care providers, who must consider safety, efficacy and possible interactions with standard therapies. Further analysis will include the results from over 400 respondents. METHODS: In 1995, 500 primary care patients presenting to a walk-in clinic with physical complaints were screened for mental disorders with the PRIME-MD. Five years later, patient surveys assessed symptom outcome, mental disorders (PRIME-MD), functional status (MOS SF-6) and whether they had been diagnosed or treated for a mental disorder. Vital status was assessed with the Social Security Index. RESULTS: Forty patients (8%) died. Among the remainder, 65% completed surveys, with follow up losses equal between patients with and without a mental disorders. The prevalence of mental disorders declined from 30% at baseline to 12%, though patients with a disorder at baseline were more likely to still have a disorder at 5 years (RR: 3.5, 95% CI: 2.0 ± 6.0). Only 32% of patients with a mental disorder at enrollment were diagnosed during the 5 years of follow up. The rate of diagnosis varied by disorder: 52% of patients with major depression, all patients with panic disorder and 66% of patients with generalized anxiety at baseline were diagnosed. For subthreshold disorders, diagnoses rates were lower: 32% for depression and 5% for anxiety. Patients with unrecognized depression (RR: 5.0, 95% CI: 1.2 ± 21.1) or anxiety (RR: 4.8, 95% CI: 1.1 ± 23.5) were more likely to still have their disorder 5 years later, compared to patients whose disorders were recognized. While most patients with minor depression or subthreshold anxiety (73 ± 83%) had their disorder at only one time point and no patient with subthreshold anxiety progressed to generalized anxiety or to panic disorder, 18% of those with minor depression progressed to major depression by 5 years. Patients whose mental disorder went undiagnosed over 5 years were more likely to have worsening of their initial symptom (RR: 3.6, 95%CI: 1.6 ± 8.4), greater stress (RR: 2.3, 95% CI: 1.9 ± 2.8), greater worry that their symptom represented a serious illness (RR: 2.9, 95% CI: 1.7 ± 4.9), a greater number of``other'' bothersome physical symptoms (p < 0.0001), and worse functional status (p < 0.0001). Outcomes among patients whose mental disorders were diagnosed during the intervening 5 years, were the same as among those without mental disorders. CONCLUSION: Patients with unrecognized major mental disorders were more likely to still be struggling with their disorder 5 years later, had less resolution of their presenting physical symptom and experienced worse health-related outcomes. METHODS: In 1995, 500 adults presenting with a physical complaint completed surveys that assessed symptom characteristics, functional status (MOS SF6) and mental disorders (PRIME-MD). Follow up surveys assessed symptom outcome, functional status and satisfaction at 2 weeks, 3 months and 5 years. RESULTS: Patients averaged 43 years in age (18 ± 92), were 52% female, 45% African American and 49% white. Patients presented with a myriad of complaints that we collapsed to 13 categories. Musculoskeletal symptoms were the most common category, present in a third and half had some sort pain complaint. Twenty ± one percent had experienced their symptom less than 3 days, 40% less than a week, 68% less than a month; 46% had seen a previous doctor for the same problem. Most patients experienced improvement, 70% at 2 weeks, 79% at 3 months and 75% at 5 years. There was no relationship between type of symptom and likelihood of improvement. However, there was good correlation between improvement in symptoms between each of the three time points. (Spearman's rho:0.40 ± 0.43). Among patients whose symptom had improved by 3months, most (83%) were still improved at 5 years. Conversely among those reporting no improvement, 52% were still not improved at 5 years. There was no relationship between likelihood of symptom improvement and age, sex, or baseline functional status. Patients without symptom improvement had worse functional status (p < 0.001), more illness worry (p < 0.001), a greater number of``other'' bothersome symptoms (p = 0.003) and were more likely to have an active mental disorder at 5 years (RR: 1.3, 95% CI:1.02 ± 1.7). Independent predictors of improvement included improvement at 3 months, symptom less than 3 days in duration at presentation and no patient worry that the illness could be potentially serious at presentation. CONCLUSION: Patients with symptoms present for more than 3 days at presentation,who are worried their symptom may be serious or who have not improved by 3 months are likely to still have problems with their symptom at 5 years. The 1994 AHRQ Low Back Pain Clinical Guidelines recommended treatment with tylenol in patients less than 55 years of age with no historical or physical examination findings suggestive of more serious causes. Nonsteroidal use was discouraged. Muscle relaxants, narcotics and routine radiographs were deemed inappropriate. Our purpose was to assess the impact of these guidelines on clinical practice. METHODS: Patients greater than 17 but less than 55 years in age, being seen in primary care settings (FP, IM, GP) with low back pain, without an inflammatory or secondary diagnosis to explain their back pain were abstracted from the National Ambulatory Medical Care Survey. Data from 1991 ± 1993 (prior to release of the 1994 AHRQ), were compared to data from 1995 ± 1997. RESULTS: A total of 11,854,653 encounters met criteria and were included in this analysis. The mean age of patients was 37 years, 57% were male and 34% of the visits were injury related. Prior to the guidelines, 0.33% of patients with back pain were treated with acetaminophen, 39% with a nonsteroidal, 27% with muscle relaxants and 4% with narcotics. Twenty-seven percent were referred for physiotherapy and 16% had an Xray associated with the visit. After the guideline release in 1994, acetaminophen use increased to 3%, muscle relaxant use declined to 17% and referrals to physiotherapy declined to 22%. Nonsteroidal (40%), muscle relaxants (20%), narcotic use (6%) and the rate that Xray's were obtained (19%) were unchanged. CONCLUSION: The AHRQ guidelines increased acetaminophen use, but only to 3%. Physicians were less likely to prescribe a muscle relaxant and were less likely to refer for physical therapy. There was no impact of the guidelines on clinician use of nonsteroidals, narcotics or radiographic evaluation. PURPOSE: Clinicians are often reluctant to recommend alcohol consumption among those with a history of stroke. We therefore sought to examine the relationship between alcohol intake and the risk of total and cardiovascular mortality in men with a previous history of stroke. METHODS: The study population consisted of the 104,353 physicians who comprised the enrollment cohort of the Physicians' Health Study. We focused our analyses on the 1,320 men who indicated a history of stroke at baseline and provided self-reported data on alcohol consumption. Cox proportional hazards was used to model the association between alcohol and mortality after adjustment for major lifestyle and clinical risk factors. Alcohol consumption was classified into four categories, with those reporting rarely/never used as the referent group. RESULTS: During a mean follow-up of 4.6 years, 369 men died, of whom 267 died from cardiovascular disease. Multivariate relative risks of total mortality were decreased in men with a history of stroke who consumed < 1drink per week, 1 ± 6 drinks per week, and at least 1 drink per day, at 0.88(0.60,1.28), 0.64(0.48,0.85) and 0.71(0.54,0.94) respectively (p, linear trend 0.028). Multivariate relative risks of cardiovascular mortality in men with a history of stroke were also decreased in men who consumed < 1 drink per week, 1 ± 6 drinks per week, and at least 1 drink per day, at 0.89(0.58,1.36), 0.56(0.40,0.79), and 0.64(0.46,0.88) respectively (p, linear trend 0.008). Adjustment for lifestyle and other clinical risk factors did not significantly change the risk estimates for total or cardiovascular mortality as compared with age-adjusted models. Because only fifty-five men died as a result of stroke, we were underpowered to examine this association, but the risk estimates were nonsignificantly lower among men consuming at least 1 drink per week.\nCONCLUSION: These data suggest that alcohol may reduce the risks of total and cardiovascular mortality in men with a history of stroke. More data are needed to confirm or refute these results. PURPOSE: Historically, it has been suggested that the symptoms associated with benign prostatic hyperplasia can be aggravated by infrequent sex. We evaluated this by assessing the cross-sectional association between frequency of ejaculation and lower urinary tract symptom severity and general self-perceived health among participants in the Olmsted County Study of Urinary Symptoms and Health Status among Men. METHODS: In 1989 and 1990, 2,115 Caucasian men between the ages of 40 and 79 years were recruited from a random sample of Olmsted County residents (55% response rate). These men completed a self-administered questionnaire that assessed lower urinary tract symptom severity with questions similar to those in the American Urological Association Symptom Index (AUASI) and completed a similar questionnaire biennially thereafter through 1998. In 1994, a question was added to assess the frequency of ejaculation during the previous month. This was answered by 81 percent of participants in that round. RESULTS: Overall, men who reported ejaculating at least once a week were less likely to have moderate-severe (AUASI > 7) symptoms than men reporting no ejaculations (Odds ratio=0.62, 95% Confidence Interval=0.51, 0.75). There was a similar association with health-related quality of life (Poor-good vs. very good-excellent, OR=0.45, 95% CI=0.37, 0.60). The association with symptom severity did not exist within age decade, however. The odds ratios for ejaculating at least once a week were 0.93, 0.79, 1.21 and 1.12 for men in their forties, fifties, sixties and seventies, respectively. Thus, after adjusting for age, the odds ratio was 0.95 (0.75, 1.21). The odds ratio for health-related quality of life, however, remained virtually unchanged (OR=0.47, 95% CI=0.37, 0.60). CONCLUSION: While there has been speculation about the adverse effects of abstinence on prostate health, these cross-sectional data suggest that frequency of ejaculation has no effect on lower urinary tract symptoms; the apparent protective association appears to be an artifact caused by the confounding effects of age. This is not the case, however, for the cross-sectional association with quality of life. PURPOSE: Re-hospitalization after in-patient treatment for community-acquired pneumonia (CAP) is common. The aims of this study were to examine reasons and identify predictors of rehospitalization (rehosp). METHODS: This analysis was performed as part of the randomized, controlled trial,`D issemination of Guidelines for Pneumonia Length of Stay,'' implemented in 7 hospitals in Pennsylvania from February 1998 to March 1999. The trial assessed the effect of a guideline intervention to reduce the length of stay (LOS) for patients hospitalized with CAP. Inclusion criteria included a clinical diagnosis of pneumonia and a chest radiograph with a new pulmonary infiltrate. Severity of illness was quantified using the Pneumonia Severity Index (PSI), a predictor of 30-day mortality in patients with CAP. For this study, 2 physicians independently reviewed the records of patients re-hospitalized within 30 days and afterwards reached a consensus on reasons for rehosp. Demographic, clinical, laboratory, and radiological data were used to identify reasons for rehosp. Categorical and continuous variables were assessed using the Chi-square test and the Student's t-test, respectively. Any variable significant at the p < 0.05 level was entered into a multiple logistic regression model. RESULTS: Of the 608 patients (283 in the intervention arm and 325 in the control arm), 69 patients (11%) were re-hospitalized within 30 days. There was no significant difference in readmission rates between the 2 groups. The mean time to rehosp was 8.49 (+/À 0.96) days. The reasons for rehosp were CAP-related (n=14), combination of CAP and comorbid condition (n=3), or comorbid condition alone (n=52). The kappa statistic, assessing inter-rater reliability, was 0.784. The major comorbid conditions requiring rehosp were cardiovascular (n=19), neurologic (n=6), pulmonary (n=6), gastrointestinal (n=5), genitourinary (n=5), and others (n=11). Significant predictors of rehosp included age (p < 0.001), unemployment status (p = 0.04), congestive heart failure (p < 0.001), coronary heart disease (CAD) (p < 0.001), ventricular arrhythmias (p = 0.014), chronic obstructive pulmonary disease (COPD) (p < 0.001), chronic oxygen treatment (p = 0.001), and PSI (p = 0.05). Significant predictors in the multiple logistic regression model included CAD (odds ratio 3.2 with a 95% confidence interval of 1.8 ± 5.5), COPD (2.5, 1.5 ± 4.2), and unemployment status (2.4, 1.1 ± 5.3). CONCLUSION: This study describes the incidence and reasons for re-hospitalization in patients with CAP and identifies subsets of patients that could be targeted for interventions to decrease re-admissions after CAP. The value of hospice lies in its ability to provide continuous quality care until the time of death. However, some patients are discharged from hospice, usually because they no longer meet hospice criteria or choose more aggressive care. We sought to determine differences between patients who are discharged vs. those who die in the hospice setting. METHODS: We identified 48 patients who were discharged from a local hospice organization between the years 1996 and 2000. We then selected 47 control cases, matched on primary diagnosis and month of admission, who died while in the same hospice organization. Data were collected on these patients through restrospective chart review. RESULTS: Of the 95 patients, 66% were female, 87% were Caucasian, 37% were married and 74% had Medicare as the primary payor source. The mean age of admission was 70 years, mean length of stay 50 days, and mean Karnofsky status on admission 30%. The most common primary diagnosis was cancer (38%), followed by neurologic disease (14%) and dementia (14%). At the time of admission, 43% received care at home, 40% in assisted living or a nursing home (AL/NH), and 17% in the hospice facility. At the time of discharge/death, 33% were at home, 41% in AL/NH, and 26% in the hospice facility. The reason for discharge was stabilization of condition in 54%, revocation of hospice benefit in order to pursure more aggressive treatment in 13%, and transfer to different hospice in 11%. Discharge disposition was to a different hospice in 11%, to AL/NH in 48%, and to home in 23%. The discharged patients vs. deaths did not differ in age, sex, race, payor source, site of hospice care, admission weight, level of cognition, severity of pain, level of mobility at admission, number of ADL's performed at admission, or Karnofsky status at admission. Having a primary caregiver was also not significant. Compared to patients who died, those discharged had a longer length of stay (mean 62 and 38 days, p=0.05), were less likely to be married (25% and 50%), and less likely to be bedbound at discharge (23% and 76%, p = 0.000). CONCLUSION: Patients discharged alive from hospice, compared to case controls matched on diagnosis and date of admission who died in hospice, had similar demographics and level of functioning at the time of admission. The presence of a primary caregiver did not alter outcome and being married was associated with death. We speculate that hospice provides additional social contact or improved medical care that prolongs the life of some patients. PURPOSE: Recognizing patients' expectations is considered as an important objective for primary care physicians. However, few studies have investigated the influence on such matter of race and/or ethnicity, specially among immigrants. The aim of the study was to investigate whether patients' expectations among immigrants and natives are different and if physicians can perceive them. METHODS: The design is a two month pre-consultation patient survey and post-consultation physician survey (matched pairs) in an academic primary care center. Subjects were natives (Swiss French-speaking people) and immigrants (ex-Yougoslav, European, French-speaking African and other) consulting without appointment. The main outcome measures were patients' expectations (14-item questionnaire: receiving a prescription, a sickness certification, reassurance, seeing a specialist, etc...), patients' perception of their health status, physicians' perception of their patients' expectations (14-item questionnaire) and agreement between patient and physician. Questionnaires were available in 3 languages (French, Serbo-Croat and Albanian). RESULTS: 343 patient and 333 physician questionnaires were analyzed. Patients were separated into 5 groups: Swiss (n=167), European (n=50), Ex-Yugoslav (n=37), African (n=62) and other (n=27). Immigrants (n=176) do not consult more quickly than native patients and initiate a treatment at home as often (n=ns). However, more immigrants perceive their health as bad (22.7% vs 5.4% p < 0.01) and ask their doctor for reassurance (84.3% vs 62.7% p < 0.01). More Ex-Yugoslav patients expect to be referred to specialists (60% vs 30% p < 0.01) and more African patients wish to receive medication than Swiss patients (83% vs 59% p < 0.01). These results are confirmed by a stepwise logistic regression including independent variables such as age, sex, civil status and education. Finally, we found a poor agreement between patients' expectations and their perception by physicians, regardless of patients' origin (k < 0.36) CONCLUSION: Our study suggests that patients' expectations may differ according to their origin and that physicians certainly have a poor perception of their patients' expectations in an outpatient emergency setting, regardless of patients' origin. However, further investigation is needed to analyze whether the physicians' lack of knowledge of their patients' expectations influences the latter's satisfaction and the quality of medical care. shown that vaginal administration of hormones decreases UTI risk in post-menopausal women with recurrent UTIs, but evidence supporting a similar effect of oral hormones is mixed. We assessed the effects of oral hormone treatment in a randomized trial and evaluated potential risk factors for UTI frequency among post-menopausal women. METHODS: 2,763 postmenopausal women were enrolled in the Heart and Estrogen/progestin Replacement Study (HERS), a randomized, double-blinded, controlled trial to evaluate hormone treatment for the prevention of coronary heart disease events in women with established coronary heart disease. Participants were randomly assigned to daily 0.625 mg of conjugated estrogens plus 2.5 mg of medroxyprogesterone acetate or placebo and followed for a mean of 4.1 years. We analyzed results for 1,318 women randomized to hormone therapy and 1,336 women randomized to placebo who had complete data. Participant demographic information, reproductive history, sexual activity, vaginal symptoms, urinary incontinence symptoms, overall health status and chronic medical illnesses including diabetes were assessed at baseline and each annual visit. Number of UTIs diagnosed by a physician in the previous year was assessed by self-report at each annual visit. A proportional-odds model for repeated ordinal measures was used to test the treatment effect at all annual visits together, and risk factors for UTI were assessed using generalized estimating equation logistic models. RESULTS: UTI frequency tended to be a little higher in the group randomized to hormone treatment, although the difference was not statistically significant [Odds Ratio (OR) 1.2; 95% confidence interval 1.0 ± 1.4]. Among the 116 women who reported multiple UTIs in the previous year at baseline, there was no difference in the odds of UTI for the oral hormone vs. placebo group [OR 1.1 (0.7 ± 1.9)]. The average incidence of UTI at each annual visit was 12.3%. Among women with at least one UTI during a reporting period, 32.2% had two or more UTIs in the previous year. In multivariate analysis, risk factors significantly associated with UTI included diabetes requiring medications [OR 1.6 (1.3 ± 2.0)], urinary urge incontinence symptoms [OR 1.5 (1.3 ± 1.7)], frequent vaginal itching or dryness [OR 1.4 (1.1 ± 1.7)], history of child-birth [OR 1.4 (1.0 ± 1.9)], and self-reported poor health [OR 1.4 (1.2 ± 1.6)]. UTI in the previous year was strongly associated with incident UTI [single UTI: OR 6.9 (5.8 ± 8.1); multiple UTI: OR 18.2 (14.0 ± 23.5)]. After controlling for diabetes status, fasting blood glucose level was not associated with risk of UTI. Sexual intercourse was not a risk factor for UTI. CONCLUSION: Oral hormone therapy did not reduce the frequency of urinary tract infections. Potentially modifiable risk factors to decrease risk of urinary tract infection in postmenopausal women are different from those for younger women, and include diabetes, vaginal symptoms, and urge incontinence. PURPOSE: Thyroid dysfunction increases with age, is more common among women, and among whites is associated with lipid changes. There is limited information on racial differences in thyroid dysfunction and the association with cholesterol abnormalities. METHODS: Health ABC is a cohort of well-functioning whites and blacks, ages 70 ± 79, recruited for a population-based study in 1997. We analyzed thyrotropin (TSH) and serum cholesterol test results for 2,742 participants performed at the second annual visit. Free thyroxine (FT4) levels were performed on those with abnormal TSH results. Medical history and physical measurements were gathered at baseline and the second annual visit. The relationship between TSH and total cholesterol was analyzed with multivariable linear regression adjusting for multiple covariates. RESULTS: 267 (9.7%) participants were taking thyroid hormone medication and 223 (83.5%) of these men and women were biochemically euthyroid. Among those not taking thyroid hormone, 2,364 (95.5%) participants were euthyroid. Subclinical hypothyroidism was the most prevalent disorder among all race/gender groups; white women had a significantly higher prevalence of subclinical hypothyroidism than black women (4.5% vs. 1.6%, p = 0.004). Mean cholesterols were highest for those who were biochemically hypothyroid and lowest among those who were hyperthyroid. After adjustment for age, gender, race, body-mass index, smoking, alcohol use, diabetes, hormone-replacement therapy and thyroid hormone use, a TSH > 7.0 ulU/ml was associated with a 18 mg/dl increase in cholesterol and a TSH < 0.1 ulU/ml, with a 21 mg/dl decrease and did not differ between black and white participants. CONCLUSION: Healthy community-dwelling elderly blacks have a lower prevalence of thyroid dysfunction compared to whites. The association between increased TSH and increased cholesterol is similar in blacks and whites. PURPOSE: Atrial Fibrillation (AF) is the most common sustained arrhythmia, and is the cause of significant morbidity. Risk factors which increase the incidence of AF are: increasing age, ischemic heart disease, valvular heart disease, cardiomyopathy, congestive heart failure and chronic lung disease. AF may also occur in a normal heart. Ibutilide, a class III antiarrhythmic agent, has emerged as an effective intravenous agent for treatment of AF to normal sinus rhythm (NSR). However it is associated with a 4% chance of ventricular arrhythmias. In addition, Ibutilide remains a very expensive medication often requiring a second dose. Oral Propafenone, a class IC agent, has been shown to be an effective treatment for recent-onset AF in patients without structural heart disease. It is an inexpensive medication usually administered as an oral medication in the dose range of 150 to 300 mg. Recently it was shown that a single dose of 600 mg of Propafenone, was successful compared to control, in converting recent-onset AF to NSR. Although the effectiveness of both oral Propafenone and Ibutilide, individually, in converting recent-onset AF to NSR has been well established, the comparative studies of these agents are lacking. The aim of this study is to evaluate in a randomized fashion the efficacy of Intravenous Ibutilide versus a single 600 mg dose of oral Propafenone in treatment of recent-onset AF. METHODS: All eligible patients with recent-onset AF/atrial Flutter (AFL) were identified and placed in the telemetry unit. Recent-onset AF was defined as arrhythmia lasting less than two weeks duration. The onset of AF was established based on symptoms such as palpitation, dizziness, chest pain or electrocardiogram (ECG) finding. If timing of onset of AF/AFL was unclear or greater than 48 hours, before attempting cardioversion, patients underwent a transesophageal echocardiogram (TEE). All patients were started on heparin and their prothrombin time (PTT) was maintained at 1.5 ± 2.0 times control. If patients had a rapid ventricular rate of greater than 100 bpm, AV nodal blocking agents were used to control the ventricular response. Exclusion criteria included: age less than 18 year old; hemodynamically unstable patient; previous myocardial infarction; known coronary artery disease; unstable angina; NYHA functional class greater than 1 heart failure; ventricular rate less than 60 bpm; hypokalemia (less than 3.5 mEq/L); hyperkalemia (greater than 5.5 mEq/L); untreated thyroid disease; recent GI bleeding; history of adverse drug reaction; contraindication to heparin; second or third degree atrioventricular block; bifascicular block; Complete bundle branch block; sick sinus syndrome; other antiarrhythmic therapy used within 8 hours prior to the enrollment in this study. Patients were monitored, TSH was checked and all electrolytes including potassium and magnesium were supplemented prior to drug administration. After it was determined that AF/AFL has been stable with a documented duration of greater or equal to one hour, eligible patients were randomly assigned to receive a single 600 mg dose of oral Propafenone or an intravenous infusion of 1 mg of Ibutilide over 10 minutes. A second infusion of Ibutilide was given 10 minutes after completion of the first infusion if conversion did not occur. Serial ECG's were performed following drug administration. A 12-lead ECG was performed immediately upon cardioversion to NSR. Cardioversion was identified as a stable sinus rhythm lasting for at least one hour. In case of persistent AF/AFL, patients underwent electrical cardioversion after the 24-hour observation period. RESULTS: We identified a total of 100 patients with recent-onset AF/AFL. Of the 100 patients 37 were eligible for enrollment in this study. 19 patients (51%) converted to NSR spontaneously. 18 patients were randomized to either receive Propafenone(8) or Ibutilide(10). 6 of 8 patients converted to NSR following Propafenone(75% conversion rate) with a mean time to conversion of 4.5 hours. 7 of 10 patients converted to NSR following Ibutilide (70% conversion rate). No significant difference in conversion rate between drug arms. All patients except for one required two doses of Ibutilide for conversion. Mean time for conversion to NSR with Ibutilide was 2 hours. No significant ventricular tachyarrhythmias were noted. CONCLUSION: Propafenone is a safe, inexpensive and well-tolerated agent for the acute conversion of recent-onset AF/AFL to NSR, with the same efficacy as Ibutilide. In select patients(e.g. patients with no structural heart disease) Propafenone may be safe to use as an outpatient basis.\nemail to communicate with their doctor. These patients were more likely than those unwilling to use email to be younger (mean age 47.1 vs 55.4), have higher incomes ( > $75,000, 32.2% vs 17.0%) and to report better health (self assessed 10pt scale, 7.2 vs 6.5, All p < .01). Patients and physicians reported a number of barriers to use of email: 46.2% of patients who were email users`w ould rather speak to a real person'' and 31.5% thought email``would take too long'' but only 11.1% were worried about privacy at work or home. 47.4% of physicians were afraid they would be overwhelmed with patient emails, 30.2% did not feel they had enough time to check patient emails, and 44.8% felt patient email would add rather than substitute for other tasks. More physicians than patients thought that email could improve the patient-physician relationship (67.0% vs 54.3% of patient email users, p < .05) but physicians were more concerned than patients over security issues (43.2% vs 33.4% of patient email users, p < .05). CONCLUSION: Patients and their physicians are increasingly connected to email but few patients are connecting to their doctors. Our results suggest that patients perceive different barriers to use of email than their physicians. Patients may be more concerned about whether email will substitute for phone calls or visits versus concerns about privacy. Physicians appear to be optimistic about the role of email in improving the patient-physician relationship, but have concerns about privacy and workload. Research is needed to address these concerns about the efficiency, effectiveness and appropriate use of electronic patient-physician communication.\nRESPOND TO STROKE SYMPTOMS. G. Kefalas 1 , S. Hazelett 1 , K. Hua 2 , K.R. Allen 1 , G.C. Wickstrom 3 ; 1 Summa Health System, Akron, OHIO; 2 Northeastern Ohio Universities College of Medicine, Rootstown, OHIO; 3 Summa Health System, Akron, OH PURPOSE: Lack of knowledge of stroke signs and symptoms is prevalent, even among stroke survivors. This lack of knowledge can translate into a delay in seeking treatment at the onset of stroke symptoms. Such delays limit the effectiveness of current treatments for stroke. It is assumed that the education stroke survivors receive on an acute stroke unit is sufficient to ensure that they will recognize and properly respond to stroke symptoms after discharge. This assumption has not been tested. METHODS: This study examined stroke survivors' knowledge of stroke symptoms and their anticipated response to these symptoms at the time of discharge from an acute stroke unit and after either a 3 month post-discharge comprehensive team management intervention or usual post-discharge care. This was a secondary analysis of data from a randomized controlled pilot study measuring the overall impact of the post-discharge intervention and included 96 patients discharged from an acute stroke unit. The investigator-generated``stroke knowledge test'' assessed 1) how patients would respond to specific stroke symptoms (i.e., call 911, call their Dr, call a friend, take care of themselves), 2) whether they could correctly label varied symptoms as stroke-related, and 3) if they would call 911 if they knew they were having a stroke. RESULTS: Overall, the percentage of correct responses declined over three months in the control group but increased in the intervention group. At three months 91% of intervention patients and 85% of controls recognized numbness in an extremity as a stroke symptom, and, of these, significantly more intervention patients (p = 0.02) reportedly would have called 911 if they experienced such numbness. Likewise, 100% of intervention and 97% of control patients recognized sudden speech difficulties as a stroke symptom at three months, but significantly more intervention patients would have called 911 if they experienced these speech difficulties (p = 0.002). Lastly, 94% of intervention patients and 88% of controls recognized sudden onset of dizziness as a stroke symptom at three months, but significantly more intervention patients (p = 0.005) would have called 911 if they experienced such dizziness. When asked at three months what they would do first if they knew they were having a stroke, 86% of intervention patients and 74% of controls said they would call 911. CONCLUSION: This study showed that stroke education incorporated in a comprehensive post-discharge intervention improves post-discharge knowledge of stroke symptoms compared to education on the stroke unit alone, and, in as much as self-reported anticipated behavior correlates with actual performance, results in more patients calling 911 at the onset of stroke symptoms. These results suggest that in-hospital education alone may not be as effective as that combined with education delivered in an outpatient setting. They also suggest that the emphasis of educational efforts should be on the appropriate response to stroke symptoms, not just on stroke symptom recognition. PURPOSE: In the PURSUIT trial, eptifibitide was found to reduce the risk of acute myocardial infarction (AMI) and mortality (composite) from 15.7% to 14.2% (relative risk reduction [RRR] = 9.6 percent) in patients presenting with non-ST elevation acute cardiac ischemia (ACI). A published cost-effectiveness analysis, based only on the more favorable United States (US) results of the trial, found that the cost-effectiveness ratio for eptifibitide for this indication was $16,491 per year of life saved. We sought to estimate the cost-effectiveness of eptifibitide based on the results of the entire trial and the expected risk of AMI/death in a community based-sample of patients. METHODS: Using logistic regression equations to predict patient-specific risks of AMI and death and assuming a constant RRR of 9.6 percent for AMI and death, we estimated the costeffectiveness of routine use of eptifibitide across a population of 2,780 consecutive patients with non-ST elevation ACI who were admitted to hospital, but were not treated with eptifibitide. RESULTS: Predictions were obtained on 2,766 (99.5 %) of patients who met inclusion criteria. Of these patients, 2.0 percent died at 30 days and 12.2 percent had the composite outcome of AMI or death. Given these overall risks and a constant RRR, the average cost per year of life saved in our sample was estimated to be $41,832 per year of life saved. Moreover, adjusting each patient's risk of AMI and death using individual patient characteristics, only 6 percent of patients were at sufficiently high-risk to warrant therapy under a threshold of $20,000 per year of life saved. More than half of admitted patients were at such low risk for a bad outcome, even without therapy, that the marginal cost-effectiveness in these patients was greater than $50,000 per year of life saved. According to our analysis, one in twenty admitted patients were at insufficient risk to warrant therapy even under a threshold of $100,000. CONCLUSION: Given a constant RRR, use of eptifibitide is economically attractive in highrisk patients, but less attractive in low risk patients. Strategies to risk stratify patients to optimize clinical and economic outcomes should be considered. PURPOSE: It has been demonstrated that primary angioplasty (PTCA) in high-PTCA-volume hospitals is more effective in reducing mortality in reperfusion-eligible patients with acute myocardial infarction (AMI) than thrombolytic therapy. However, the benefits of PTCA disappear completely at low-volume hospitals. These findings support the strategy of bypassing low-volume community hospitals to deliver reperfusion-eligible patients with AMI to highvolume cardiac centers. However, bypassing community hospitals, or transferring patients from their emergency departments, causes delays that may nullify the expected benefits of PTCA. Moreover, immediate thrombolytic therapy leads to excellent outcomes in most patients. Thus, it is not clear which patients might benefit from PTCA, in the face of additional transportrelated delay. METHODS: We used the results of ten published trials and meta-regression techniques to assess the relationship between the treatment benefit of PTCA (in terms of the reduction in mortality rate in a trial) and procedure-related time delay (i.e. the difference between the median``door-toneedle'' time with thrombolytic therapy and the median``door-to-balloon'' time with PTCA in a trial). The magnitude and statistical significance of the relationship was estimated using weighted least squared regression, weighting each trial by the inverse of the variance of its log odds ratio. We also assessed the relationship between treatment benefit and mortality risk (using the mortality rate in the control group treated with thrombolytic therapy as a measure of baseline risk). RESULTS: A statistically significant trend demonstrates that the treatment effect of PTCA decreases across trials as procedure-related delay increases (p = 0.014). The regression line crosses the x-axis at 48 minutes, suggesting that, where the median procedure-related delay is greater than about three quarters of an hour, PTCA may be no more effective than thrombolytic therapy and may be harmful, even at the high volume centers represented in these trials. Additionally, all trials in which the baseline mortality risk of the patients was moderate to high (reflected by a mortality rate in the thrombolytic treatment arm of greater than 5 percent), showed treatment benefit, while four out of five trials with low mortality risks demonstrated no benefit for PTCA, and potential harm. CONCLUSION: Our meta-regression suggests that high-risk patient populations get substantially greater benefit from PTCA than low risk patients with AMI. It also suggests that the benefits of PTCA, on average, are nullified where the procedure-related delay is about 45 minutes or more. How this``time interval to mortality equivalence'' varies with a patient's mortality risk, and with the time elapsed from symptom onset, requires modeling on individual patient data. PURPOSE: Studies suggest that health status influences patient satisfaction, but little work has examined the influence of general physical functioning versus disease severity health status measures on satisfaction. Using data from a survey of diabetic patients, our objectives were to examine 1) the association between health status and patient satisfaction using two different health status measures and 2) whether the associations differed with different dimensions of satisfaction. METHODS: We surveyed 2000 patients receiving diabetes care across four Veterans Integrated Service Networks in fiscal years 1998 and 1999. Diabetes severity was measured using diabetes-related components of the Total Illness Burden Index (TIBI), a measure of diabetes complications and co-morbidities. Physical health status was measured by the Physical Function Index (PFI10) from the Short Form 36. Using satisfaction with overall quality as the dependent variable, we constructed two separate multiple linear regression models that examined the association between physical health status (PFI10 or TIBI) and overall satisfaction, controlling for gender, race, income, education, age, number of primary care visits, and presence of mental health diagnosis. We re-ran these models using a scale of satisfaction with patient-provider communication as the dependent variable. RESULTS: 70% of eligible patients responded to the survey (n=1314). These patients reported low levels of physical function (PFI10 mean(SD) = 46.5(30.2)), and high levels of diabetes severity (TIBI mean(SD) = 40.5(18.8)). Overall, these patients were moderately satisfied, with 65% percent reporting excellent or very good quality of care. Lower levels of physical functioning were associated with less satisfaction (p < .01), but generic physical health status explained less than 1% of the variation in satisfaction (model r-squared =.03). In contrast, diabetes-related severity and co-morbidities were more strongly associated with lower levels of satisfaction (p < .001), with the TIBI explaining 4% of satisfaction variation (model r-squared =.05). Results were similar when we examined satisfaction with patient-provider communication. CONCLUSION: In this diabetes population, diabetes-related disease severity and comorbidities explained a greater portion of the variance in satisfaction than did a measure of physical functioning. Adjusting only for general physical function in studies comparing satisfaction among individual providers or organizations may not be sufficient if patients of these providers have similar physical functioning but differing degrees of disease severity. PURPOSE: Although attention to substance abuse is considered important in the care of the homeless, published data do not indicate whether homelessness carries a worse prognosis following detoxification. We studied the association between housing status and substance abuse relapse following detoxification (detox). METHODS: Subjects underwent inpatient detox for alcohol, cocaine or heroin from 6/97 to 3/ 99. Baseline interviews assessed substance use, homelessness during the 6 months prior to detox, and demographic, health and psychosocial variables. Six months later, subjects reported recurrent substance use (relapse) and post-detox treatment history. Survival analysis (proportional hazards regression) was used to assess the association between homelessness, other clinically relevant predictors, and tune to relapse follow4ng detox. We specified a single interaction term, homelessness with Inpatient Sobriety Stabilization Program (ISSP) exposure. ISSP's are voluntary, short-term transitional sobriety-oriented facilities, often called``holding programs.'' RESULTS: Of 254 subjects (54% of 470) available at 6 months, 72% reported relapse, occurring a median of 31 days after detox. Forty-nine percent of subjects had reported 1 homeless night before detox. Follow-up rates did not significantly differ between homeless and nonhomeless subjects. There was no difference in time to relapse for subjects stratified by housing status or by varying durations of homelessness. Controlling for demographic and psychosocial variables, subjects with full-time employment were at significantly lower risk of relapse ISSP-(Hazard Ratio=0.71, 95% CI .50 ± .98). There was no main effect for homelessness (p=.19) but there was a significant interaction between homelessness and ISSP exposure (p=.04). The Table shows the proportion relapsed at 6 months, broken down by housing status and ISSP exposure, and helps to demonstrate the interaction. Homeless persons who used ISSP's had the lowest relapse risk. In Navajo women, a population at high-risk for type 2 diabetes mellitus, depot medroxyprogesterone (DMPA) is a common contraceptive due to its ease of administration. However, recent evidence suggests that DMPA contraception may lead to weight gain and independently decrease insulin sensitivity. The objective of this study was to determine if DMPA was associated with development of diabetes in Navajo women. METHODS: We conducted a clinic-based case-control study. Eligible subjects were Navajo women aged 18 ± 50 years who had seen a healthcare provider at a Navajo Area Indian Health Service clinic at least once in 1998. Diabetic cases (n = 284) and non-diabetic controls (n = 570) were matched by age. Medical records were reviewed to determine contraception use before the diagnosis date of diabetes in each case and its matched controls. RESULTS: DMPA users were more likely to develop diabetes than patients who had used combination estrogen-progesterone oral contraception only (OR 3.8, 95% CI [1.8 ± 7.9]). The greater odds persisted after adjustment for body mass index (BMI) (OR 3.6, 95% CI [1.6 ± 7.9]). When DMPA use of 3 months vs. 4 ± 11 months vs. 12 or more months was compared, risk of diabetes was associated with longer use(p = 0.02). Longer DMPA use was also associated with greater BMI; each additional month of use was associated with a 0.12 kg/m2 greater BMI (95% CI 0.001 ± 0.24). CONCLUSION: DMPA contraception was associated with greater risk of diabetes compared to combination oral contraception, and risk was greater with longer use and persisted after adjustment for body mass index. Additional research is needed to confirm these results and elucidate the mechanism, but DMPA may have this previously unrecognized side effect that could influence choice of contraceptive method, especially in women at high risk for diabetes mellitus. PURPOSE: Diabetes mellitus affects an increasingly younger population. Although over 10 million women in the United States use oral contraceptives (OCs), the association between diabetes and current OC use in young adult women is unclear. We studied the associations between 1)current OC use and 2)glucose levels, insulin levels, and diabetes in young adult women. METHODS: Female participants (n=2,787) in the Coronary Artery Risk Development in Young Adults study (CARDIA), a multi-center, prospective observational study of African-Americans and whites, were aged 18 ± 30 years at enrollment. OC use, fasting glucose, fasting insulin levels, and diabetes diagnosis were assessed at study examination years 0, 7, and 10. Current OC use was defined as OC use at each time of examination; non-use (the reference group) was defined as combined past OC use and never use at each examination. Using generalized estimating equations, we analyzed the cross-sectional associations between 1)current OC use and 2)fasting glucose, fasting insulin, and presence of diabetes.\nRESULTS: Current OC users differed from non-users on several factors. Current users were younger, had a lower mean body mass index (BMI), lower smoking prevalence, lower parity, and higher mean total cholesterol at all exams. By year 7, current users were more highly educated and by year 10 current users were more likely to be white and were more physically active than non-users. In unadjusted analyses, current use was associated with lower fasting glucose levels [À4.9 mg/dl, 95% CI (À5.7, À4. PURPOSE: Advanced lung cancer is a disease with little hope of long term survival. The two treatment options for patients are chemotherapy or supportive care. Several factors may influence the decision to undergo treatment. One poorly understood factor is the influence of a patient's faith on how they make medical decisions. We compared the effect of faith on treatment decisions among doctors, patients (PTs) and their caregivers (CGs). METHODS: 100 PTs with newly diagnosed advanced (stage IIIB) or metastatic (stage IV) lung cancer, their CGs, and 257 medical oncologists were interviewed separately using a standardized questionnaire. Participants were asked to rank, from 1 to 7, the factors that influenced their treatment decisions: (1)cancer doctor's recommendation, (2)faith in God, (3)ability of treatment to cure disease, (4)side effects, (5)family doctor's recommendation, (6)spouse's recommendation, (7)children's recommendation. Rankings were compared between the 3 groups of participants using restricted simultaneous ordered logistic regression models. Comparisons between those PTs who ranked faith as a high priority (1 or 2), and those PTs who ranked faith as a low priority (3 ± 7), were made using t-tests and chi-square tests. RESULTS: All 3 groups ranked the oncologist's recommendation as the most important factor. However, PTs and their CGs ranked faith in God second, while physicians placed it last (p < 0.001). The ability of the treatment to cure disease and side effects were important to all groups. PTs and CGs ranked their family doctor, spouse or child's recommendation as less important. Physicians were generally in agreement with PTs and CGs with the exception of physicians feeling the spouse's input was more important than did the PTs or CGs. PTs who placed a high priority on faith in God (n=53) were more likely to be African American (40% vs. 19%; p < 0.05). Irrespective of race, PTs who placed a high priority on faith in God were less likely to have graduated high school than those who ranked faith lower (47% vs. 72%; p < 0.001). There was a trend for PTs who ranked faith high to leave all decisions regarding treamtent to their oncologist. CONCLUSION: PTs and CGs are in complete agreement on the factors that are important in deciding on treatment options for advanced lung cancer but differ substantially from doctors. All 3 agree that the single most important factor is the oncologists's recommendation. However, faith in God is an extremely important factor for PTs and CGs but not physicians. PTs who ranked faith high are disproportionately African American and less likely to have graduated high school. Medical decision making incorporates tangible probabilities and utilities, (i.e. morbidity, mortality, side effects and costs of differing treatment options), into decision models that aid in a patient's treatment choice. This study suggests the previous model of decision making may be oversimplified. To our knowledge this is the first study to demonstrate that, for some, faith is an extremely important factor in medical decision making, more so than even the efficacy of treatment. If faith plays an important role in how some PTs decide treatment, and physicians are unaware of or do not account for it, the decision making process may be unsatisfactory to all involved. Future studies should clarify how faith impacts on individual decisions regarding treatment. Alcohol consumption decreased in all treatment arms. The 77 subjects (36%) who sustained a 30% decrease in drinks/month from baseline through 12 months had significant improvement in the SF-36 Mental Component Summary score (P = 0.037) and Physical Component Summary score (P = 0.058) and had fewer alcohol-related consequences (P < 0.001) when compared to those who did not sustain a decrease. In addition, the 34 subjects who sustained a decrease to non-hazardous levels (defined as < 16 drinks/week for men and < 12 drinks/ week for women) through 12 months had significant improvement in the SF-36 Physical Component Summary score (P = 0.002) and had fewer alcohol-related consequences (P = 0.047) when compared to those who continued a hazardous level of use. CONCLUSION: Outpatient problem drinkers who sustain a reduction in alcohol consumption have improved health-related quality of life and fewer alcohol-related adverse consequences. Benefits are seen across a wide spectrum of alcohol problems. These findings provide additional motivation for primary care physicians to identify patients with alcohol problems and to initiate intervention. Our overall response rate was 34.6%. We found that over half of the residents who responded had ever fallen asleep while driving, and 21.9% of respondents were involved in an automobile accident during residency. Over half of the respondents had received a moving violation, most often for driving fast and driving to or from clinic or the office. There was no correlation between frequency of call and hours of sleep to incidence of motor vehicle accidents and receiving a citation; however, there was a higher incidence of falling asleep while driving in patients who were on call every third night and slept fewer than four hours a night (p = 0.001 for both). If they did have an accident, it was most likely on a post-call day returning from the hospital. 89.9% of the residents got four or fewer hours of sleep on a typical on-call night. CONCLUSION: Residents-in-training are at high risk for motor vehicle accidents and falling asleep while driving when they are post-call and have less sleep. Mechanisms for reducing resident fatigue post-call such as a night-float system may reduce these incidences. . Descriptive statistics, t-tests, and ANOVA (SPSS version 10) were used to describe the population and determine associations between the variables of interest. RESULTS: 66/82 (80%) individuals from 14 PoPCRN sites consented to and were able to participate. 56% were female, 89% were white, 39% were cared for at home, and 53% had a cancer diagnosis. The median age was 76 years, median Karnofsky score 50%, and median time between hospice admit and interview was 49 days. MQOL responses (range 0 ± 10; 0=bad, 10=good) indicate that these patients had few physical symptoms, but the ones they had were problematic (single troublesome symptom score=4.4). The MQOL subscale scores indicate that these patients were less troubled by psychosocial and existential issues than by physical symptoms: Total MQOL score=7.1, Physical well-being score=6.0, Physical symptom overall score=5.9, Psychological symptom score=7.1, Existential well-being score=7.4, Support score=8.6. There were no significant associations between age, marital status, gender or length of hospice care and any of the QOL scores. A Karnofsky score < 50% was associated with worse Existential well-being (6.9 vs. 7.9, p=0.017) while a cancer diagnosis was associated with greater Existential well-being (7.8 vs. 6.9, p=0.047). Being cared for at home and a cancer diagnosis were both associated with a greater sense of Support ( From pharmacy data, we categorized the type of prescribed ART for > 50% of treatment time in each year into 4 intensity levels: none; 1 ART; 2+ ART but not highly active ART (HAART); or HAART including a protease inhibitor or non-nucleoside reductase inhibitor. Changes in ART from the base year to the next were classified as: suboptimal (ie, continuing or increasing to 1 ART or changing from a more intense to a less intense regimen); acceptable (ie, continuing or increasing to 2+ non-HAART drugs); or optimal (ie, continuing or increasing to HAART). Indicators were also created for visits (N=0, 1 ± 3, 4+) to a provider with a NYS contract to offer HIV-focused care in exchange for higher Medicaid payment. Each patient's outpatient care pattern in the base year was categorized as: regular medical care alone ( > 35% of visits to one provider); regular substance abuse treatment alone (6+ months with one provider); both; or neither. We estimated ordinal logistic regression models of ART pattern corrected for clustering of observations. RESULTS: Temporal changes in ART were: suboptimal (44%), acceptable (20%), and optimal (36%). Adjusted odds ratios (AOR) of greater intensity of ART were higher for persons with PURPOSE: Patients with limited English proficiency who are seen by language discordant providers may communicate through a variety of interpretation methods. We studied the effect of translation method on satisfaction among Spanish-speaking patients at a hospital-based walkin clinic serving primarily indigent patients. METHODS: All Spanish-speaking patients and a random selection of 10% of English-speaking patients presenting to the walk-in clinic at Denver Health Medical Center between July and October 2000 were approached for study entry. Participants completed a self-administered post-visit questionnaire assessing demographics, general health, and satisfaction with clinic visit.`L anguage concordant'' patients were defined as Spanish-speaking patients seen by Spanishspeaking providers and English-speaking patients. These patients were compared with`l anguage discordant'' patients who used AT&T interpreters, family, or other interpreters for translation. Patients were considered``satisfied'' if they rated their satisfaction as very good or excellent. Evaluation of satisfaction was by multiple logistic regression, controlling for age, sex, race, language spoken, education, and health. RESULTS: Of the 536 study participants, 67% were Spanish-speaking. Among Spanishspeaking patients, 42% were seen by language concordant providers. For patients who did not see language concordant providers, translation was through ATT telephone interpreters (19%), family members (23%), and other interpreters (16%). Language concordant and language discordant participants did not differ in age, sex, race, insurance status, or education level. Language concordant patients were less likely to be Hispanic (59% vs 95%, p=0.001) or describe their health as poor or fair (29% vs 51%, p=0.001). Language concordant patients and patients using ATT telephone interpreters reported identical overall visit satisfaction (77%), while patients using family or other interpreters were significantly less satisfied (54% and 49%, p < 0.01 and p=0.007, respectively). Compared to language concordant patients, patients who had family members translate were less satisfied with their provider in regards to listening to health concerns (62% vs 85%, p=0.003) and discussing sensitive issues (60% vs 76%, p=0.02). Patients who used other interpreters were less satisfied with provider skills (60% vs 83%, p=0.02), manners (71% vs 88%, p=0.02), listening (54% vs 85%, p=0.002), explanations (57% vs 84%), p=0.02), answers (57% vs 84%, p=0.05), and support (63% vs 84%, p=0.02). CONCLUSION: Patients utilizing less formal translation methods, such as family members and ad hoc interpreters, are less satisfied with care than those seeing language concordant providers or using AT&T telephone interpretation. METHODS: Women ages 35 ± 50 without a history of breast cancer were recruited from the waiting room of a university internal medicine practice. Following a baseline questionnaire about breast cancer risk factors, and benefits/harms of mammography, 179 women were randomized to one of three videos. For women ages 40 ± 49 who undergo 10 years of mammography, the videos described the number of lives extended(1/1000), the number of false positives expected(300/1000), and the number of women expected to worry after a false positive mammogram screening(100/1000). Information was numerically equivalent in each video but presented in a positive frame(1 woman would have her life extended) a negative frame(999 women would not have their lives extended) or a balanced frame(positive and negative information given). Participants completed a follow-up questionnaire immediately after viewing the video. RESULTS: Participants were predominately insured(80%), white(60%) and high school graduates(63%). At baseline, 3% endorsed the correct response(1 per 1000 women screened for 10 years would live longer because they had mammograms) and 76% thought 100 or 500 women would live longer. After the video, 53% endorsed the correct response with no differences by video (p=.21). For the number of false positives expected, 15% gave the correct response at baseline, but at followup the correct responses were given by more women in the negative(65%) and balanced(65%) frames than in the positive frame(40% P < .01). The results were parallel for the number expected to worry after a false positive (62%,56%, and 32% for negative, balanced and positive frames respectively). A majority of respondents in all three groups thought the benefits of mammography were more important than the harms before(75 ± 80%) and after seeing the the video(77 ± 81%). CONCLUSION: Women's baseline knowledge of the benefits of mammography was overly optimistic. All three videos improved women's knowledge of the benefits/harms of mammography. Despite this improvement in knowledge and exposure to varying frames of information, women's perceptions of the benefits/harms did not change. To determine if``numeracy '' affects women's ability to learn information about the potential benefits and harms of mammography. METHODS: In the waiting room of a university internal medicine practice we recruited 179 women ages 35 ± 50 without a history of breast cancer and randomized them to one of three videos. For women ages 40 ± 49 who undergo 10 years of mammography, the videos described the number of lives extended(1/1000), the number of false positives expected(300/ 1000), and the number of women expected to worry after a false positive mammogram screening(100/1000). Participants completed a questionnaire immediately after viewing the video. Information presented in each video was numerically equivalent, but to obtain the correct answers, subtraction was required for at least one question in each video. Numerate women were defined as being able to correctly note that 40% was equivalent to a 4 out of 10 chance. RESULTS: Participants were predominately insured(80%), white(60%), high school graduates(63%), and over half were numerate(65%).The table shows the percentage of correct responses for numerate and non-numerate women when the answers were given in the video compared to when subtraction was required to get the correct answers.\nCONCLUSION: Most numerate women were able to learn numeric information from the video but had difficulty manipulating this information if they were not given the correct response in the video. Non-numerate women were unable to learn numeric information even when given the correct response. Although women in their forties are supposed to be informed of the benefits/harms of mammography to make decisions about screening, many women may have difficulty understanding this information if it is relayed numerically. The sample consisted of a primarily well educated group of 30 ± 50 year old (75%) professional men (37%) and women (63%). Almost all of the sample had been born in China (93%), claimed Mandarin Chinese as their native language (88%), and had resided in the United States an average of 8.9 (SD=7) years. Although most of the respondents had health insurance (93%) and more than half (69%) had a primary care provider, only 37% stated that they would have a routine check-up with this provider if they``felt fine.'' However 54% of the females in our sample reported having annual pap screening and breast exam by their physicians. Over half of the sample (63%) reported problems with understanding medical terms. Less educated respondents were more likely to be hesitant to see the doctor due to language difficulties (p = 0.001) and more likely to use western medicine for a cold (p = 0.023). Respondents with lower incomes were also more likely to be hesitant to see the doctor due to language difficulties (p = 0.036) but respondents with higher incomes were more more likely to have a primary care physician (p < 0.001). CONCLUSION: Although our sample was well educated and had adequate access to health care services, the inability to understand medical terminology may be a prime contributor to the under-utilization of health care services. A more diverse and larger sample is needed to further assess the utilization patterns from all socioeconomic levels. In addition, health literacy levels of Chinese Americans need to be assessed in order to provide culturally competent health care to this population. PURPOSE: Group A -hemolytic streptococci (GABHS) are cultured in 10 ± 25% of adults with sore throat. GABHS is the main cause of sore throat requiring antibiotic therapy. The Infectious Disease Society of America recommends either penicillin or erythromycin as first-line agents to prevent complications and reduce symptoms. Our goals were: 1) define the rate and type of antibiotics given to patients with sore throat by community primary care physicians over a ten-year period, and 2) determine predictors of antibiotic prescription and use of nonrecommended antibiotics for sore throat. METHODS: The National Ambulatory Medical Care Survey (NAMCS) collects data from office-based physician practices in the United States including patient demographics, patients' reasons for visit, physician diagnoses, and medications prescribed. Using NAMCS from 1989 to 1998, we analyzed 2,141 adult primary care visits with a chief complaint of sore throat. We estimated national antibiotic prescription rates over time and determined both the most frequent diagnoses made and the most frequent antibiotics prescribed. We evaluated significant predictors of antibiotic prescription and non-recommended treatment using multivariate logistic regression. RESULTS: An estimated 6.8 million adult patients each year made acute visits for sore throat to office-based, primary care physicians between 1989 and 1998. The most common diagnoses were acute pharyngitis (42%), acute upper respiratory tract infection (21%), acute tonsillitis (8%), and streptococcal infection (6%). Antibiotics were prescribed to 73% (95% CI 70 ± 76%) of patients. The most common antibiotics prescribed were aminopenicillins (23% of visits), cephalosporins (14%), penicillin (14%), and erythromycin (10%). Over ten-years the proportion of patients receiving any antibiotic decreased while the proportion of patients receiving non-recommended treatment increased ( 1989 ± 1992 1993 ± 1995 1996 ± 1998 NHANES included 157 women with a personal history of breast cancer. Eleven of these survivors (7.0%) were currently using HRT. CONCLUSION: A surprisingly large fraction of breast cancer survivors use HRT. The concordance between two estimates obtained using very different methods argues against random or data error. Other possible explanations include: 1) the patient opts to accept the putative risks of recurrence in an informed manner, 2) the prescriber does not know that HRT is contraindicated in breast cancer, 3) the prescriber rejects the theory of hormone induced recurrence, 4) the prescriber does not know the patient is a breast cancer survivor, or 5) the patient self-prescribes. We believe that HRT use in breast cancer survivors is a sentinel event that may represent medical error. In stage 1, multi-pattern regressions were performed to impute PCS and MCS using SF-12 items only (simple model), and then using SF-12 items plus patients demographics and comorbidities (enhanced model) in an attempt to reduce bias from the simple model. Distribution and variation of the imputed SF-12 values were evaluated. Based on confidence bands of the imputed SF-12 values, a cut point of number of missing SF-12 items was selected for using the simple or enhanced model to impute SF-12 PCS and MCS. In stage 2, patients with no missing SF-12 items who also had demographic and comorbidity data were randomly classified into 11 subgroups. Permutations of missing patterns (missing 1 to 11 out of the total 12 items) on SF-12 items were generated to simulate missingness (missing 1 to 11 SF-12 items) for these patients. The imputed PCS and MCS were compared to the observed values for these patients separately by the identified cut point. The analysis was performed separately for chronic and non-chronic subgroups. RESULTS: Results: In stage 1, the standard errors of means of imputed summary scores increased almost monotonically by the number of missing SF-12 items, ranging from 0.11 to 4.55 for PCS and 0.09 to 2.18 for MCS in the non-chronic subgroup, and from 0.28 to 5.84 for PCS and 0.24 to 5.62 for MCS in the chronic subgroup. A significantly consistent increase of the width of confidence bands (about 50% in standard error) occurred between 5 and 6 items missing. Of the 13,438 patients, 10,766 (8949 non-chronic and 1817 chronic) patients had non-missing data for all SF-12 items as well as demographic and comorbidities. Productmoment correlations between the imputed and the observed scores were large, ranging from 0.73 to 0.97. The correlations from imputation with the enhanced model were consistently higher than the correlations from the simple model. The increments of correlations by the enhanced model were statistically significant for patients with more than 6 missing SF-12 items (7%, P < 0.05). CONCLUSION: Conclusion: For patients with partially missing SF-12 items, direct imputation of summary SF-12 physical and mental scores is efficient. Adding patients' demographics and comorbidity information to the imputation model for PCS and MCS can improve the imputed value and reduce bias. The enhancement of adding patients' demographics and comorbidities to the imputation models for PCS and MCS is necessary and important for patients with 6 or more SF-12 items missing. PURPOSE: Most clinicians rely on the history to make an accurate diagnosis and prescribe appropriate treatment. However, as increasing administrative and financial pressures shorten the outpatient clinical visit, the opportunity to take a careful history is becoming more abbreviated. This study prospectively evaluated whether accurate history taking, under precisely controlled conditions, actually leads to better outpatient clinical practice. METHODS: We prospectively collected data in the primary care general internal medicine clinics at two VA-university teaching hospitals. Greater than 95% of residents and attendings consented to participate Ð and 20 were randomly selected as subjects. Eight unannounced standardized patients (SPs) were introduced into the clinic of each selected provider presenting four common conditions: COPD, Low Back Pain, diabetes mellitus and coronary artery disease (i.e., 2 SPs per condition). Thus, every physician saw an identical set of cases. All visits were new patient appointments. Visits were scored by the SP using a closed-ended checklist of quality criteria; other criteria were assessed by abstracting the medical record. Scoring was based on explicit criteria derived from national guidelines and expert panels. Separate scores were generated for History Taking, the Physical Examination, Testing (laboratory and imaging studies), and Diagnosis plus Treatment. Scores were expressed as a percentage of criteria that were correct. Multiple linear regression models were used to predict if better history taking led to more accurate diagnosis and treatment plans, more correct testing, and appropriate physical examinations. The models controlled for case, site, and level of training. RESULTS: Physicians who scored higher on history taking (that is, took more accurate histories) made more accurate diagnoses and more appropriate treatment decisions (p = 0.006). By contrast, more accurate physical examinations and diagnostic test ordering did not predict better diagnosis and treatment (p > 0.05). The quality of diagnosis and treatment varied between the two sites (p < 0.001) but not by level of training or case. Taking an accurate history did predict the appropriateness of the physical examination (p = 0.004), but it did not predict testing (p > 0.05). CONCLUSION: Under controlled conditions, where case mix was specified by experimental design, our results show that accurate history taking predicted when residents and attendings were more likely to make the correct diagnosis and prescribe the best treatment. While accurate history taking also guided the physical examination, the examination itself did not lead to subsequently higher process scores. The site effects were also strong. These findings demonstrate that successful completion of the history effectively predicts of diagnostic and treatment accuracy. Emphasis on development of history taking skills in residency programs, and adequate visits time allocations in practice settings, should contribute to higher quality clinical care. were able to recall their PCP's name. When asked,``When you are sick and need care right away, how quickly do they see you?'' 70% answered the same day or the next day. Regarding ED usage, 49% of patients called triage before coming to the ED; those with CMA insurance were less likely to call (42% vs 75%, p =. 04). Of those that called, 96% reported they were told to go to the ED (``callers''). ED nurses triaged 59% of``callers'' and 44% of non``callers'' to Level 1 or 2 (out of 4; 1 is high). Eighty per cent of respondents rated``How well doctor listens to you,''`D octors respect for you,'' and``Doctors ability to figure out what is the matter'' as excellent or very good. There were no significant differences in ratings between PCPs and ED physicians.\nCONCLUSION: Our small pilot survey indicates that patient knowledge of, and access to, acute primary care was very good. However a large number of patients with non-urgent conditions still didn't call before presenting to the ED. Almost all that did call were sent to the ED, even though 41% were then judged to be of lower sickness acuity by the ED triage nurses. These results suggest that our CMA and other patients need more education and/or incentives to access primary care alternatives to ED use. Our results also suggest that our triage nurses could benefit with further training on appropriate ED referral. We hypothesized that physically active PAD persons with no exertional leg symptoms would have better leg functioning than other leg pain groups. METHODS: Study participants were 454 men and women with PAD identified from three medical centers in Chicago. Participants were categorized into one of six mutually exclusive leg symptom groups: Group 1:No exertional leg pain/active (walked > 6 blocks in the last week) (13%); Group 2: No exertional leg pain/inactive ( < = 6 blocks walked last week) (6%); Group 3: Atypical exertional leg pain and walk through leg pain (8%); Group 4: Intermittent claudication (37%); Group 5: Atypical exertional leg pain/stop walking with leg pain (17%); Group 6: Leg pain on exertion and rest (19%). Functional measures included six-minute walk, four meter walking velocity, time for five chair rises, and standing balance. All statistical analyses were adjusted for multiple comparisons using Bonferroni method. RESULTS: Group 1 and Group 3 had significantly less severe PAD, as measured by the ankle brachial index, than the other groups. Compared to all other groups, Group 6 had the highest prevalence of lower extremity arthritis, disc disease, diabetes, and depression. In general, functional performance was poorest in Group 6 and best in Groups 1 and 3. Compared to Group 1, Group 6 had slower walking velocity (0.75 m/sec vs. 0.89 m/sec, p < 0.05), achieved shorter distance in the six-minute walk (972 feet vs. 1201 feet, p < 0.05), had slower time for rising from a seated position five times (14.2 sec vs. 11.3 sec, p < 0.05), and was less able to hold the tandem stand position for ten seconds (32% vs. 51%, p < 0.05). Compared to Group 1, Group 4 was more likely to stop during the six-minute walk (38% vs. 16%, p < 0.05) and walked fewer blocks in the last week (35.7 vs. 53.0, p < 0.05). Group 2 had slower walking velocity than Group 1 (1.02 vs. 1.22 meters/sec p < 0.01). CONCLUSION: Comorbid disease may contribute to the nature of leg symptoms reported by PAD patients. Clinicians can use the leg symptom categories defined above to gauge the degree of functional impairment associated with PAD. PURPOSE: Understanding the natural history of weight gain on a population basis may be a critical step towards developing effective clinical or public health interventions. While the marked rise in overweight and obesity prevalence in this country has been well documented by the serial cross sectional data of the US National Health and Nutrition Examination Survey (NHANES), longitudinal description of the trend is lacking. This study assesses the development of body mass of a cohort of young US adults and identifies predictors including age, gender and ethnic group that may have implications for targeted intervention. METHODS: The National Longitudinal Survey of Youth (NLSY79) is a nationally representative sample of over 12,000 persons aged 14 ± 22 years at baseline, with oversampling of minority ethnic groups. Body mass index (BMI = kg/m2) was calculated from self-reported height and weight data at 12 sample points over 17 years.`Overweight' and obese' were defined as BMI > 25 and > 30, respectively. Mean BMI was calculated by age group, gender and ethnicity. In this study, unadjusted data are used to describe trends in BMI, and to calculate incidence of overweight and obesity using survival analysis. RESULTS: BMI trends were strongly related to age, race and gender. Women's BMI increased steadily with age, with average BMI reaching``overweight'' status by age 26 in Black women, age 29 in Hispanic women and age 35 in all others. The average BMI of Black women was in the obese range before age 40. Ethnic differences in weight prevalence were evident by age 17. Survival analysis quantified the incidence rate of obesity as 2.5 times faster for Black women as for Non-Hispanic/Non-Black women (CI 2.2 ± 2.8) and 2.1 times faster for Hispanic women versus those who were Non-Hispanic/Non-Black (CI 1.8 ± 2.4). Male average BMI showed less dramatic age trends and a different ethnicity pattern: Hispanic men consistently had the highest mean BMI values. Hispanic men developed obesity 2.0 times faster (CI 1.7 ± 2.3) and Black men developed obesity 1.8 times faster than Non-Black/Non-Hispanic men (CI 1.6 ± 2.0). CONCLUSION: This large longitudinal survey shows marked differences in the rate of weight accumulation for different ethnic groups, evident at a very young age. This pattern persisted through young adulthood with excess weight, on average, increasing gradually with age. To alter the trajectory of obesity in this country, interventions should target young adults, and focus on those of minority ethnic groups. have been shown to be as effective as antidepressant medications for treating patients with major depression. The success of these methods may be related in part to the use of specific counseling techniques. These analyses assess the extent to which primary care clinicians and mental health specialists use CBT techniques to treat primary care patients with major depression. METHODS: Cross-sectional descriptive evaluation of counseling received by 567 patients meeting criteria for 1-year major depressive disorder based on a structured diagnostic interview. The 567 enrolled participants are from a consecutive sample of patients attending one of nine participating primary care practices in California (3 based in a single academically-affiliated VA medical center and 6 in independent medical centers from 1 region of Kaiser Permanente). Participants completed an interviewer-administered survey of health perceptions and utilization patterns. Patients reported the frequency of visits to medical and mental health providers and the extent to which their providers used four different CBT techniques. These included helping the patient reduce negative thinking, encouraging enjoyable activities, assisting with problemsolving, and helping the patient feel better about their life as it is. RESULTS: Patients reported an average of 3.2 visits (median=1.0, range 0 ± 144, 61.3% with no visits) to medical providers in which they discussed their personal or emotional problems and 4.5 (median=0, range 0 ± 96, 60.7% with no visits) visits to mental health specialists. Of those patients with at least one visit to a medical provider during the past six months, between 42.1% and 57.6% per therapeutic technique reported that their medical providers had used it. The range was 78.9% to 84.9% per therapeutic technique for patients visiting mental health specialists. The most prevalent therapeutic technique was encouragement of more enjoyable activities. 59.3% and 85.5% of patients reported receiving counseling that included at least two of the four therapeutic techniques from medical and mental health providers, respectively. CONCLUSION: Although the mental health specialists used therapeutic counseling techniques more often than did the primary care clinicians in these practices, primary care clinicians used them more often than we expected given the competing demands of generalist practice. This optimistic result suggests that further evaluation of the efficacy and effectiveness of primary care clinician therapeutic counseling in improving outcomes is warranted. Little is known about the predictors of alternative medicine use in the veteran population. This study investigates whether veterans' sociodemographic characteristics, selfreported health status, health-related beliefs, and satisfaction with their VA health care providers, are related to their use of alternative medicine. METHODS: Participants included patients attending 7 VA general internal medicine clinics participating in the VA Ambulatory Care Quality Improvement Project (ACQUIP), a multicenter randomized controlled trial to test the effectiveness of an information feedback system on improving health status and satisfaction outcomes. This analysis is based on a random subsample of 1034 patients who also received the ACQUIP Health Beliefs Scale. This scale assessed patients' attitudes towards conventional medicine and adherence to health-conscious lifestyles. Patients' satisfaction with their provider was measured with the 12-item humanistic component of the Seattle Outpatient Satisfaction Questionnaire. Self-reported health status was measured with the mental, physical, and pain subscales of the SF-36. Additional data included patients' age, gender, ethnicity, level of education, and income. Alternative medicine use was determined with a questionnaire assessing the use of the following: biofeedback/relaxation, chiropractic, acupuncture, massage, herbs, homeopathy, naturopathy, spiritual healing, folk remedies, and other. Multiple logistic regression techniques were used to evaluate the association between alternative medicine use within the past year (yes or no), sociodemographic characteristics, health beliefs, and patient satisfaction. RESULTS: 495 patients returned the Health Beliefs Scale (48% response) and were included in this analysis. 124 patients (25%) reported the use of at least one alternative medicine therapy in the past year, with the most common modalities including herbs (13.5%), chiropractic (7%), and massage (6%). In multivariate analyses adjusted for sociodemographic characteristics and selfreported health status, the following were associated with alternative medicine use: more education (OR 1.5; 95% CI 1.3 ± 1.9); better physical health (OR 1.044; 95% CI 1.009 ± 1.081); and more pain (OR 1.022; 95% CI 1.005 ± 1.039). After adjustment for sociodemographic factors and subjective health status, alternative medicine use was associated with the following health-related attitudes and behaviors: believing more in the value of non-medical treatments (OR 1.4; 95% CI 1.1 ± 1.9); adhering to health-conscious lifestyles (OR 1.7; 95% CI 1.3 ± 2.2); and actively seeking health information from non-medical sources (OR 1.6; 95% CI 1.3 ± 2.1). In adjusted analyses, there were no significant associations between alternative medicine use and attitudes towards conventional medicine or patient satisfaction. CONCLUSION: A quarter of VA general internal medicine patients in this study reported the use of alternative medicine in the past year. Patients who used alternative medicine had more education, and reported better physical health but more pain. Alternative medicine users also believed more in the value of non-medical treatments, reported adherence to health-conscious lifestyles, and reported obtaining more health information from non-medical sources. Similar to previous studies, negative attitudes towards conventional medicine and lower levels of patients' satisfaction with their VA providers did not appear to influence use of alternative medical modalities. Procedures Ð Using established criteria, potentially inappropriate medications were identified in the hospital formulary and assessed by the central pharmacy database for frequency of use. The hospital admissions database was used to identify the total number of elders hospitalized in the previous year. The two databases were merged, sorting by patient age, non-intensive care unit location, and our defined inappropriate medications. A medical error rate was calculated by dividing the total of inappropriate medications dispensed by 100 patient-days. Patient days were calculated by length-of-stay data also obtained from the hospital admissions database. The study was repeated in the subsequent 6-months to assess for secular trends and the stability of the error rate. RESULTS: A total of 11,797 doses of inappropriate medications were dispensed to 15,011 patients aged 70 and over during a 12-month period. The most common of these prescribed medications were meperidine (3,652 doses), amitriptyline (1,736 doses) , and diazepam (1,644 doses) . The medical error rate was 0.12 per day, or 12 errors per 100 patient days. Findings for the subsequent 6 months were similar. CONCLUSION: The results of our study are troubling though not surprising. Academic teaching hospitals often require all patient orders to be written by resident physicians, with variable levels of senior supervision. Educational interventions targeted at internal medicine residents have the potential to reduce the medical error rate in elderly hospitalized patients. References of key articles were also reviewed for related studies. Studies were included if they enrolled postmenopausal women and reported deep venous thrombosis (DVT), pulmonary embolism (PE), or VTE as either part of a predetermined outcome or as a reportable adverse event related to HRT. Another Medline search from January 1991 to June 2000 identified studies addressing SERMs and VTE risk. Ten HRT studies (two randomized controlled trials, seven case-control studies, and one cohort study) and five SERM randomized controlled trials met inclusion criteria. Two investigators abstracted data on participants, intervention, VTE rates, and confounders. We used the Bayesian data analysis framework for the meta-analysis of HRT studies. The SERMs were not included in the meta-analysis. RESULTS: HRT was associated with an increased risk of VTE in current users (RR=2.34; 95% Cl, 1.83 ± 2.94). Five studies showed a significant increase in VTE with HRT. The highest VTE risk was reported with the first year of URT use (OR =2.9 to 6.7). Two of the SERM randomized controlled trials reported an increased risk of VTE (RR= 1.91 to 3. 1). CONCLUSION: Postmenopausal HRT is associated with an increased risk of VTE. SERMs may be similarly associated with VTE risk. These findings are pertinent to clinicians and patients considering HRT.\nBREAST CANCER SCREENING IN AMERICAN SAMOAN WOMEN. S. Mishra 1 , F. Hubbell 1 , P. Luce 2 ; 1 University of California, Irvine, CA; 2 National Office of Samoan Affairs, Carson, CA PURPOSE: Little is known about breast cancer screening practices or predictors of age-specific screening for Samoan women. We determine population-based estimates of utilization of clinical breast exam (CBE) and mammography by Samoan women and examine predictors of recent age-specific utilization of screening exams. METHODS: Through systematic, random sampling procedures, we identified and interviewed 720 adult (!30 years) Samoan women residing in American Samoa, Hawaii, and Los Angeles. Multivariate logistic regressions were performed to determine independent predictors for recent age-specific screening RESULTS: Only 55.6% of women (!30 years) had ever had a CBE and 32.9% of women (!40 years) had ever had a mammogram. Furthermore, only 24.4% and 22.4% of Samoan women (!40 years) residing in Hawaii and Los Angeles, respectively, had an age-specific mammogram within the prior year. Independent predictors of age-specific CBE screening included age, education, health insurance, ambulatory visit, and being a resident of Hawaii or Los Angeles; and those for mammography included ambulatory visit and awareness of screening guidelines. CONCLUSION: Population-based estimates of age-specific breast cancer screening among Samoan women are lower than nation objectives and those reported for other minorities. Targeted efforts that address doctor patient communication on preventative behavior, improved access to health care services (especially in American Samoa), and focused educational awareness programs are needed to improve the dismal screening rates observed in this indigenous population. PURPOSE: Controversy exists on whether postmenopausal hormone replacement therapy (HRT) can lessen or avert cognitive impairment that may lead to dementia. The purpose of this study is to examine the association of HRT use and impaired cognition. METHODS: Postmenopausal women (n=1403) participating in the population-based Beaver Dam Eye Study and the Epidemiology of Hearing Loss Study in Beaver Dam, Wisconsin were included in these analyses. Data were from the 10-year follow-up for the eye study and the concurrent 5-year follow-up for the hearing study (1998 ± 2000) . HRT use was assessed by questionnaire and confirmed by participants' presentation of prescribed medications at the eye examination. Cognitive impairment is defined as less than 24 points, or 80%, on the minimental status exam, or doctor-diagnosed Alzheimer's disease. Odds ratios were obtained from logistic regression models. RESULTS: The average age of the women was 69 years (range 53 to 97). Twenty-five percent of participants were current HRT users. The average age of current users was 65 years; the average age of non-users was 72 years. More current users completed high school than nonusers, 91% and 78%, respectively. In preliminary analyses, the overall prevalence of cognitive impairment was 6%. HRT users were less likely to be cognitively impaired than non-users (5.7% vs 0.5%, p = 0.0003). After adjusting for age, education, and depression (as measured by Short Form-36 mental health subscale), the odds of being cognitively impaired were not statistically significantly different for HRT users versus non-users (OR = 0.40; 95% CI = 0.14 ± 1.15, p = 0.09). CONCLUSION: These preliminary analyses suggest that postmenopausal HRT use may be associated with a slighter lower odds of having impaired cognition. However, this association may be marking selection bias, as the relationship is no longer statistically significant after adjusting for the confounding effects of age, education, and depression. Longitudinal studies, adequately controlling for these confounders, are needed to determine the effect of HRT use on cognition. PURPOSE: Assessing patient satisfaction with care received is important to measure as it is a primary goal of providers and clinic practices. Studies done, to date, have been limited in that most measure patient satisfaction with experiences related to medical student education and have been retrospective in design. We designed a prospective study to find the differences in patient satisfaction with outpatient care provided by continuity clinic resident physicians compared to satisfaction with care provided directly by faculty physicians in the same practice. METHODS: During one week in June 2000, 1107 adult and pediatric clinic patients completed a patient satisfaction questionnaire following their visit, during which the patient was either seen by the faculty member or by the resident physician year 1 through 4. The data was collected from 5 primary care clinics; pediatrics, internal medicine and a combined medicine/ pediatric clinic. Four clinics were in community practices and one clinic was an academic hospital based practice. The questionnaire comprised of 26 questions addressing service characteristics of the office staff, provider and facility. Ten questions assessed satisfaction of care provided by the doctor. Differences in results for faculty vs. resident patients were analyzed for the entire set of collected responses, as well as for each clinic. RESULTS: 1772 total patients were seen during the study week. Sixty-three percent of the questionnaires were completed (1107); 824 from faculty patients and 283 from visits with the resident. 42% of the faculty patients and 24% of the resident patients saw the doctor they wanted to see on the day of the visit. 67% of the faculty patients and 60% of the residents patients``strongly agreed'' that the resident answered questions in a way the patient could understand. 52% of faculty patients and 43% of the resident patients indicated that the time spent with the physician was``excellent''. 62% of the faculty patients rated thoroughness of the exam as excellent versus 50% of the resident visits.47% of the faculty visits and 36% of the resident visits rated the care received overall as``excellent''. All differences reached statistical significance with p < 0.05. A similar distribution of small differences, all indicating better satisfaction with care by faculty occurred in all 5 participating practices. CONCLUSION: Patients were more satisfied with care received by faculty as compared with residents. Difficulty for the residents patients to schedule an appointment with their physician of choice is an important barrier to improving patient satisfaction. Overall access to and use of HIV care is lower for Hispanics than for whites, but little is known about how these vary within the diverse Hispanic population in the US. We examined access to, use of, and unmet need for care among HIV+ Hispanics according to level of acculturation, language in which subjects chose to complete surveys, citizenship status, gender, education and insurance status among the 415 Hispanic patients (15% of all subjects) in the HIV Costs and Services Utilization Study (HCSUS). METHODS: HCSUS is a nationally representative study of HIV-infected persons in care. Weights were used to adjust for sampling, multiplicity, and non-response. Standard errors were corrected for the multistage sampling design. Use of care was measured in four dimensions: ambulatory care, emergency care, hospital care and mental health services. Overall access was measured with a six-item scale (alpha = 0.72) asking about urgent care, emergency care, hospital care, clinic location, costliness of services, and access to specialists. Unmet need for care was measured by five items asking about foregone medical care due to: inability to get time off work, being too sick to seek care, lack of transportation, needing to care for someone else, or insufficient money to pay for care. All results reported are significant at a p < 0.05 level. RESULTS: In separate multivariate analyses controlling for CD4 count, Hispanics who were US citizens reported greater use of emergency care (Odds Ratio [OR]=1.92), hospital care (OR=2.59) and mental health services (OR=1.92) than non-citizens. Hispanics who were more acculturated (OR=1.76), who completed an English-language survey (OR=2.43), who were US citizens (OR=1.85), who were more educated (OR=1.93) and who were insured (OR=3.54) reported greater access to care. Women reported less use of ambulatory care (OR=0.66) and mental health services (OR=0.63) and worse overall access (OR=0.63) than men. Women were more likely to report unmet need for medical care (OR=2.17) than men (due to any reason of the five reasons asked about). CONCLUSION: Significant variation exists among HIV+ Hispanics in access to and use of care by language preference, acculturation, citizenship status and gender. Interventions to improve access for Hispanics should prioritize those Hispanics in greatest need. University of Pittsburgh, Pittsburgh, PA PURPOSE: Community-acquired pneumonia (CAP) is the leading infectious disease cause of death in the United States. Although risk factors for short-term mortality are widely studied, there is very little data on the risk factors for long-term mortality after surviving CAP. The aim of this study is to examine the predictors of long-term mortality after initial CAP. METHODS: This analysis was a follow-up of the 4 United States sites of the Pneumonia Patient Outcomes Research Team (PORT) prospective, multicenter cohort study. Baseline demographic and clinical data, including comorbid illnesses, laboratory and radiology results, were obtained for all patients. Long-term mortality was determined using the National Death Index. Patients were enrolled between October 1991 and March 1994, and mortality information was obtained up to December 1998. Charlson's comorbidity scale was used to assign a comorbidity score for preexisting comorbid conditions and age. All patients who died within 90-days of enrollment were excluded from the analysis in order to study the long-term mortality of those surviving after the acute illness. Statistical modeling included the use of Kaplan ± Meier survival plots and the log rank test for categorical variables, and univariate Cox proportional hazard modeling for continuous variables. Any variable that was statistically significant (p 0.05) was then entered into a step-wise forward multivariate Cox proportional hazard model. RESULTS: Of the 1555 subjects enrolled at the 4 U.S. centers, 1419 subjects survived past 90days after enrollment. Of those patients 472 (33.3%) died by December 1998. During the first year after enrollment 29.2% of patients died, in the second year 19.1%, third year 16.1%, fourth year 10.0% and !fifth year 25.4%. Significant baseline predictors of long-term mortality were being DNR (hazard ratio 2.1 with 95% confidence interval of 1.5 ± 2.9), poor nutritional status (1.9, 1.3 ± 2.8), pleural effusion (1.4, 1.1 ± 1.8), steroid use (1.6, 1.3 ± 2.1), nursing home residence (10.1, 3.2 ± 32.1), Charlson's score of 1 or 2 (2.5, 1.6 ± 4.0), Charlson's score 3 or 4 (5.8, 3.9 ± 8.9), and a Charlson's score !5 (14.6, 10 ± 21.5). Significant protective factors against mortality included !college education (0.6, 0.5 ± 0.8), female gender (0.7, 0.6 ± 0.8), and the symptom of fever (0.7, 0.6 ± 0.9). CONCLUSION: This study demonstrates that there is a significant long-term mortality burden after CAP, and that age, education, gender, comorbidity, nutritional status and nursing home status are important predictors of long-term mortality after CAP. PURPOSE: Although studies of tea consumption and mortality among healthy adults report conflicting results, the effect of tea consumption on patients with acute myocardial infarction is unknown. METHODS: In a prospective cohort study, we followed 1935 patients (601 women and 1334 men) hospitalized with a confirmed acute myocardial infarction between 1989 and 1994 at 45 community hospitals and tertiary care centers in the United States, as part of the Determinants of Myocardial Infarction Onset Study. During hospitalization, participants reported their usual intake of caffeinated tea during the year prior to infarction to trained interviewers. We excluded 35 patients with missing information on tea intake. We analyzed survival through 1995 using Cox proportional hazards regression, controlling for age, sex, body-mass index, previous myocardial infarction, diabetes, hypertension, education, income, current and former smoking, alcohol use, regular exertion, acute infarct complications, previous medication use, and receipt of thrombolytic therapy. RESULTS: Of the 1900 eligible patients, 1019 consumed no tea, 615 consumed less than 2 cups daily (moderate use), and 266 consumed 2 or more cups daily (heavy use). Heavy tea drinkers were older, less obese, and more likely to be female than non-drinkers, but their sociodemographic and clinical characteristics were otherwise similar. During a median followup of 3.8 years, a total of 313 patients (16%) died. Compared to non-drinkers, total mortality was lower among moderate tea drinkers (hazard ratio 0.78; 95% confidence interval (CI), 0.60 ± 1.01) and heavy tea drinkers (hazard ratio 0.65; 95% CI, 0.44 ± 0.97; p for trend = 0.01). When modeled continuously, the hazard ratio for each additional daily cup of tea was 0.89 (95% CI, 0.78 ± 1.01). The effect of tea was similar in men and women, in younger and older patients, and for total and cardiovascular mortality. Controlling for coffee intake did not change the results. CONCLUSION: Self-reported tea consumption during the year prior to acute myocardial infarction is associated with improved survival following infarction. PURPOSE: With recent changes in the financing of health care, home care patients may be discharged from home care prematurely, placing them at risk for adverse outcomes. Little is known about what happens to patients after home care discharge. We examined the frequency of adverse outcomes within 3 months of home care discharge, and identified factors associated with their occurrence. METHODS: Patients admitted to a single home care agency between April and September 2000 were invited to participate in the study. We collected data by chart review and telephone interview. We followed patients for 3 months after home care discharge and collected information on 5 adverse outcomes: death, readmission to home care, nursing home placement, emergency room use, and hospitalization. As potential correlates of adverse outcomes, we analyzed patient demographics, reason for home care, dependency in activities of daily living (ADLs) and instrumental activities of daily living (IADLs), cognitive function, depression, number of medications, caregiver support, and living alone. We developed a logistic regression model for adverse outcomes within 3 months of home care discharge. RESULTS: Of 217 eligible patients, 180 (83%) agreed to participate in interviews. To date, 155 have been discharged from home care (85% remained home, 13% were hospitalized, and 2% were transferred to hospice). We obtained 3-month outcomes on 108 (84%) of the 129 patients who remained at home after home care discharge. Of these patients, 60% were female, 78% were white, 74% were !65 years of age (mean age 74 years), and 72% had Medicare or Medicare managed care as their primary insurance. The median home care length of stay was 23 days. At the time of home care discharge, 57% had ! 1 ADL dependency; 95% had ! 1 IADL dependency; 37% lived alone; and 16% had no caregiver. The most common diagnoses were congestive heart failure (25%), chronic pulmonary disease (28%), diabetes (28%) and cancer (30%). Within 3 months of home care discharge, 34 patients (32%) had one or more adverse outcomes: 5% died, 4% were readmitted to home care, 26% had an emergency room visit, and 15% were hospitalized. Patients with ! 1 ADL dependency were more likely to have an adverse outcome compared to those with no ADL dependency (42% vs. 17%, p = 0.007). In a multivariable model including patient age and sex, factors associated with adverse outcomes within 3 months of home care discharge were ! 1 ADL dependency (Adjusted Odds Ratio 3.9 [95% CI: 1.3, 11.5]) and a history of cancer (4.7 [1.7, 12.9] ). CONCLUSION: Home care patients are at substantial risk for adverse outcomes within 3 months of home care discharge. Those with functional dependence or cancer are at highest risk. Further research is necessary to determine whether additional home care services would prevent adverse outcomes in these high risk patients. To determine if Enhanced QTc Dispersion occurs in ischemia during pharmacologic stress testing. This may serve as a non-invasive method to detect ischemia in patients suspected of having CAD. BACKGROUND: QTdispersion (Qtd), defined as the interlead variability in QT intervals on a 12 lead surface electrocardiogram (EKG) reflects regional inhomogeneity of ventricular repolarisation. Increased QTd has been reported in patients with ischemic heart disease (IHD) during exercise and after myocardial infarction (MI). Pharmacological coronary vasodilatation is known to alter regional myocardial blood flow. However, the effect of redistribution of coronary blood flow during pharmacological coronary vasodilatation on myocardial function is unclear. We hypothesized that increased disparity of myocardial blood flow can lead to nonhomogenous regional ventricular repolarisation. METHODS: We examined the effect of pharmacologic coronary vasodilatation on QTd in 81 subjects who received dypiridamole during myocardial stress imaging study (imaging). Patients with history of MI in the past 2 weeks, electrolyte imbalance, cardiac arrhythmia, long QT syndrome, those on pro-arrhythmia agents were excluded. Routine EKGs were performed at rest (0 Mts) and peak stress ( 7 ± 9 Mts after dipyridamole infusion). All EKGs were interfaced to a personal computer equipped with a digitizing tablet consisting of a magnifier used to enhance the accuracy of measurements. QT intervals were measured in all 12 leads and corrected for heart rate (QTc) using Bazett's formula. Intra & interpersonal variations were minimized by repeated blinded measurements. Patients were divided into three groups based on clinical probability and the results of the myocardial perfusion scan. 25 subjects with high pre-test probability for IHD and high risk reversible defects on imaging (reversible group) were compared with 33 other subjects with low clinical probability and normal imaging study (``normal'' group). 12 patients with low risk fixed defects on imaging and variable probability (``fixed'' group) were also studied. Mean Qtcd values at rest and during pharmacologic stress testing were compared in each group. Statistical analysis was done using t test. Results are expressed in mean+ SD.\nImaging results ( * n) * QTcd at rest Examination Survey, 1988 ± 1994 , a stratified, multistage sample of the US civilian non-institutionalized population. The study involved detailed home interviews, a careful physical exam, laboratory and other measurements. The study sample comprised 5,063 subjects with HTN. Ethnicity indicators were non-Hispanic Whites (NHW), non-Hispanic Blacks (NHB) and Mexican ± Americans (MA). Socioeconomic indicators were years of education and the poverty ± income ratio (PIR). The outcome measures were treatment (self-report), adherence (self-report) and control of HTN (SBP < 140 and DBP < 90). Treatment measures were medication prescriptions, counseling to restrict salt or sodium, Counseling to lose weight, or use of other methods like exercise, restrict alcohol intake, and to change diet. Adherence measures were self-report of taking medications, report of salt or sodium restriction, attempts to lose weight, and report of other approaches like exercise, alcohol restriction, and to change diet. BP control was measured by taking the mean of 6 BP measurements taken on 2 different days. Multivariate regression analyses, while accounting for the complex sampling design by using the appropriate sampling weights, adjusted for age, sex, access to health care, obesity, and urbanization. Significant results (p < .05) are reported as means or as odds ratios (OR) with the referent category being NHW, PIR less than or equal to 1 or < 12 years education. RESULTS: There were no racial or socioeconomic differences in likelihood of prescription of medicines or weight reduction counseling. NHB were more likely to be counseled for salt restriction (OR 1.4) . Subjects with > 12 years education were more likely (OR 1.5) to be counseled to exercise, restrict alcohol intake, and to change diet. In subjects who were counseled, NHB (OR 1.6) and subjects with > 12 years education (OR 1.6) were more likely to report salt restriction. NHB were also more likely to attempt to lose weight (OR American adults over age 25 who completed the household questionnaire and laboratory exam in NHANES III. All respondents were asked:``Have you ever had your cholesterol checked?'' If respondents answered yes, they were asked:``Have you ever been told by a doctor that your cholesterol level was high?'' and``Because of your high cholesterol, have you ever been told by a doctor to take prescribed medication?'' If a prescription medication was taken, the interviewer asked to see the medication container. Bivariate and multivariate analyses were used to examine the impact of race and ethnicity on cholesterol screening and on taking a cholesterol lowering medication. Unweighted counts and weighted percentages and odds ratios are presented. Odds ratios are adjusted to account for age, gender, income and, education, insurance status, comorbid illness and regular source of health care. RESULTS: African Americans and Mexican Americans were significantly less likely than whites to report ever having their blood cholesterol checked (Table 1) . Of those who were told to take medication, African Americans and Mexican Americans were significantly less likely to be taking a cholesterol-lowering agent ( Table 1 ). The mean total serum cholesterol (from the laboratory exam) of those who reported being told their cholesterol was high was 234 mg/dL as compared to a mean of 198 mg/dL of those who were not told their cholesterol was high (p < .001).\nCONCLUSION: African Americans and Mexican Americans are less likely to report serum cholesterol screening than whites. Even when identified as requiring medication, African Americans and Mexican Americans were less likely than whites to be on cholesterol lowering agents. These disparities in primary prevention are likely to lead to an increase in the risk of coronary artery disease in these populations. To describe the outcome of the Institutional Review Board (IRB) process from a multi-site QI evaluation and to examine participation rates as a function of recruitment and informed consent procedures. METHODS: We analyzed participation rates from a patient survey and medical record abstraction from 1504 individuals with either congestive heart failure or diabetes treated at 13 clinical sites in the United States that had implemented an evidence based QI intervention. We used bivariate analyses to assess the effect of recruitment procedures and informed consent requirements on participation rates. RESULTS: Recruitment procedures mandating patient permission to be contacted about the study varied by clinical site (Table 1 ). Among the sites that required advanced permission, only 58% (584/1012) of potentially eligible participants granted permission to be contacted about the study. Participation rates varied significantly by the type of recruitment and informed consent procedures required ( Table 2) . CONCLUSION: We found substantial variation in provisions for subject recruitment and informed consent. These differences significantly impacted participation rates, and possibly the generalizability of this multi-site QI evaluation study. To describe the demographic characteristics of participants who completed a mailed survey as compared to those initial non-responders who completed the same survey over the telephone. METHODS: To better understand barriers to care for abnormal cervical cytology, we performed a cohort study of all women with an abnormal pap smear who received care at Kaiser Permanente Los Angeles Medical Center between July 1998 and October 1999. We administered a mailed survey with telephone follow-up for non-responders between April and August 2000. Potential participants were mailed an introductory letter and survey in both English and Spanish with a self-addressed stamped return envelope. If no response was obtained within three weeks, a second letter and survey were mailed. If no response was received within three weeks, a trained bi-lingual interviewer called potential participants and offered to complete the survey over the telephone. The survey asked questions regarding patient satisfaction, health beliefs, cancer knowledge, and socio-demographics characteristics. We used bivariate and multivariate analyses to describe the population characteristics of respondents who replied by mail as compared to those who completed the survey by telephone. RESULTS: Of the 1049 potentially eligible participants, 733 women completed the survey for an overall response rate of 70%. Thirty one percent (n = 226) of the surveys were received after the first mailing, 25% (n = 185) were returned after the second mailing, and 44% (n = 322) were completed over the telephone. Sixty seven percent of those who completed the survey in Spanish were obtained by telephone, compared to only 37% of the surveys completed in English. (p < .001) In multivariate analysis, Latinas who completed the survey in Spanish and African American women were significantly more likely to complete the telephone survey than their white counterparts. Telephone respondents were younger, had lower household incomes, and were significantly less satisfied with their health care than those who responded to the mailed survey. Women who completed the telephone survey had more misconceptions about cancer and were more likely to report no knowledge of having an abnormal pap smear. Harvard University, Boston, MA PURPOSE: Pharyngitis is the second most common presenting complaint in the primary care office. Since rheumatic fever has become uncommon in the United States while rapid streptococcal antigen test technology has improved and concerns over drug side effects have grown, we examined cost-effective diagnosis and treatment of patients with suspected GABHS pharyngitis. METHODS: We constructed a decision analysis model from a societal perspective to examine the short-term cost-effectiveness of five strategies for the management of adult patients with pharyngitis: 1) Observation without testing or treatment, 2) Empiric treatment with penicillin, 3) Throat culture using a two-plate selective culture technique 4) Optical immunoassay (OIA) followed by culture to confirm negative OIA tests, or 5) OIA alone. We obtained data on test characteristics and event probabilities from published studies. We estimated utilities from a published patient survey which used a time-tradeoff technique to measure perceptions of outcomes (mild penicillin reaction, severe penicillin rxn, acute rheumatic fever) expressed in pharyngitis day equivalents. Based on published studies in which patients assigned a utility of 0.95 to other common symptoms such as diarrhea and dyspepsia, we estimated phayngitis to be associated with a utility of 0.95. We estimated costs from our hospital's financial systems, prior publications, and Medicare reimbursement rates. We converted all costs to year 2000 dollars. In sensitivity analyses, we varied values for probabilities and costs across ranges that included all published values or across ranges of 50 ± 200% of our baseline estimates. RESULTS: At a baseline prevalence of GABHS pharyngitis of 9.7%, a culture alone strategy was both most effective and least expensive, resulting in an average of .2618 quality-adjusted life days (QALDs) lost and an average cost of $6.19 per patient. For our base-case analysis, average costs in dollars, effectiveness in lost QALDs, and incremental cost-effectiveness were as below (see table) . Our results were sensitive to the prevalence of GABHS, so that OIA followed by culture was most effective at a prevalence greater than 17%. Observation was least expensive at a prevalence less than 3% and empiric treatment least expensive at a prevalence greater than 75%. As the probability of allergic reactions was varied throughout a possible range from 0 to 5%, OIA with culture, culture, and observation were all optimal strategies. Our results did not change with variations in costs of diagnosis or treatment. CONCLUSION: We found that observation, culture, and two rapid antigen test strategies for diagnostic testing and treatment of suspected GABHS pharyngitis in adults were all very similar in terms of both effectiveness and costs, although culture was least expensive and most effective at our institution's GABHS prevalence of 9.7%. We did not find empiric treatment to be most effective or least expensive at any prevalence of GABHS seen in adult populations. METHODS: A population-based telephone survey of 1205 women from a combination of random-digit-dial and targeted listed household samples from lower income census tracts of Washington, D.C. was conducted from January ± March, 2000. Valid and reliable measures of specific aspects of primary care and of the physician ± patient relationship were used. RESULTS: The survey response rate was 85%. Socioeconomic characteristics of the respondents reflected success in reaching the population sought. Four attributes of primary care (continuity, organizational accessibility, comprehensiveness of services, and coordination of specialty care) were important positive correlates of all aspects of the patient ± physician relationship (trust, compassion and comprehension), regardless of insurance, demographics, socioeconomic or health status. For example, women with the highest level of comprehensiveness of services at their usual source of care, were 11 times as likely to trust their physician (p < .01) and 6 times as likely to find their physicians compassionate and communicative (p < .01), compared to those with the lowest level of comprehensiveness. Higher organizational accessibility was strongly associated with greater trust, compassion and communication aspects of the physician ± patient relationship. (OR 3.2, OR 7.4 and OR 6.9 respectively, p < .01 for each). CONCLUSION: Women in ambulatory care systems that most strongly exhibit the features of primary care report stronger physician ± patient relationships than women whose usual sources of ambulatory care lack those primary care features. Ambulatory care systems which are organized to be more accessible to consumers, which permit patients to see the same clinician for their visits, which provide more comprehensive services, and which allow clinicians to coordinate specialty services are more likely to foster strong relationships between their patients and physicians, and to have patients who are more satisfied with those relationships. We excluded studies based on previously published decision analyses. The articles were reviewed by 2 investigators who were blinded to author and journal. We extracted information necessary to determine if the search strategies and databases searched were reported, if reference lists were reviewed for additional studies, if a systematic effort was made to find relevant unpublished studies, and if an a priori selection criteria or any validity grading system was used. RESULTS: We identified 257 articles through our Medline search and 36 met our eligibility criteria. The search strategy, including databases searched, was reported in 9 (25%) of the articles. Reviewing the reference list for additional studies was reported by 5 (14%) of the articles. Though no article reported a systematic effort to locate unpublished studies, 3 articles did report using data from unpublished sources. Two studies (6%) reported using a selection criteria and one study (3%) a grading system for the quality of the evidence. CONCLUSION: Our study shows that only 25% of CDAs published in major medical journals reported their process for selecting and evaluating the data they used. This is very concerning in view of the importance these items play when readers must appraise the validity of a CDA.\nAlthough it is possible that most studies were very rigorous in their selection of data, the lack of a clear report in the methods section makes it difficult for readers to identify stronger CDAs from ones that are less evidence-based. Our limitations include the fact that some of these data elements can only be obtained from databases like SEER, but all of these studies included probabilities that were obtained from the medical literature. We believe that the addition of evidence selection and appraisal in CDAs can strengthen these studies and give readers more confidence to evaluate their results. SD 3.8) ; the median number of months with the same counselor was 12. We found a high level of interest in quitting: 81% expressed the desire to quit, and 71% had plans to quit within the next six months. Sixty-nine percent agreed at least somewhat that counseling may aid people with nicotine addiction, and 73% that smoking cessation should be discussed in counseling sessions. However, 56% reported that their counselors had never asked about smoking. Over two-thirds reported that counselors never or rarely 1) discussed the adverse health effects of nicotine (68%), 2) advised them to quit (68%), nor 3) discussed smoking cessation options (72% We collected data on risk factors for coronary artery disease and type of surgery to be performed during the preoperative evaluation. All patients had CBC, routine chemistry, coagulation studies, electrocardiogram, and chest x-ray according to the protocol followed in the ambulatory surgery unit. A chart review conducted at least a month after the surgery was done to assess the incidence of postoperative cardiovascular complications. RESULTS: Average age of patients studied was 57, with a range of 28 ± 86 years. There were 108 men and 173 women. 30% had an abnormal electrocardiogram, 61% had hypertension, 30% had diabetes, 37% patients had at least one other risk factor for coronary disease, and 53% had two or more cardiac risk factors for surgery. The type of surgery included a variety of ambulatory procedure. The most common were eye 21%, breast 15%,cholecystectomies/ laparatomies 10%, hernias 7%, and ENT 5%. Overall outcome There were no patients in this cohort group who had postoperative cardiac complications. CONCLUSION: The incidence of cardiac complications after low-risk ambulatory procedures is low, even in the patients with risk factors. Additional testing is very unlikely to add to the ability to predict risk in this group of patients. À.13 ** À.21 ** À.08 * À.18 ** À.20 ** +.26 ** Fat Intake À.07 À.08 * À.06 À.08 * À.10 ** +.10 * Fr/Veg Intake À.14 * À.13 ** À.11 ** À.16 ** À.12 * +.18 ** Manage Stress À.33 ** À.31 ** À.15 ** À.32 ** À.25 ** +.38 ** Tob(Smokers only) À.23 À.11 À.12 À.03 À.03 +.12 Phys Activity À.19 * À.22 ** À.13 ** À.22 ** À.20 ** +.28 ** * P < .05; ** P < .001 PURPOSE: Diabetes mellitus is associated with abnormal autonomic function. Impaired glucose tolerance and diabetes mellitus are related to increased mortality. Abnormal heart rate recovery (aHRR), a measure of autonomic dysfunction, is also known to be associated with increased mortality. Whether impaired fasting glucose (IFG) is associated with aHRR has not been characterized. METHODS: 5190 healthy adults without medically treated diabetes (mean age 45, 39 % women) enrolled in the Lipid Research Clinics Prevalence study underwent exercise testing. Low physical fitness was defined according to the lowest quartile of peak METs. HRR was defined as the change from peak heart rate to that measured after 2 minutes of recovery, an abnormal value was < 42 BPM. RESULTS: 504 subjects (10%) had IFG and 131 (3%) were diabetics. An aHRR was found in 1699 (33%) adults; 1196 (23%) were unfit. Increasing levels of fasting plasma glucose (FPG) were strongly associated with aHRR (see figure) , even at glucose levels < 110 mg/dl. The association between increasing FPG and poor physical fitness was weaker. FPG remained an independent predictor of aHRR after adjustment for standard risk factors and resting heart rate (RHR) (adjusted OR for increase of glucose of 500/glucose by 1, 1.17, 95% CI 1.05 ± 1.30, chisquare = 9, P = 0.003). There was a strong interaction between FPG and RHR for prediction of aHRR. The association between FPG and aHRR was very strong with RHR 80 BPM (P for interaction = 0.0008); no association was present with RHR < 80 BPM. CONCLUSION: 1. FPG is strongly and independently associated with aHRR, even at nondiabetic levels. 2. The association between FPG and aHRR is substantially affected by resting heart rate. Internists. When practicing physicians are asked about the areas of medical practice where they feel the least prepared by their formal medical education and the most uncomfortable in clinical practice, they frequently cite scenerios around controlled drug prescribing. These include acute / chronic / and malignant pain management, management of anxiety vs. depression, management of insomnia, identification and management of addictive dissorders, and opioid and benzodiazepine pharmacology. Pilot data exist to suggest that the prescribing of controlled drugs in primary care clinic settings bear little if any resemblence to current practice recommendations. As part of a controlled clinical trial of alcohol screening and interventions in a primary care population, we performed a chart review in the Resident and Attending Primary Care Medical Clinic of a large urban teaching hospital. In an effort to assess controlled drug prescribing practices, part of the chart review included assessing whether there was addiction screening information in the patient record when controlled drugs were prescribed. METHODS: A total of eight hundred charts were randomly chosen from the three Internal Medicine Clinic resident firms as well as from the Attending clinic. Charts were categorized based upon 1) whether controlled drugs were prescribed at any time in the 12 months prior to the audit, 2) the type of controlled drug prescribied, 3) wheter there was any evidence of an alcohol or drug abuse histroy documented in the chart, and 4) the result of that history if documented. The charts were abstracted by trained research assistants using a data template and supervised by the Project Director and the Project Administrator. RESULTS: A total of 768 charts were completely reviewed, of which 135 indicated some prescribing of controlled drugs (17.6%). The percentage of patients receiving controlled drug prescriptions was not different based upon the gender of the patient (18%of men and 17%of women), and the type of controlled drugs showed no gender differences. 45%of women and 35%men had no evidence of a substance use history having been taken in their charts, and 3%of women and 13%of men had charts that documented hazardous or harmfull use of alcohol or drugs. Of the 70 male pateints prescribed controlled drugs, they represented 17.6%of those wit hno substance use assessment, 18.8%of those with a low risk substance use assesment, and 17%of those with active substance use problems documented in their charts. For the 65 women patients prescribed controlled drugs, they represented 18.5%of those with no substance use assessment, 14.7%of those assesed as low risk, and 33.3%of those wit hchart documentation of active addictive problems. There were no differences between the controlled drug prescribing patterns of attendings versus residents. CONCLUSION: The rate of prescribing controlled drugs and the appropriateness of prescribing decisions is becomming more and more closely monitored, bot hby regulatory agencies as well as advocay groups. Little is known about the controlled drug prescribing practices of practicing physicians, but they self-report it as an area of great un-ease. Although guidelines are not well established in this area of practice, there is general agreement that ambulatory controlled drug prescribing is relatively contraindicated in the face of active or past substance abuse on the part of the patient. This pilot data contains concerning information to suggest that a lack of any documentation of substance use histroy is common place when prescribing controlled drugs in primary care. Worse, the precentage of patients receiving controlled drug prescriptions who have a documented active substance abuse problem, is as high as those who receive a prescrition without any history taken, and atlease as high as those who both have an apropriate history AND whose hostory indicates that they are a low risk population.\nOminously, attending prescribing practice seems identical to house staff practice patterns. PURPOSE: Infliximab is an antibody to tumor necrosis factor-alpha. This drug has in several studies been shown to be effective in treating patients with inflammatory and fistulous Crohn's disease (CD) refractory to conventional therapy. However, treatment with infliximab is expensive and has associated toxicities. In addition, the mean duration of response to infliximab is approximately 12 weeks which necessitates re-dosing at regular intervals. Therefore identifying predictors of rate of response to infliximab could be of great benefit in selection of patients for this treatment. Furthermore, identifying factors that are associated with a prolonged duration of response could allow for modification of these factors to achieve a longer duration of response in treated patients. The purpose of this study was to identify factors predictive of rate and duration of response to infliximab in patients with CD. METHODS: 100 patients with refractory CD (59 with inflammatory CD and 41 with fistulous CD) and at least 3 month of follow-up following infliximab infusion were evaluated (mean follow up = 9 months; range = 3 ± 17 months). Clinical-response, duration of response, smoking history, gender, race, duration of disease, concurrent immunosuppressive or steroid use, age at diagnosis and age at infusion were analyzed. RESULTS: Rate of response: 67%of patients responded to infliximab. Rates of response were significantly higher in patients with fistulous disease (80%) compared to those with inflammatory disease (58%); p < 0.02. 77%of non-smokers responded compared to 49%of smokers (p < 0.004). The favorable response in non-smokers applied mostly to patients with inflammatory disease (p < 0.001). Concurrent use of immunosuppressive medication was also associated with higher rates of response in inflammatory disease(p < 0.007). Duration of response: There was a statistically significant longer duration of response among non-smokers compared to smokers both for inflammatory and fistulous disease. Among patients with inflammatory Crohn's disease, 87%on concurrent immunosuppressive medication had a duration of response longer than 2 months compared to only 45%not on any immunosuppressants (p < 0.04 The social norm for a larger body image may support the propensity for overweight and obesity, which has a prevalence of 66%, in black women. The goal of this study was to determine a psychometrically stable and socially acceptable Figure Rating Scale (FRS) for assessing body image in black women. METHODS: The study sample (n = 50)was selected sequentially from black women (mean age 52.3+/À 10 years) who were being screened for a larger randomized trial of nutrition and exercise education in urban black churches (Project Joy). Three standard published FRS were compared with a new culturally specific scale, the Reese FRS, developed from digitized photographs of black women and modified according to recommendations from focus groups. All four FRS consisted of nine ordered body images increasing in size from very thin to obese. Distributions of respondent' s selections on all scales were examined relative to anthropometric measures, including body mass index, BMI(weight in kg/m2) and categories of obesity and overweight (National Expert Panel on Overweight and Obesity Guidelines). Cultural identity, based on questions adopted from an African American Acculturation scale, and its relationship to FRS preference was also assessed. RESULTS: Body weight distribution for US black women over 40 years matched that found in NHANES III. All four FRS performed similarly and correlated significantly with BMI, r=À 0.70 to À 0.75, p < 0.0001. The percentage of obese women who identified with one of the three largest images in the FRS was 22%on three scales and 38%on one; this is markedly below 56%of women who were obese based on the national guidelines. Overall, 44%of the women preferred the new Reese FRS; among those women with cultural identity scores in the upper quintile, 72%preferred the new FRS. CONCLUSION: Overall, for all FRS, there is considerable overlap among images selected for all weight categories. There is a strong preference for the culturally specific Reese scale, especially among women with strong cultural identity scores. This new FRS appears to be more socially acceptable. The failure of a large percentage of obese black women to identify themselves as obese needs further investigation. PURPOSE: Evidence supports the effectiveness of collaborative care for depression in primary care, incorporating clinician education, proactive care management, and collaboration with mental health specialists. Little is known, however, about the influence of proactive care management on processes and outcomes of care for depression. The purpose of these analyses is to assess adherence to specific aspects of the nurse care management protocol, and evaluate the effects of adherence on quality of care and intermediate outcomes.\nMETHODS: We used a randomized encouragement design to study the effects of assisting 6 managed care organizations to implement collaborative care for depression in 30 experimental and 16 control practices. We identified a total of 920 depressed patients and referred them to their clinical practice site' s collaborative care program. Access to the program was through a nurse care manager. Care managers recorded all patient contacts on pre-structured forms. Patients completed self-administered surveys at baseline and every six months. Our evaluation uses bi-variable and multi-variable regression analyses. RESULTS: Overall, 73%of patients completed initial assessment visits. There was considerable variation among sites in adherence to the care management protocols, e.g., assessment of patient treatment preferences at the initial visit ranged from 18%to 100%at different sites (73%of patients assessed overall).\nIn bivariate regression analyses, greater performance of care management processes was strongly associated with higher quality of depression care, including the degree to which patients received and adhered to appropriate anti-depressant and psychotherapy regimens. Performance also related to improved levels of patient knowledge, probable depression, and active coping. When nursing assessment, proactive follow-up, and other self-management support processes were included together in regression analyses, the number of follow-up contacts had the greatest effect on patient outcomes (p < 0.001). CONCLUSION: Practices assisted in implementing collaborative care models for depression achieved varying levels of adherence to the care management design. Of all the processes studied, the amount of proactive follow-up had the strongest effect on care and outcomes.\nPRIMARY CARE PHYSICIAN DEPARTURE: EFFECTS ON HEALTH CARE QUALITY. A.G. Pereira 1 , S.D. Pearson 1 ; 1 Harvard Medical School/Harvard Pilgrim Health Care, Boston, MA PURPOSE: Discontinuity of care is an important health policy issue, but few data exist to assess its effects on health care quality or outcomes. This study evaluated measures of health care quality in patients whose primary care providers (PCPs) left their medical practice. METHODS: This study was performed in a large, multi-site multispecialty group practice associated with a single insurer. We used a controlled pre-post design to compare measures of health care quality received by patients whose PCPs left the practice (LEAVEmds) with the measures of health care quality in patients whose PCPs did not leave the practice (STAYmds). For all patients, we compared rates of preventive care, and in patients with hypertension, we compared blood pressure control. The practice had a standardized process for reassigning patients of departing PCPs. Patients were eligible for analysis if their insurance and care within the practice was uninterrupted over a four-year study period. LEAVEmds were matched to eligible STAYmds by age, sex and practice site. RESULTS: During the study period, nine PCPs, caring for 3,931 patients, voluntarily left the group practice. These LEAVEmds were matched to 16 STAYmds, caring for 8,009 patients. Mean age of LEAVEmd patients and STAYmd patients was the same: 46 (13); 76%of the LEAVEmd patients were women, as compared to 80%of the STAYmd patients (p < 0.01). During the two baseline years prior to LEAVEmd departures, there were no differences in quality of care measures between the two groups of patients. In the two years following PCP departure there was no difference in rates of Pap smears among women aged 25 ± 65 (88%vs. 88%, p = 0.85), or in mammography in women 50 ± 65 (90%vs. 93%, p = 0.09). Rates of fecal occult blood testing for colon cancer in all patients aged 50 ± 65 were lower among LEAVEmd patients (51%vs. 56%, p = 0.02). There was no difference in the proportion of patients with hypertension who experienced a rise of !10%in mean systolic and/or diastolic blood pressure (6.0%vs. 7.0%, p = 0.53). CONCLUSION: Overall, PCP departure from this group practice was not associated with substantial decreases in rates of several important preventive screening measures for their patients who continued to receive care within the practice. Among patients with hypertension, we found no difference in rates of blood pressure control. Further research is needed to address two questions: first, whether these findings are consistent with the experiences of patients in a variety of practice settings, and second, whether PCP discontinuity may adversely affect subgroups of patients with other chronic conditions. PURPOSE: Helical computed tomography (CT) is commonly used to diagnose pulmonary embolism (PE), although its operating characteristics have been insufficiently evaluated. Therefore, we aimed to assess the performance of helical CT. METHODS: Two-hundred and ninety-nine consecutive patients admitted to the emergency ward for clinically suspected PE, with a D-dimer level above 500 \"g/L (ELISA assay) were included. The diagnosis of PE was established by a validated algorithm including clinical assessment, lower limb compression ultrasonography, lung scan and pulmonary angiography. The CT scans were read by radiologists blind to all clinical data 3 months after image acquisition. RESULTS: Pulmonary embolism was present in 118 patients (39 %) in the study population. In 12 patients (4%), the CT scan was inconclusive, of whom two had a PE. Among patients with a conclusive CT, sensitivity of CT was 70%(95%CI 62 to 78), specificity was 91%(95%CI 86 to 95), positive predictive value was 84%(95%CI 76 to 91) and negative predictive value was 82%(95%CI 76 to 87). Sensitivity of a strategy adding lower limb ultrasound to helical CT increased to 79%(95%CI 71 to 86), and associating a lung scan in patients with normal ultrasound, helical CT being performed in patients with a nondiagnostic lung scan, would have a sensitivity of 95%(95%CI 89 to 98) and a specificity of 93%(95%CI 88 to 96). Interobserver agreement was high (kappa coefficients 0.82 to 0.90). CONCLUSION: Helical CT should not be used as a single test in suspected PE, but it could replace angiography in combined strategies including ultrasound and lung scan. PURPOSE: Helical computed tomography (CT) is increasingly used in the diagnosis of suspected pulmonary embolism, alone or in combination with other diagnostic tests, such as lung scan, lower limb ultrasound (US) or plasma D-dimer measurement (DD). Therefore, we assessed the cost-effectiveness of including helical CT in the diagnosis of suspected pulmonary embolism. METHODS: We performed a formal cost-effectiveness analysis by a decision model. Probabilities for test characteristics and clinical outcomes were obtained from the literature. Cost estimates were derived from our hospital's database. We compared 1) CT as a single test, and 2) inclusion of CT in a sequential strategy resting on clinical assessment, DD, US, lung scan and pulmonary angiography, either as a substitute for angiography or for lung scan. These strategies were compared with a reference strategy in which all patients with a nondiagnostic lung scan underwent an angiogram. Outcome measures were costs per patient, 3-month quality-adjusted survival, and incremental costs per quality-adjusted life years (QALYs) gained. RESULTS: Helical CT as a single test is associated with a 1%higher mortality than the reference strategy, and higher costs ($3,439 per QALY compared to $3,202 per QALY for the reference strategy). Replacing angiography by CT in the sequential strategy, was the most costeffective strategy ($2,447 per QALY gained). Replacing lung scan by CT was also cost-effective ($2,700 per QALY gained) provided an angiogram was performed in patients with a high clinical probability and a negative CT. Omitting DD measurement from the strategies increased costs but did not change effectiveness. The results were stable over the entire range of values tested in sensitivity analysis. CONCLUSION: Helical CT as a single test in suspected pulmonary embolism is not costeffective. However, CT may be cost-effective when included in combined diagnostic strategies.\nTRUST OF PHYSICIANS AND SATISFACTION AMONG MINORITY PATIENTS WITH ISCHEMIC HEART DISEASE. L.A. Petersen 1 , T.C. Collins 2 , N.R. Kressin 3 ; 1 VAMC, Houston, Texas; 2 Baylor College of Medicine; 3 Boston University, Bedford, Massachusetts PURPOSE: Studies show that African ± Americans (AAs) are less satisfied with their health care. Since AAs have historically experienced bias within the health care system, and recent work suggests bias on the part of physicians toward minority patients, the goal of this study was to assess racial differences in patients' trust and satisfaction with their physicians regarding decision-making for coronary disease treatment. METHODS: We prospectively enrolled 431 males with nuclear imaging studies graded as positive for cardiac ischemia (71.6%self-reported white, 28.4%self-reported AA) at 5 VA hospitals (Houston, Atlanta, St. Louis, Pittsburgh, Durham). We collected survey data using the published Trust in Physician scale, Seattle Angina Questionnaire, and new items developed from focus groups. RESULTS: There was no difference in mean age of the groups. AAs were less likely than whites to be``completely satisfied'' that everything possible is being done for their cardiac symptoms (44.4%vs 51.4%, respectively; P = 0.05). There was a trend toward more AAs reporting dissatisfaction with explanations provided by the physician (14.8%vs. 8.0%; P = 0.12). Though all patients had positive imaging studies, equal percentages (33.4%vs 33.8%; P = 0.98) thought they``did not have heart disease''. There were no racial differences in the percentage reporting that their heart condition was``not at all serious'' (3.2%vs 3.1%; P = 0.97). There were no racial differences in the percentage of patients who preferred to leave all treatment decisions to their doctors, who agreed with statements that doctors knew the patient' s medical and personal situations, or who reported that doctors were respectful or were concerned about them. Despite racial differences in overall satisfaction with medical care, there were no differences in the percentage who agreed that they``trust the doctor' s judgments about medical care'' (85.3%vs 88.7%; P = 0.17) or who stated that the doctor was``well qualified to manage medical problems'' (83.0%vs 87.5%; P = 0.79). CONCLUSION: Though we found racial differences in satisfaction with care and with physician ± patient communication, there were no racial differences in patients' trust of their physicians. Further work should assess whether there are racial differences in the construct of trust not captured with current measurement scales as well as develop strategies to improve physician ± patient communication and satisfaction with care.\nPURPOSE: H 376/95 is a novel, oral direct thrombin inhibitor that shows predictable pharmacokinetics and has not shown clinically significant food or drug interactions. Anticoagulants such as warfarin are used to lower the risk of stroke in patients with nonvalvular atrial fibrillation (NVAF). In the SPORTIF II study, the tolerability and safety of three doses of H 376/95 were compared with warfarin in NVAF patients. METHODS: This was a randomized, parallel-group study of NVAF patients who had at least one additional risk factor for stroke. The primary outcome was the number of adverse events, e.g., bleeding and thromboembolic events. The duration of treatment was 12 weeks, during which three groups received H 376/95 (n = 187) 20, 40 or 60 mg bid, given double blind. In a fourth group, warfarin (n = 67) was managed and monitored according to normal routines, aiming for an INR of 2.0 ± 3.0. RESULTS: A total of 257 patients were randomized to treatment and 254 patients received study drug. The median age was 70 years (range: 39 ± 95). In addition to NVAF, all patients had one additional risk factor for stroke, 75%had two additional risk factors, and 42%had three or more additional risk factors. The number of minor bleeds was comparable and low in all four groups. There was only one major bleed (vaginal) observed in a patient receiving warfarin and none in the H 376/95 groups. Out of 67 patients in the warfarin group, two patients had transient ischaemic attacks. Of the 187 patients in the H 376/95 groups, one patient had a transient ischaemic attack and one patient had an ischaemic stroke; both patients were in the H 376/95 60-mg bid group. CONCLUSION: Fixed doses of H 376/95 up to 60 mg bid were well tolerated, without the need for dosage adjustment or coagulation monitoring, during a 3-month treatment period in NVAF patients with medium-to-high risk for stroke and systemic embolism.\nin mortality is a discrepancy in the rate of follow-up of abnormal Pap smears among African ± American women. The objective of this study is to determine the demographic factors associated with a delay to follow-up of abnormal pap smears. METHODS: Eligible subjects were a sample of women aged 18 years and older with an abnormal cervical cytology report between February 1999 ± April 2000 screened at an academic medical center or one of three neighborhood health centers. We obtained information on subjects from a registration database and a pathology database. We excluded subjects who did not have a race category specified (17%). We defined adequate follow-up conservatively, as cytology or pathology within 4 months for dysplasia (low or high grade squamous intraepithelial lesion or carcinoma in-situ) and within 7 months for atypia (atypical squamous or glandular cells of unknown significance). We analyzed differences in follow-up rates by race, age, insurance status, source of care (academic medical center or neighborhood health center), and type of pap abnormality (dysplasia or atypia). RESULTS: Of the 345 subjects, 57%were African American, 16%were Caucasian, and 26%were from other racial/ethnic groups. 49%of the subjects were under the age of 30. 62%were screened at the medical center and 38%at the neighborhood health centers. Only 18%of the subjects had private insurance. Overall, only 53%of the subjects had follow-up for an abnormal pap smear either by additional cytology or pathology within 4 months for dysplasia (50%) or 7 months for atypia (56%). 55%of African ± American women, 55%of Caucasian women, and 48%of women from other racial/ethnic groups received follow-up (not significant). The follow-up rate of 45%for subjects less than 30 was significantly lower than that of subjects aged 30 or older (61%, p-value 0.003). No differences in follow-up rates were seen according to the subject's race, insurance status, source of care, or type of pap abnormality. CONCLUSION: In a low income, predominantly minority population, the rate of follow-up for abnormal pap smears was low, only 53%. This may explain the higher mortality rate seen in African ± American women. Younger women were less likely to receive adequate follow-up. Race, source of referral, insurance status, and type of pap abnormality were not associated with follow-up rates in this cohort. Patients were also asked to express their preferences regarding physician involvement in their religious/spiritual life. Descriptive statistics were tabulated. Associations between patient characterisitics and patients' preferences for specific physician spiritual behaviors were assessed using bivariate and multivariate techniques. RESULTS: Two hundred-ninety nine patients were surveyed. Patients' ages ranged from 19 to 86 years. Fifty six percent were male, 46%were African American, 51%white, and 63%belonged to Protestant denominations. Fifty three percent had annual incomes of $20,000 or less. Fifty nine percent of African American patients (AA) felt it was important that their physician have strong spiritual beliefs compared with 40%of whites (W)(p < .001); 45%of AA vs 32%of W would want a different physician if their doctor did not believe in God or a higher power (p < .001). Forty five percent of AA felt their health would improve if their physician prayed for them vs 32%of W (p = .03). Fifteen percent of AA were willing to give up time spent on medical problems to discuss spiritual issues with their physicians while only 4%of W agreed with this viewpoint (p = .001). In bivariate analysis, significant associations with patient desires for more physician religious involvement in medical encounters (p < .05) were AA race,older age, education less than college, and higher score on the SWB scale. In logistic regression, only AA race remained significantly associated with willingness to give up time spent on medical care in exchange for spiritual discussion (OR 3.8, 95%CI 1.3 ± 11) . AA race (OR 1.9, 95%CI 1.1 ± 3.4) and higher SWB score (OR 1.8, 95%CI 1.3 ± 2.4) were associated with patients' desire for physicians to have strong religious beliefs. Patients who would change physicians if theirs did not believe in God/higher power were more likely to be AA (OR 2.1, 95%CI 1.1 ± 3.9), have higher SWB score (OR 1.5, 95%CI 1.0 ± 2.1) and education less than college (OR 1.9, 95%CI 1.1 ± 3.4). AA race (OR 2.0, 95%CI 1.1 ± 3.5) and being married (OR 2.1, 95%CI 1.2 ± 3.7) were associated with the belief that physician prayer would improve patients' health. Age, sex and physical function had no significant associations. CONCLUSION: Physician religious beliefs and behaviors are more important to AA patients than to white patients. Physicians should be aware that religious discussions might be important for effective communication with AA patients in making medical decisions. PURPOSE: It has previously been reported that many patients would like their physicians to engage in prayer with them as part of their medical care. We studied the relationship of care settings to this preference and physicians' willingness to respond to patients' religious/spiritual needs.\nMETHODS: We surveyed patients and physicians at 7 medical centers in 4 states (NC,GA,FL,VT). For the patient questionnaire, a trained research assistant administered a 112-item survey that included questions pertaining to demographics, health status, functional status (SF-36), and spirituality assessment (SWB scale). Patients were also asked to express their preferences regarding physician involvement in their religious/spiritual life. The physician questionnaire was administered to residents and primary care practitioners through the mail. Descriptive statistics were tabulated. RESULTS: Two hundred ninety-nine patients and 444 physicians were surveyed. Patients' ages ranged from 19 to 86 years. Fifty six percent were male, 46%were African ± American, 51%white, and 63%belonged to Protestant denominations. Fifty three percent had annual incomes of $20,000 or less. Of the physicians, 45%were practicing physicians and 55%were residents/fellows. Seventy three percent were internists and 21%family practitioners. Forty five percent of physicians were of Protestant denominations. Twenty percent of patients wanted their their physicians to pray with them during a routine office visit, but only 5%of physicians wanted to pray with their patients under the same circumstances. However, 55%of physicians said they would pray with their patients during a routine office visit if the patients requested. If patients were hospitalized and near death, 52%wanted their physicians to pray with them compared to 26%of physicians who felt this appropriate. This number increases to 77%of physicians if a patient requests such prayer behavior. CONCLUSION: Patients' preferences for prayer in medical settings increases in settings associated with more severe illness. Physicians' willingness to engage in prayer with patients rises in a parallel manner but does not match the level of patients' desires unless prayer by a patient is requested. These data suggest that patients should communicate their feelings about prayer to their doctor if they truly desire their physician' s participation. PURPOSE: Alcohol is known to affect HIV risk behaviors and adherence to antiretroviral medications, but its prevalence among HIV-infected persons and effective screening approaches are less well described. Our objective was to determine the prevalence of a history of alcohol problems in patients entering primary care for HIV infection and to assess the positive predictive value of the CAGE questionnaire for alcohol abuse or dependence. METHODS: Between 7/97 and 10/00, HIV-infected patients presenting to a multidisciplinary clinic for evaluation and linkage to primary care were assessed for alcohol problems using the standard threshold of 2 or more positive responses to the CAGE questionnaire. The predictive value of the CAGE was evaluated by administering the CIDI-SAM, an interview for DSM-IV lifetime diagnoses of alcohol abuse and dependence, to a sample of those with a positive CAGE screening test. RESULTS: Among the 715 patients who spoke English or Spanish, mean age was 38.6 years; 70%were male; 50%were Black, 25%Latino, 23%White, 2%other. Primary HIV risk factors were injection drug use 47%, heterosexual 37%, and men who have sex with men 16%. Most (673/715, 94%) were evaluated for alcohol problems: 41%(279/673) had a positive CAGE screening test (2+); 7%(47/673) had 0 or 1+ CAGE scores, but were deemed to have had an alcohol problem by physician clinical judgment. Of the 326 patients with reports of alcohol problems, 116 underwent a diagnostic interview: 80%(93/116) met DSM-IV criteria for diagnosis of lifetime alcohol dependence, 14%(16/116) for lifetime alcohol abuse, and 6%(7/ 116) did not meet criteria for either diagnosis. In the group of patients with 2 or more positive responses to the CAGE questionnaire (n = 102), 95%(97/102) met DSM-IV criteria for alcohol dependence or abuse. In the group not identified by the CAGE but by clinical judgment (n=14), 86%(12/14) met the same criteria. CONCLUSION: Nearly half of patients presenting for HIV care in an urban clinic had a history of past or present alcohol problems, and the positive predictive value of the CAGE questionnaire was 95%for lifetime diagnoses of alcohol abuse or dependence. Given the prevalence of alcohol problems in this population, the potential impact of such problems on treatment, and the high predictive value of this simple screening tool, alcohol screening should be routinely implemented for all patients initiating HIV medical care. PURPOSE: Previous research suggests that diabetic patients with low literacy have lower disease-specific knowledge. We sought to examine the prevalence of low literacy among high-risk diabetic patients and determine if literacy level affects the change in knowledge or average hemoglobin AIC (HBAIC) after a comprehensive intervention to improve diabetes care. METHODS: Using a well-validated literacy instrument, the Rapid Estimate of Adult Literacy in Medicine (REALM), we screened 91 diabetic patients who were referred for enrollment in a comprehensive, pharmacist-based diabetes care program in a university internal medicine clinic. The intervention involved direct verbal teaching, simple diagrams, and phone-based follow-up to help patients better manage their blood sugar levels. We obtained diabetes knowledge scores and HBAIC values at entry and after 3 ± 4 month follow-up. We then analyzed the effect of literacy level on change in knowledge and HBAIC from baseline to follow-up using nonparametric Wilcoxon rank sum tests. RESULTS: Mean age of subjects was 58 (range 27 ± 87); 63%were female, 65%were African ± American, and 38%completed high school. Average duration of disease was 9years (range 0 ± 35). Low literacy, defined as a reading level below 9th grade (REALM < 61) was present in 78%of patients (32%grade 3 or below, 22% grade 4 ± 6, 24%grad 7 or 8). The effect of literacy on the response to the intervention is shown in the Table. CONCLUSION: Low literacy is extremely common among high-risk adult patients with diabetes. Our intervention improved knowledge and outcomes for patients with high and low literacy, but the effect was greater for patients with higher literacy. To prevent disparities in health outcomes, specific interventions to assist low literacy patients should be developed. We prospectively evaluated patients with any viral symptoms (temp > 1008F, headache, fatigue, sore throat, myalgias, night sweats, rash, oral ulcers, or diarrhea) and any risk for HIV infection (sex or drug use) in the prior two months. Primary infection was defined as ELISA negative and RNA positive; chronic infection was defined as ELISA/Western Blot positive and RNA positive. RESULTS: 1010 patients who presented to the UCC with viral symptoms were screened. Of 413 interested and eligible patients, 375 (91%) enrolled and had antibody and RNA testing. HIV infection was diagnosed in 9 (2.4%, 95%CI: 1.1%, 4.5%). Primary infection was diagnosed in 4 (1.1%, 95%CI: 0.3%, 2.7%) and chronic infection in 5 (1.3%, 95%CI: 0.4%, 3.0%). Fever was associated with a higher prevalence of primary HIV infection (7.0%, OR = 24.8, p < 0.0001). Symptoms associated with a higher prevalence of chronic HIV were oral ulcers (5.7%, OR = 6.8, p = 0.018) and diarrhea (4.2%, OR = 12.3, p = 0.0047). The four patients with chronic HIV and available CD4 counts all had very advanced disease (CD4 < 50/uL). CONCLUSION: Undiagnosed primary and advanced chronic HIV infection are prevalent in patients presenting with viral illness to an urban urgent care clinic. Testing for both should be recommended for these patients. PURPOSE: Coronary artery calcium (CAC), a marker of coronary artery disease (CAD), may be detected and quantified using electron beam computed tomography (EBCT). Proposals have been made to use the CAC score to guide cholesterol-lowering therapy, but the costs and benefits of this strategy have not been quantified. We performed a cost-effectiveness analysis to determine whether measurement of CAC with EBCT is a cost-effective method of targeting patients for cholesterol-lowering drug therapy. METHODS: A Markov model was constructed to estimate the incremental cost-effectiveness, over 10 years, of five different strategies:``Treat none, ''``Treat all, ''or test with EBCT and treat if the CAC score is greater than 0 (``Test and treat>0\"), 100 (``Test and treat>100\"), or 400 (``Test and treat>400\"). Overall rates of CAD events were based on Framingham risk equations. Distributions of CAC scores given risk factors were calculated from the literature. Summary relative risks for CAD events in each of four CAC score strata (0, 1 ± 100, 101 ± 400, > 400) were calculated with a random-effects model using data obtained from meta-analysis of articles identified through a systematic search of the literature. Therapy with HMG-CoA Reductase Inhibitors (STATINS) was assumed to reduce CAD event rate by 31%. Cost data were derived from literature review. Two base-case analyses were performed. Case 1 was a 50 year old man with low density lipoprotein cholesterol (LDL) of 175, high-density lipoprotein cholesterol level (HDL) of 45, and no other CAD risk factors. In Case 1, STATINS would not be recommended according to the National Cholesterol Education Program II (NCEP II) guidelines. Case 2 was the same 50 year old man with high cholesterol, but also Stage I hypertension, for whom STATINS would be recommended by NCEP II. RESULTS: Meta-analysis: Four studies met inclusion criteria. Relative risks (with 95%confidence intervals) for CAD events were 1.0 (reference), 3.9 (1.7 ± 8.8), 7.9 (2.5 ± 24.4), and 9.9 (2.8 ± 34.6), given CAC scores of 0, 1 ± 100, 101 ± 400, or > 400, respectively. Cost-effectiveness analysis: In each case, the``Test and treat ''strategies were more costeffective than the``Treat all ''strategy. This result held true while the cost of EBCT was less than $2400 (base case $400/scan), and the cost of STATINS was more than $150/year (base case $849.84/year). It was also insensitive to reasonable variation of other parameters, including the cost of CAD events, overall risk of events, relative risk given CAC score stratum, relative risk reduction with STATINS, and distribution of CAC scores. The``Test and treat > 400'' strategy failed by extended dominance in both cases. In Case 1, the incremental cost-effectiveness ratios (C/E' s) of all``Test and treat ''strategies, though lower than``Treat all,'' were greater than 300,000 $/life-year compared with``Treat none'' (the NCEP II recommendation). Given a reasonable C/E threshold of 50,000 $/life-year, this result was also insensitive to variation of parameters. In Case 2, the C/E of``Treat all'' (the NCEP II recommendation), in comparison to the``Test and treat > 0'' strategy, was 1,600,000 $/life-year. The per-year cost of STATINS would have to be less than $160/year, or the cost of EBCT greater than $2400, in order to make the NCEP II standard (``Treat all'') cost-effective or dominant in comparison to the optimal`T est and treat'' strategy. Note that the NCEP II standard (``Treat all'') in comparison with`T reat none'' also did not meet reasonable C/E standards, consistent with a recent costeffectiveness analysis of STATINS for primary prevention of CAD (Ann Intern Med 2000;132:769 ± 779). CONCLUSION: Screening of patients with EBCT who would not otherwise qualify for cholesterol-lowering therapy, based on NCEP II guidelines, does not meet reasonable costeffectiveness guidelines. On the other hand, using EBCT to identify patients at very low risk for CAD events among patients who would otherwise be treated with STATINS may be a costeffective strategy in selected patient populations. Research on the utilization of screening mammography for women has consistently shown that women are not taking advantage of recommended screening services. Most of the research on breast cancer screening has utilized retrospective crossectional survey designs, convenience samples and has not controlled for important confounding variables. These studies have limited themselves to describing characteristics of patients who do not get screened or listing patient-reported reasons for underutilization. Studies have implicated cost, insurance status and socioeconomic status as strong predictors of who will follow through with screening. METHODS: Randomized, unblinded, controlled interventional design. Consecutive clinic attendees who were > 40 years of age and eligible for a screening mammogram were enrolled in the study (N = 334). Patients were randomized within resident patient panels to be offered free screening mammograms or to be offered mammograms without mention of cost. Residents used a standardized script to offer all elegible patients a mammogram. Those who agreed were given appointments prior to leaving the clinic. The hypothesis was that those patients who were assigned to receive a free mammogram would be more likely to follow through with their mammogram appointment. RESULTS: Baseline characteristics between the control and intervention group were similar. Overall compliance rates were similar for both groups. Fifty four percent of the control group and 63%of the intervention group completed mammograms. This difference was not significant (chi square = 2.88; p = .237). Black women were 23% less likely than their non-black counterpoints to complete the mammogram (p = .069). There was no association between completion of mammogram and age, education, family history of breast cancer or type of insurance. CONCLUSION: Offering free screening mammograms and eliminating many of the known barriers to screening was not sufficient in this population to encourage compliance with screening mammography. PURPOSE: Low health literacy, a hidden obstacle to adequate health care, has significant impact on health services utilization. We set out to determine the association between health literacy of medical inpatients and subsequent emergency room visits, and hospital readmissions. METHODS: The study is a prospective cohort study with 3 months of follow-up, and took place in the medical service of an urban, teaching, hospital in Providence, Rhode Island. Patients admitted to the medical service at Rhode Island Hospital were eligible to participate in the study. A total of 293 hospitalized patients were approached over a seven week period for participation in the study, of which 161 (55%) completed the instrument. Patients who agreed to participate completed the short version of the Test of Functional Health Literacy in Adults (sTOFHLA), which was administered by a bilingual research assistant. RESULTS: Of the participants, 54%were female, and the mean age was 59 years old. 126 (78%) of the participants scored in the adequate health literacy range, which was defined as a score of 23 ± 36 (maximum score = 36). 35 (22%) patients scoring in the marginal health literacy range (17 ± 22), and those scoring in the inadequate range (0 ± 16) were combined into one group as low health literacy. Three months after discharge from the hospital, the hospital computer records were reviewed for the main outcomes-number of emergency room visits and hospital readmissions. There were 22 emergency room visits in the 126 patients with adequate literacy and 15 emergency room visits in the 35 patients with low health literacy (mean number of emergency room visits 0.17 (95%CI 0.082 ± 0.267) vs. 0.43 (95%CI 0.092 ± 0.765), p = 0.04). Patients with low health literacy had 2.5 times more visits to the emergency room than patients with adequate literacy. There were 52 hospital readmissions in the 126 patients with adequate literacy and 25 readmissions in the 35 patients with low health literacy (mean number of readmissions 0.413 (95%CI 0.237 ± 0.589) vs. 0.714 (95%CI 0.346 ± 1.083), p = 0.12). CONCLUSION: In our study of a medical inpatient population, patients with low health literacy had an increased number of emergency room visits within three months of discharge when compared to patients with adequate health literacy. Future studies should be focused on interventions aimed at patients with low health literacy to improve health services utilization. Higher * 10.5% 8.4% À2.1% (À3.5%, À0.7%) À1.1% (À2.5%, +0.4% Lower ** 9.6% 8.5% À1.1% (À1.6%, À0.5%) * Higher literacy is !9th level (n=19); ** Lower literacy is < 9th grade (n=72) ACCESS TO CARE ISSUES IN A PUBLIC HOSPITAL URGENT CARE CLINIC. S. Prock 1 , H. Batal 1 , S. Majeres 1 , L. Lasater 1 , R. Lundgren 2 , J. Adams 1 , P. Mehler 1 ; 1 Denver Health Medical Center, Denver, CO; 2 Colorado Prevention Center, Denver, CO PURPOSE: To describe access to care issues in a population of patients seeking care at a public hospital urgent care clinic. METHODS: 20%of patients presenting to the Denver Health Medical Center urgent care clinic (UCC) between June 15 and August 11, 2000 were randomly approached to participate in a pre-visit interview, offered in both English and Spanish. Patients were asked standardized questions regarding physical and mental health, their reasons for accessing care in the UCC, barriers to health care, history of prior Emergency Department (ED) and UCC usage, reasons for delaying health care, chronic medical conditions, and prior receipt of preventive health services. Study patients' charts and admission records were abstracted for the number of prior visits and documentation of receipt of preventive health services at our institution, ethnicity, age, gender, and insurance status. Data were analyzed using multiple logistic regression. RESULTS: There were 1006 patients surveyed with a refusal rate of 20.3%. The study patients did not differ from the total population who accessed care during this time period in regards to age, insurance status, ethnicity, or gender. A regular source of care, other than an ED or UCC, was identified by only 37.1%of patients. Those patients with insurance were 1.7 times more likely to note a regular source of health care (p = 0.0046) than those without insurance or those on the state indigent care discount program. Hispanic (p = 0.0075) and black (p = 0.04) patients were more likely than white patients to note a regular source of health care. Older patients (p < 0.0001)and female patients (p < 0.0001) were also more likely to report a regular source of health care. Patients accessing care in the UCC were likely to have delayed accessing care, 71%had been sick for more than two days, with 49%noting that their current medical problem had been present for a week or longer. Those patients without insurance or on the state indigent care program were more likely to report a delay in seeking care (p = 0.0279). Among those who delayed care for more than two days, 26.4%reported that a lack of insurance contributed to their delay. CONCLUSION: Patients presenting to a public hospital urgent care clinc often delay care and often do not have a regular source of health care. Having a regular source of care was associated with age, gender, ethnicity, and insurance status. Patients without insurance were more likely to report delaying health care.\nH. Quan 1 , J.E. Arboleda-florez 2 , G.H. Fick 1 , H.L. Stuart 2 , E.J. Love 1 ; 1 University of Calgary, Calgary, Alberta; 2 Queen's University, Kingston, Ontario PURPOSE: Only a few small studies have explored the association between various physical illnesses and suicide in the elderly and they have produced inconsistent results. Thus, we undertook this larger study to more definitively assess the association between elderly suicide and physical illness. METHODS: This case-control study included all suicides (920 cases) and motor vehicle accident deaths (1,050 controls) for 1984 ± 95 in the Province of Alberta, Canada among Alberta residents aged 55 years and older. We reviewed Medical Examiner records and extracted sociodemographic information. Then, deterministic linkage was used to link subjects to the Alberta provincial health care registry to determine personal identifiers. Those identified in the registry (1766, 90%) were linked with hospital discharge and physician claims data. We extracted coded diagnoses for each subject in the two years prior to the date of suicide or accident. The accuracy of the administrative data diagnoses was assessed through patient chart review in a sub-set of the study population, revealing kappa values ranging from 0.70 for prostatic disorder to 1.0 for cancer. RESULTS: Compared to the motor vehicle accident victims, the elderly who committed suicide were more likely to be men (78.6% vs. 62.2%), aged 55 ± 64 years old (50.6% vs. 37.5%), unmarried (46.4% vs. 41.0%), white (96.4% vs. 91.7%), residents of high median income areas (43.7% vs. 26.9%) or urban areas (58.4% vs. 40.5%), and to have a history of depression (37.0% vs. 6.2%) or other psychiatric illnesses (57.1% vs. 26.0%). The two groups were similar in the proportion of unemployed (80.9% vs. 82.8% currently). When controlling for sociodemographic characteristics and psychiatric disorders, the elderly who committed suicide were more likely to have had cancer (odds ratio [OR]: 1.73, 95% confidence interval [CI]: 1.16 ± 2.58) and prostatic disorders excluding prostate cancer (OR: 1.70, CI: 1.16 ± 2.49) than were motor vehicle accident victims. Among those who were married, chronic pulmonary disease was more frequent among the elderly who committed suicide than among motor vehicle accident victims (OR: 1.86, CI: 1.22 ± 2.83). Ischemic heart disease, cerebrovascular disease, peptic ulcer disease, and diabetes mellitus were not independently associated with elderly suicide. CONCLUSION: Cancer, chronic pulmonary disease, and prostatic disorders appear to be associated with suicide among the elderly. Physicians and other clinicians should consider assessment and monitoring of patients' suicidal tendencies when such conditions are present. PURPOSE: Hypothyroidism (HYPO) is associated with increased atherosclerosis in autopsy studies. Higher plasma homocysteine (Hcy) levels, even within the normal range, are an independent risk factor for cardiovascular disease. A few studies have shown that patients with HYPO have increased Hcy levels. Some have suggested that this may be due to the slightly increased creatinine (Cr) level in HYPO but other factors affecting Hcy status were not examined. Therefore, we assessed Hcy, vitamin and Cr levels together in patients with HYPO. Because little comparative information exists for patients with hyperthyroidism (HYPER) and none for patients with subclinical hypothyroidism (SC-HYPO), these conditions were also studied. METHODS: Hcy, thyroid stimulating hormone (TSH), free thyroxine (FT4), vitamin B12 (B12), folate and Cr levels were measured in 11 (2 men, 9 women), 10 (2 men, 8 women), and 19 (5 men, 14 women) patients with HYPO, SC-HYPO and HYPER respectively. RESULTS: Mean (SD) Hcy level was higher in HYPO than in HYPER (9.63.0 vs 8.42.7 \"mol/l) but not significantly so (p = .26) and only 1 patient had an abnormal Hcy (normal in women = 4.4 ± 12.1 \"mol/l). Hcy in SC-HYPO (10.32.2 \"mol/l) was also higher than in HYPER (p = .055). Plasma Hcy did not correlate with either TSH or FT4 levels. Although Cr levels were higher in HYPO than in the other groups (p = .0003), the Cr level did not correlate with Hcy. On the other hand, folate levels were significantly lower in HYPO than in HYPER (9.26.0 vs 17.611.2 \"g/l, p = .013); folate levels were also lower in SC-HYPO (8.9 3.2 \"g/l; p = .005). The folate levels correlated inversely with Hcy levels in the entire population (p = .001) and within the HYPO group (p = .06). B12 levels showed no significant patterns in thyroid disease. CONCLUSION: Our data show that Hcy levels are higher in HYPO than in HYPER but not significantly so. While Cr levels were significantly higher in HYPO than in HYPER, our data suggest that Cr levels are not the major determinant of Hcy changes in thyroid disease. Instead, folate status appears to play an important role in thyroid disease that was not appreciated because vitamin and renal status had not been examined together until now. Finally, our data raise the possibility that early changes in Hcy levels similar to those in HYPO may appear in SC-HYPO. If confirmed by our therapeutic trials, these data may provide an additional reason for intervention in SC-HYPO to mitigate atherosclerotic risk. PURPOSE: Female patients are less likely to undergo cardiac procedures after a myocardial infarction (MI) than male patients. We examined the use of cardiac catheterization and coronary revascularization post-MI to determine whether sex disparities in procedure use were associated with physician sex or were more pronounced when a patient and physician were of different sexes. METHODS: We evaluated data from the Cooperative Cardiovascular Project, a sample of Medicare beneficiaries hospitalized for MI in 1994 and 1995. Patients age 65 years and older with a confirmed MI who presented directly to the hospital with no prior history of revascularization (n = 111,319) were linked with physician data provided by the American Medical Association and evaluated for the use of cardiac catheterization (CATH) and coronary revascularization (REVASC, by PTCA or CABG) within 60 days of admission for MI. Separate multivariable logistic regression analyses were employed to ascertain the influence of patient sex and physician sex for CATH and REVASC use adjusting for patient sociodemographic characteristics, illness severity, physician factors (specialty, age, race, practice type), and hospital characteristics. A patient sex/physician sex interaction term was incorporated in multivariable analysis in order to test whether differences in procedure use were greater when a patient and physician were of different sexes. RESULTS: Female patients were less likely to undergo CATH (39.4% vs. 52.1%, p = 0.001) and REVASC (25.8% vs. 36.7%, p = 0.001) than male patients, while patients treated by male physicians were more likely to undergo CATH (45.9% vs. 39.5%, p = 0.001) and REVASC (31.4% vs. 25.8%, p=0.001) than patients treated by female physicians. Female patients remained less likely to undergo CATH (odds ratio [OR]: 0.82, 95% confidence interval [CI] 0.80,0.85) and REVASC (OR 0.80, 95% CI 0.77,0.82) in multivariable analysis, regardless of the treating physician' s sex. In contrast, patients treated by male physicians were more likely to undergo CATH (OR 1.15, 95% CI 1.07,1.23) and REVASC (OR 1.14, 95% CI 1.06,1.22) than those treated by female physicians, regardless of patient sex, in adjusted analysis. Differences in CATH (p = 0.66) and REVASC (p = 0.32) use were not greater when a patient and physician were of different sexes. CONCLUSION: Though male patients and patients treated by male physicians were more likely to undergo cardiac catheterization and coronary revascularization post-MI, differences in procedure use were not greater when a patient and physician were of different sexes, suggesting that factors other than sex bias account for sex differences in cardiac procedure use. PURPOSE: Patient satisfaction instruments are frequently used to measure the quality of care provided by physicians and practices. Because these instruments are used for diverse purposes, however, they often become long and difficult for patients to complete, especially those from vulnerable populations. We attempted to reduce the 63-item Veterans Administration (VA) Ambulatory Customer Care Satisfaction Survey into a concise instrument focused on patients' concerns. METHODS: Patients were recruited sequentially at two VA Medical Center clinics and two medical clinics at a nearby University. Patients were first asked an open-ended question,``What is important to you regarding your outpatient clinic visits?'' They were instructed to provide comments based only on their ambulatory care, not inpatient or specialty care. Comments with similar themes were grouped into categories. For the second part of the task, they were shown a list of 15 randomly chosen items from the VA instrument and asked to rate each on a 5-point scale from``not at all important'' to``extremely important.'' A range of content and complexity was represented within each set of 15 items. RESULTS: A total of 496 patients participated. Most were middle-aged (mean age = 50.5, sd = 17.8), male (67.1%), and African ± American (59.9%); 38.7%had a high school education or less. A total of 879 comments were provided about their outpatient clinic visits. The majority of patients gave at least one (80.6%) or two comments (53.8%). The five topics volunteered most frequently were the wait time for appointments (31%), doctor ± patient interactions (14%), getting good service and care (11%), overall speediness (11%), and friendliness/courtesy by all staff (10%). However, when patients rated a list of existing items, the average rating for every item was above 4.0 (i.e., between``very important'' and``extremely important'').\nCONCLUSION: Reducing the number of items in patient satisfaction questionnaires is complex. Patients are willing to specify what is important to them in their ambulatory care, however, most patients volunteer only one or two issues, and these issues vary across patients. When shown a partial list of items from an existing instrument, ceiling effects limit the patients' ability to reveal the relative value of potentially competing items. The disparity in results with the two methods suggests researchers need to be aware of how existing instruments were generated, think about the ability and willingness of patients to discriminate among multiple items with similar themes, and find ways to capture what is important to patients without overburdening them.\nCOST-EFFECTIVENESS OF CANCER SCREENING IN THE ELDERLY. J.S. Rich 1 , J.D. Birkmeyer 1 ; VA Medical Center, White River Junction, VT PURPOSE: Although the benefits of continuing to screen for cancer in the elderly are unknown, the elderly are screened. We developed a Markov model to estimate the costeffectiveness of continuing to screen for cancer beyond 70 years of age. METHODS: For each of three cancers (breast, cervical and colon), we estimated the marginal cost-effectiveness of continuing to screen average risk elderly men and women who had previously undergone regular screening. All-cause and cancer-specific mortality data were obtained from the National Center for Health Statistics and the Surveillance Epidemiology and End Results survey. Assuming that the benefits of screening seen in younger patients extend to the elderly, we used a reduction in cancer-specific mortality of 27% for biennial mammography and 33% for annual fecal occult blood testing (FOBT) to model screening benefits. We assumed a 70% reduction in cervical cancer mortality to model benefits with triennial screening Pap smears. Three costs were included: cost of the screening test, cost to evaluate an abnormal, and the cost of cancer care. We assumed that there were no harms with screening. RESULTS: The table below shows the incremental gains in life expectancy and costeffectiveness of continuing to screen the elderly for cancer. The values for the 70 ± 79 year old interval are compared to stopping screening at age 69; the values for the 80 years and over interval are compared to stopping screening at age 79.\nCONCLUSION: Under assumptions that are very favorable to screening, continuing to screen for cancer beyond 80 years of age does not appear to be cost-effective. Although screening for breast and colon cancer in 70 ± 79 year olds may be cost-effective, the gains in life expectancy are small and may be outweighed by potential harms.\nAT HOME WITH HEART FAILURE & TRADE: EFFECT OF STANDARDIZED TELEPHONIC CASE MANAGEMENT FOR HEART FAILURE. B. Riegel 1 , B. Carlson 1 , Z. Kopp 2 , B. Lepetri 2 , A. Unger 3 ; 1 Sharp HealthCare, San Diego, CA; 2 Pfizer, Inc., New York, NY; 3 Science Applications International Corporation, Reston, VA PURPOSE: Case management that promotes heart failure (HF) self-care is thought to decrease the need for hospitalization but few randomized, controlled clinical trials have tested the approach. Since much of the effectiveness of case management depends on the unique abilities of the provider, decision-support software from Pfizer, Inc.,``At Home with Heart Failure TM '', was used to standardize care. METHODS: A prospective randomized controlled clinical trial was conducted to evaluate the effectiveness of software-supported telephonic case management in decreasing acute care resource use. 281 physicians from two hospitals in So. California were matched on specialty and practice size and randomized to intervention or usual care. Patients who were cognitively intact and spoke English or Spanish were identified during a HF hospital admission; 358 patients of the randomized physicians were included. Mean age was 72 years, 51% female, 56% unmarried, 72% class III or IV. The intervention group (n = 130) was telephoned within 5 days after hospital discharge and thereafter at a frequency guided by the software based on patient symptoms, knowledge, and needs. On average, patients received 17 calls (median = 14) from the case manager over the 6-month intervention period. Printed educational materials were mailed. Physicians were notified of patient progress in writing and telephoned as needed. Care for patients in the usual care group (n = 228) was not standardized. T-tests were used to test the hypothesis of equal mean acute care resource use in the two groups. RESULTS: Resource use was consistently lower in the intervention group: 6-month all-cause hospitalization rates were 27% lower (.62 vs. .86, p = 0.03), HF hospitalization rates were 48% lower (.22 vs. .41, p = 0.005), HF hospital days were 46%lower (1.1 vs. 2.1, p = 0.04). Multiple readmissions were 43% lower (.13 vs .23, p = .025). CONCLUSION: Standardized case management using telephonic decision-support software provided in the early months after a HF admission can augment care and significantly reduce acute care resource use. PURPOSE: In 1999, the Institute of Medicine reported that medical errors kill between 44,000 and 98,000 patients each year. There is little information on physician and public attitudes regarding disclosure of medical errors. METHODS: One thousand Colorado physicians were randomly selected to receive a mail survey describing three medical error scenarios (one minor, one moderate and one serious). The same three scenarios were presented to five hundred Colorado residents via telephone survey. For each scenario, the physicians and the public were asked if the error should be disclosed to the patient. The moderate error scenario was slightly modified from one previously tested and reported by M. Hingorani et al (BMJ 1999; 318:640 ± 1) . RESULTS: Response rates were 57%for physicians and 82%for the public. The public was less likely to desire disclosure of all three errors compared to physicians (p < 0.01) especially for the moderate error (p < 0.001) and the serious error(p < 0.045). Retired physicians (vs. active physicians, p < 0.05) and the over 65 public (vs. public under 65, p < 0.004) would not disclose the error for at least one of the scenarios. Primary care physicians (Internal Medicine, Family Medicine, Pediatrics, and General Practice) were more likely to disclose all three errors (p < 0.04) than non-primary care physicians. Physician response was not influenced by gender, years in practice, practice location or previous malpractice litigation. Desire for disclosure of the moderate error was roughly comparable for Colorado residents (88%) and United Kingdom respondents (92%). Gender, race, education, and income did not influence public response. CONCLUSION: Overall, 87% of Colorado physicians would disclose all medical errors to patients. The Colorado public was less inclined to disclose errors than physicians. Retired physicians and senior citizens were less likely to disclose one or more errors. Primary care physicians relative to all other physicians were most likely to disclose all three errors.\nOBESITY AND ACTIVITY SELF-PERCEPTIONS AND BARRIERS AMONG OLDER, LOW-INCOME HIGH FUNCTIONING WOMEN. C.S. Ritchie 1 , B.A. Stetson 1 , K. Adams 1 , E. Rucker 1 ; 1 University of Louisville, Louisville, KY PURPOSE: Obesity is increasingly recognized as a highly prevalent condition among older women and is associated with multiple comorbidities. Physical activity is an important component of weight reduction and attempts to improve function. We sought to identify perceptions regarding physical activity among older, low-income high functioning women. METHODS: We performed prompted survey completion with a group of 38 older, low-income women who volunteered as senior companions and caretakers for homebound older adults. BMI classifications are described using WHO criteria. RESULTS: Mean age: 73.81 (range 63 ± 93). Seventy percent of subjects were African American; 30% Caucasian. Nearly 65 % were overweight or obese (2.9% underweight; 32.4% normal weight; 8.8% overweight; 41.2 %obese; 14.7% morbidly obese). Of the non-obese women, 85.7% were satisfied with their activity level; compared with only 26.3% of obese women (p < .01). Despite their mobility and involvement in volunteer activities, participants cited numerous barriers to health-promoting physical activity. In contrast to common assumptions regarding social and environmental obstacles to exercise, such as family caregiving responsibilities and weather; this sample indicated that their primary barriers were cost, experiencing pain, illness or injury, safety concerns and self-consciousness. Obese women were more likely to report barriers associated with illness and injury (p < .05) and self-consciousness (p < .01). Obese women also indicated a greater dislike of solitary,``lifestyle'' exercise (p < .05). Despite a high prevalence of obesity in this group, only half of the participants reported being encouraged by their physicians to engage in physical activity. CONCLUSION: Physical limitations and obesity are significant problems even among this relatively high functioning group of older low-income women. Perceived barriers including underlying illness and self-consciousness need to be addressed when considering interventions.\nA CONTROLLED TRIAL OF COLLABORATIVE MEDICATION EDUCATION AND PHARMACEUTICAL CARE. M.S. Roberts 1 , K. Cholka 1 , J. Chang 1 , C. Amy 1 , W.N. Kapoor 1 ; 1 University of Pittsburgh, Pittsburgh, PA PURPOSE: To assess whether a multidisciplinary intervention designed to improve patient medication education and evaluate specific medication regimens can improve patient knowledge concerning medication use, increase compliance with medication use post-discharge, and decrease medication errors. METHODS: A multidisciplinary intervention was designed that included 1) a folder with medication information sheets placed at the bedside, 2) nurse-directed teaching about each medication with each administration throughout the hospital stay, 3) automatically triggered specialized teaching plans for anticoagulation, diabetes, and inhaled mediations, 4) protocoltriggered review of medications by a pharmacist to decrease administration complexity and identify potential medication-related problems (interactions, dosing problems, ADRs). The intervention was studied in 4 nursing units in a tertiary teaching hospital utilizing a non- repeated pre ± post/intervention-control design. The intervention was applied to all patients admitted to the intervention unit, and was not instituted on the control unit. Random samples of patients from both the control and intervention units before and after the implementation of the intervention were selected for participation in the evaluation component of the project. Evaluation consisted of baseline data collection and a structured survey that occurred 14 days post-discharge to assess knowledge and compliance. Multivariate logistic regression was used to test for significance. RESULTS: 2397 patients were admitted during the study period (1264 on the control unit, 1133 on the intervention). 1246 randomly selected patients (602 control, 644 intervention) were screened for participation in the evaluation component of whom 611would be responsible for their own medications post-discharge and were eligible. 302 patients agreed to participate and 239 (155 control, 124 intervention) completed the 14-day follow-up. Improvement in postdischarge assessment of knowledge regarding specific medications was significantly higher in the intervention group (7.4% for control vs 28.0% intervention, p = 0.0129), as was patient satisfaction with medication education. Protocol-initiated pharmacy consults were completed on 43 patients, of which 18 resulted in improvements in therapy. CONCLUSION: This pilot trial demonstrates that a multi-disciplinary intervention composed of nurse-directed education given at the time of medication administration linked with protocol-based consults to pharmacists and other health care providers can significantly increase patient knowledge regarding medication and satisfaction with medication education. Larger sample sizes are required to assess impact on medication error reduction, readmission and health status. ., yet little information exists on physician or public opinion regarding conclusions from that report. METHODS: We randomly surveyed 1000 Colorado physicians using a mail questionnaire and asked them to rate their agreement with several statements from the TOM report. A telephone survey of 500 Colorado residents asked respondents to rate their agreement with the same statements. Chi-square tests were used to compare proportions of physicians and the public who agreed with the statements and to assess for demographic differences by response. RESULTS: Response rates were 57% for physicians and 82% for the public. The following table shows percentages of physicians and the public who agreed with each statement:\nPhysicians 65 and older were more likely to feel that quality of health care is asignificant problem (p < 0.01), but there were no differences in physician responsesby gender, specialty, practice type, or by whether they had ever had a malpracticesuit filed against them. Of the public respondents, females, those over 65, and thosewith lower incomes were more likely to believe quality of health care is a problem(p < 0.01). Public responses did not differ by race or level of education. CONCLUSION: There is a marked disparity in public vs. physicians' perceptions of the quality and safety of health care in the U.S., as well as in the need for a national agenda directed towards reporting and reduction of medical errors. Physicians should recognize that a majority of the public believes that the quality of health care is a significant problem and that mandatory reporting of medical errors is widely supported. PURPOSE: Studies have found that African ± Americans (AA) tend to receive poorer quality care and experience poorer clinical outcomes than Caucasians (C) across a variety of medical conditions. However, little is known about racial differences in care and recovery from major depression (MD) in primary care settings. METHODS: We examined data collected as part of a clinical trial testing the effectiveness of disseminating a depression treatment guideline via an electronic medical record system (EMR). Patients presenting for routine care by a board-certified primary care physician (PCP) at a university-based clinic were screened for MD using the PRIME-MD and their PCPs were subsequently informed of this finding. Study patients had a Hamilton Rating Scale for Depression (HRS-D) score !12 and were not in treatment with a mental health specialist (MHS). The HRS-D was repeated at 6 months to ascertain recovery (HRS-D 7) . Data about depression treatment such as number of visits and discussions of depression, counseling, pharmacotherapy, and referral to a MHS, was abstracted from the EMR. Non AA/C patients were excluded from our analyses (3%) given the focus of this report on comparing differences between AAs and Cs. RESULTS: Between 4/96 and 12/98, 204 depressed patients (25% AA) met all protocol eligibility criteria and completed a baseline interview. They were cared for by 15 PCPs (100% C; 53% male; median age 39 years). At 6 months, 187 (92%) had follow-up information available including 47 (24%) AAs and 140 (73%) Cs. AA and C patients were similar at baseline on level of depressive severity (mean: HRS-D AA = 21 vs. C = 20), age (mean: 44 vs. 43), gender (female: 64% vs. 73%), employment (fulltime: 45% vs. 58%), medical comorbidity (2+ conditions: 57% vs. 59%), and history of depression (43% vs. 49%). AAs had a mean of 3.1 contacts with their PCP including 1.4 contacts discussing depression while Cs had 2.9 and 1.3 contacts, respectively over a 6-month period following study entry. PCPs documented counseling their AA patients for MD for 15%, recommending pharmacotherapy for 57%, and recommending a MHS referral for 28%. These rates were comparable for C patients (15%, 54%, and 35%, respectively). Overall, a similar proportion of AA and C patients recovered from their MD episode at 6-months (21% vs. 24%). CONCLUSION: Although inadequate caregiving and poor treatment outcomes are commonly found among patients with MD who are cared for by PCPs, we were unable to identify any disparities in care or recovery rates for MD among the AA and C patients enrolled in our clinical trial. Future studies should confirm our results and examine methods for improving the delivery of guideline-based treatments for patients of all races experiencing MD. PURPOSE: Hepatitis C viral infection represents a substantial clinical and public health burden. In New York City (NYC) with its diverse ethnic population and large proportion of immigrants, characterizing the subgroup-specific burden of HCV is important to target HCV diagnosis, treatment and prevention efforts. Our objective was to describe the clinical and epidemiologic features of HCV infection in NYC, and to assess the sub-population specific burden of HCV mortality. METHODS: We used data collected by the New York City Department of Health (NYCDOH) Vital Statistics office to identify all persons who died with a diagnosis of HCV coded as a primary or contributing cause of death between 1992 and 1997. Demographic data collected by this office was analyzed. To obtain information regarding cause of death, clinical characteristics, and risk factors for HCV infection, we performed medical record reviews on the subset who died during 1996 ± 1997. RESULTS: Between1992 and 1997, 1002 persons met the case definition for HCVassociated death. Eight hundred and ninety were identified as NYC residents. Median age at death was 55 (3 ± 97); 56% were male; 39% white non-Hispanic, 32% Hispanic, 22% black non-Hispanic, 4% Asian-Pacific Islander, 3% other and unknown. Forty eight percent were foreign born. Three hundred and nine (81%) of the 1996 ± 1997 medical records requested were received. Of these, 26 could not be reviewed because of insufficient information. Of the 283 medical records reviewed 156 (55%) had a risk factor for HCV infection reported. Of these, injection drug use accounted for 73%and blood product transfusion accounted for 26%. We calculated a median years of potential life lost (YPLL) of 12 for the entire study population. During 1997 the proportion of deaths attributable to HCV in comparison to other causes of death was 0.9% among Hispanics, 0.5% among Asian among black and 0.2% among white. CONCLUSION: Although HCV causes death among all segments of the NYC population, it has disproportionately burdened Hispanics and young adults. While additional research on incidence, prevalence, and distribution of risk factors is necessary, these data suggest that primary and secondary prevention efforts against HCV infection be directed at the most vulnerable populations. Figure 1 shows proportions of physicians'``optimal choice'' according to different specialties. Family medicine favored flex-sig + FOBT strategy, gastroenterologists-colonoscopy and internists any of these two. Only about 13% of all physician favoured FOBT strategy. Most physicians indicated patient preference (78%), clinical status (48%) and cost to the patient (41%) as determinants of using strategy other than their preferred. Factors that would, in the opinion of physicians, keep patients from undergoing colonoscopy were: apprehension of pain (77%) and embarrassment (39%) as well as patient financial cost (55%). We analyzed data from a national random-digit-dial telephone survey conducted in 1994. We measured respondents' satisfaction with their regular physicians, health care services, health insurance, and life in general, using 4-point Likert scales (4 indicating highest rating). We hypothesized that if low patient satisfaction ratings among Asians were attributable to response tendencies, we should observe a similar pattern of responses among Asians across domains related and unrelated to health care. We also asked if respondents had ever changed physicians due to dissatisfaction. RESULTS: Respondents included 627 Asian, 1005 black, 1000 Hispanic, and 1114 white adults. Asian respondents were mainly Chinese (33%), Vietnamese (32%), and Korean (32%). Asians reported lower satisfaction with their physicians and health care services than other ethnic groups, even after adjusting for demographic and health-related variables. Asians also reported lower satisfaction with their health insurance and with life in general (Table; p < .001 for all comparisons of Asians vs. non-Asians). Asians' responses to all satisfaction measures were similar and were more normally distributed than non-Asians' responses, which were more skewed toward the highest ratings. Asians were less likely than non-Asians to report having changed physicians due to dissatisfaction (24% vs. 31%, p = .001). CONCLUSION: Asians recorded lower satisfaction ratings not only for physicians and health care services but also for life in general and, despite lower reported satisfaction, were less likely than non-Asians to have changed physicians due to dissatisfaction. These findings suggest that lower satisfaction ratings among Asians are due to different response tendencies rather than worse experiences or higher expectations of health care. Further research is needed to explain this phenomenon and assess its potential impact on performance measurement for physicians and health care organizations providing care for large numbers of Asian Americans. PURPOSE: Vascular catheter-related blood stream infections are costly and associated with substantial morbidity and mortality. Central venous catheters coated with antibacterial agents have been shown to be superior to non-coated catheters. Trial results suggest, however, that central venous catheters impregnated with minocycline/rifampin, although more expensive, are clinically superior to catheters impregnated with chlorhexidine/silver sulfadiazine. It remains unclear if minocycline/rifampin catheters are cost-effective for all high-risk patients or only those requiring longer-term catheterization. METHODS: We developed a series of decision analytic models using patient-level clinical trial data to determine if minocycline/rifampin catheters are cost-effective for patients requiring various durations of catheterization. We calculated incremental cost-effectiveness ratios for patients catheterized for durations ranging from 1 to 25 days. We simulated the use of 10,000 catheters and calculated the proportion of times that the catheters were cost-effective or costsaving at each duration of catheterization. The perspective was that of the healthcare payer; the time horizon was the period of hospitalization. RESULTS: The data were too sparse to estimate cost-effectiveness for patients catheterized less than 8 days. The probability that minocycline/rifampin catheters were cost-effective or costsaving compared to chlorhexidine/silver sulfadiazine catheters in patients expected to be catheterized at least 8 days was 91%. The probability minocycline/rifampin catheters were costeffective or cost saving in patients expected to be catheterized 13 days or longer was over 95%. These findings remained relatively consistent during one-way and multi-way sensitivity analyses. CONCLUSION: The probability that minocycline/rifampin catheters are cost-effective or cost-saving increases relative to the expected duration of use. There is insufficient evidence to suggest that minocycline/rifampin catheters are clinically or economically preferable to chlorhexidine/silver sulfadiazine catheters in patients catheterized less than 8 days. However, minocycline/rifampin catheters are likely to be cost-effective for patients expected to be catheterized for periods longer than 1 week, and are clinically and economically preferable for patients expected to be catheterized for 2 or more weeks. year, with an associated mortality of 10 to 20%per episode. Though many recent studies of nosocomial bacteremia have been reported, there are limited data focusing on communityacquired bacteremia. Given the morbidity, mortality, and economic consequences of community-acquired bacteremia, we decided to: (1) describe the epidemiology and microbiology of community-acquired bacteremia; (2) determine the crude mortality associated with such infections; and (3) identify independent predictors of mortality. METHODS: This prospective study was conducted at the Seattle Division of the Veterans Affairs Puget Sound Healthcare System from January 1, 1994 to December 31, 1997. All patients with clinically significant community-acquired bacteremia or fungemia were evaluated. Data were collected on demographics, co-morbid conditions, clinical parameters, microorganisms, source of infection, and patient outcome. RESULTS: During the study period, 387 bacteremic episodes occurred in 334 patients. Staphylococcus aureus (18%), Escherichia coli (15%), and coagulase-negative staphylococci (12%) were the most commonly isolated organisms. The most frequent sources were the urinary tract, intravascular catheters, and pneumonia. Overall, almost one-third of bacteremia cases were directly related to indwelling catheters: either intravascular (20%) or urinary (10%). Approximately 14% of patients died. Patient characteristics independently associated with increased mortality included shock (OR 3.7, p = 0.02), renal failure (OR 4.0, p = 0.003), and a`D o Not Attempt Resuscitation ''order (OR 21.7, p < 0.001). The risk of death was also higher in those whose source was pneumonia (OR 6.3, p = 0.03) or an intra-abdominal site (OR 10.7, p = 0.02), or if multiple sources were identified (OR 13.4, p = 0.003). The presence of fever (temperature > 38.08C) was associated with a decreased risk of death (OR 0.4; p = 0.005). CONCLUSION: Vascular and urinary catheters were implicated in a substantial proportion of infections, emphasizing the need for appropriate and judicious use of such devices. We will likely observe an increase in the incidence of such device-related infections as healthcare expands into the outpatient setting. Fortunately, many of these infections are potentially avoidable via established infection control practices used to prevent hospital-acquired infections. Thus, strategies that have been useful in preventing nosocomial device-related bacteremia could prove successful if adapted into the outpatient setting. If this is borne out, community-acquired bacteremia may increasingly become viewed as a preventable disease. PURPOSE: Despite known interactions, the occurrence of inappropriate medication and alcohol use has not been well described. We sought to assess 1) the prevalence of potential alcohol and medication interactions in hazardous and harmful drinkers in a primary care setting, 2) the association between this medication use and alcohol consumption, and 3) whether users of medications known to interact with alcohol are counseled by physicians regarding drinking. METHODS: We interviewed current hazardous drinkers (at least one drink in the past month, and at least one positive response to the CAGE alcoholism screening questionnaire or > 14 drinks per week/ > 4 drinks per occasion for men, 7 and 3 respectively for women) just prior to a visit with a primary care physician to determine the self-reported current (30 day) prevalence of use of medications that can interact with alcohol. Alcohol consumption was assessed by trained interviewers using a validated calendar method. Immediately after the physician visit, patients reported whether there had been a discussion about alcohol with the physician during the visit that day. RESULTS: The 312 subjects were 63% male, mean age was 44, and 63%had a high school education. Subjects saw one of 41 physicians. Most (78%) reported current use of medication that can interact with alcohol: 59% used non-steroidal anti-inflammatory drugs, 44%acetaminophen, 16%antihistamines, 10% each narcotics, antidepressants, and medication for sleep, 6% anxiolytics, and 3% blood thinners (i.e. warfarin). Users of these medications drank more drinks per drinking day (median 4.0 vs. 3.5, mean 5.8 vs 4.6, p = 0.04) and binge drank more often (median 2 vs. 1 binges in the past month, mean 6 vs. 5, p = 0.03) than nonusers. There was no discussion about alcohol use with the physician for 37%of users and 40% of non-users of medications that can interact with alcohol (p = 0.68). CONCLUSION: Most hazardous and harmful drinkers in a primary care setting were using prescription and nonprescription medications that can interact with alcohol. Users of these medications drank more than non-users. Many had no discussion about alcohol with their 3.20 +/À .68 3.20 +/À .66 3.11 +/À .77 3.27 +/À .69\nNon-Asians (mean +/À SD)\n3.47 +/À .67 3.37 +/À .79 3.29 +/À .82 3.40 +/À .74 physicians. While the extent and impact of alcohol-medication interactions require further study, these results suggest that potentially dangerous alcohol and medication interactions are common and unrecognized by primary care physicians. (1966 ± 2000) , PsycLit, Cinhal, Embase, Aidsline, Healthstar, Cancerlit, the Cochrane library (clinical trials registry and the Database of Systematic Reviews), Micromedex and FEDRIP as well as references of reviewed articles. Inclusion criteria included English-language, randomized, placebo-controlled trials of antidepressant medication among adults with back pain. Data were abstracted independently by two reviewers. Two continuous outcomes, back pain severity and ability to perform activities of daily living, were measured. Study quality was assessed using the methods of Jadad and data were synthesized using a random effects model. RESULTS: Ten trials were included, the majority of which studied tricyclic antidepressants (8/ 10 studies) in the setting of chronic back pain (7/10 studies). Patients treated with antidepressants were not more likely to improve than those treated with placebo either in pain severity (Standardized Mean Difference: (SMD) 0.14, 95%CI: À 0.27,0.55) or in activities of daily living (SMD: 0.77, 95% CI: À 0.07, 1.61). Patients receiving antidepressants experienced greater side effects (24% vs. 16%, p = 0.03) than those receiving placebo.\nCONCLUSION: Antidepressants are no more effective than placebo in the management of back pain. Further randomized controlled trials of antidepressants are needed before they can be routinely prescribed for treatment of back pain. PURPOSE: Older adults vary greatly in their expectations regarding aging, with some expecting to maintain high function, and others expecting to experience functional decline. Whether having low expectations regarding aging causes older adults to seek less health care for modifiable age-associated conditions is unknown. We set out to determine whether older adults with low expectations regarding aging are less likely to think that they should seek health care for age-associated conditions associated with functional decline. METHODS: We surveyed by mail a random sample of 588 English-speaking patients aged 65 and older cared for by 20 UCLA network physicians. We measured expectations regarding aging using a recently developed and validated 38-item Expectations Regarding Aging (ERA) Survey, which includes 10 domains of expectations such as physical health, cognitive function, and mental health. Additionally, to identify beliefs regarding care-seeking, we asked participants if they felt it was``very\",``somewhat\", or``not at all ''important to seek health care for 13 ageassociated conditions such as falling, urinary incontinence and pain. Participants also reported on their chronic medical conditions, ability to perform activities of daily living, level of depressive symptoms, religiosity, and generic physical and mental health status. We then constructed a multiple linear regression model to assess the independent relationship between ERA Survey scores and beliefs regarding care-seeking, adjusting for potential confounders. RESULTS: Surveys were returned by 72% (n = 429) of those surveyed; 54% were women; the mean age was 76 years; 80% were Caucasian, 9%Latino, 6%African American; 59%reported > 2 medical conditions, and 21%reported disability. After controlling for all covariates including age, comorbidity, activities of daily living, religiosity and generic physical and mental health status, having lower expectations regarding aging was associated significantly with lower beliefs regarding care-seeking for age-associated conditions (p = .01). CONCLUSION: In this community-based sample of older adults, having lower expectations regarding aging was independently associated with placing less importance on seeking health care for age-associated, potentially modifiable conditions. These results suggest that older adults with low expectations regarding aging may not receive health care for modifiable conditions, and thus may experience preventable functional decline. METHODS: For 1984 indigent patients, adherence was modeled using multivariable linear regression with an alpha of 0.01 for statistical significance. DA was defined as either mean DA for all drugs taken by each patient or minimum DA based on the lowest adherence drug using prescription refill data from a closed pharmacy system. Additional data were drawn from a clinical data repository. RESULTS: Based on mean DA, 31%of patients took < 80% of prescribed doses. Based on minimum DA, 52% of patients took < 80% of prescribed doses for at least one drug. Increasing age, ethnicity (white), and greater medication supply per prescription were independently associated with higher mean and minimum DA. Number of drugs taken had a positive mean but negative minimum DA association. Gender, number of primary care visits, dosage schedule, and copayment were not independently associated with DA. The model explained only 6% of the variance in mean DA. CONCLUSION: In an indigent population with HBP, DM or HC, DA was associated with ethnicity, age, and quantity of drug supplied. However these factors explained only a small amount of the substantial DA variability. To target DA efforts, better predictors must be developed to identify patients most in need of adherence intervention. Prescription refill claims data could serve this purpose. Cook County Hospital, Chicago, IL PURPOSE: Because of its high prevalence and cost, preventing emergency departments (ED) visits and hospitalizations for congestive heart failure (CHF) has become the focus of extensive`d isease management ''activities. As part of a national chronic disease improvement collaborative, we sought to better understand and improve the care for patients with CHF. Because virtually all admissions in our public hospital system are the result of an emergency department visit, and our finding that approximately one in three ED visits of patients in our CHF cohort resulted in a hospital admission (unpublished data), we sought to better characterize the frequency and patterns of ED visits in this patient population. METHODS: We assembled two cohorts of patients with the diagnosis of congestive heart failure ± -1) an outpatient cohort attending one of four outpatient sessions at an public hospital general medical clinic over a three month period (7/99 ± 10/99) who had CHF listed on their PURPOSE: Low levels of functional health literacy (FHL) are common among public hospital patients with chronic conditions. Identifying patients with low FHL may improve care at the risk of disclosing a stigmatizing issue. We evaluated the acceptability and utility of FHL screening among type 2 diabetes patients (DM2). METHODS: This study was a randomized controlled trial of FHL screening. We measured FHL for all patients with the short Test of Functional Health Literacy in Adults. We enrolled 155 patients who had low FHL (scores < 23), DM2, spoke English or Spanish, and were cared for by one of 61 primary care physicians (PCPs) at a public hospital. PCPs in the intervention group were alerted prior to the visit when the patient had low FHL ; control PCPs were not. We surveyed patients regarding the acceptability of FHL screening. We surveyed PCPs regarding their satisfaction with the visit, communication techniques, and self-rated effectiveness. We also asked intervention PCPs to report their prior estimates of the patient' s FHL and the extent to which knowledge of the patient' s FHL would impact future diabetes care. Post-visit survey data were available for both patients (141) and physicians (152). Post-visit differences between intervention and control patients and physicians were examined, controlling for the clustering of patients by physicians. RESULTS: Nearly all patients (95%) felt that FHL screening was useful; of these, 99%felt it was important to share this information with their PCP. After adjusting for clustering, PCPs who received FHL feedback felt less trained and confident in caring for their patient than control physicians(p=.02), were more likely to involve patient' s family or friend in decisionmaking (p=.03), or use pictures and diagrams to promote understanding (p=.08). However, intervention and control PCPs had similar self-rated effectiveness (p = 0.66). Intervention PCPs accurately estimated the FHL of patients with inadequate FHL only 30%of the time. Intervention PCPs believed that FHL screening was clinically useful (63%of visits), would result in improved medication adherence (63%), and would improve future DM2 care (58%). CONCLUSION: Patients with DM2 and low FHL found FHL screening acceptable. PCPs, despite working in a setting with high prevalence of FHL problems, frequently overestimated patients' FHL. FHL feedback was associated with greater feelings of PCP inadequacy and use of recommended communication strategies, but no differences in physician self-rated effectiveness. Most PCPs believed FHL screening was clinically useful. Further research should explore the impact of FHL screening combined with provider communication training on patient-centered measures and clinical outcomes. RESULTS: Only 2.2%of the sample reported eating a vegetarian diet was 43%(116) of selfreported vegetarians reported eating some meat (beef, pork, poultry or fish) during the 24-hour food recall surveys. Overall, reports of vegetarian diet were more likely in women (67.8%of vegetarians v. 49.0%of controls; p < .001) and``other ''races (12.6%of vegetarians vs. 7.7%of controls; p < .05). Medical history was strongly associated with reporting a vegetarian diet. Among those who were vegetarian, 22.3%, 16.3%and 6.7%reported a history of high cholesterol, heart disease and stroke compared to 16.6%, 9.2%and 1.7%of controls, respectively (all comparisons significant at p < .01). Self-report of other risk behaviors was also significantly associated with reporting a vegetarian diet; 16.7%and 59.5%of vegetarians reported current smoking and current vigorous exercise at least two times per week compared to 23.2%and 50.9%of controls, respectively. No differences were seen between vegetarian and non-vegetarian subjects with respect to age, body mass index, household income, education level, region of country and self-reported health status. CONCLUSION: In conclusion, self-report of a vegetarian diet is uncommon among U.S. adults and is strongly associated with gender, chronic medical conditions, and the self-report of other health-promoting behaviors.\nHospital and Brown University, Providence, RI PURPOSE: We analyzed a representative sample of 19,058 visits from the 1998 National Ambulatory Medical Care Survey (NAMCS) to examine the frequency that physicians reported counseling patients about their diet. METHODS: Patients with diabetes, coronary heart disease (CHD), hyperlipidemia, obesity, and hypertension were identified using a combination of visit codes, diagnoses and current medications from forms completed by physicians after patient encounters. Independent determinants of diet counseling were evaluated using multiple logistic regression. RESULTS: Overall, diet counseling was performed during 12.3% of all visits and was more common among patients who were middle-aged (45 ± 64 years) (13.4% vs. 11.8% for other age groups; p < .05) nonwhite (14.2% vs. 12.3%; p < .01), presenting for routine chronic care or a routine physical vs. acute care (14.7% and 18.5%, respectively: vs. 9.7%; p < .01), for patients with diabetes (33.8%), obesity (55.8%), hyperlipidemia (36.2%), and hypertension (36.%) (p < .01 for all comparisons), if the physician was a primary care provider (22.0%), cardiologist (27.0%) or ostetrician/gynecologist (16.0%) vs. other providers (5.8%, p < .001), and if the patient was also counseled about physical activity (67.5% vs. 11.5%; p < .001). These effects persisted after multiple logistic regression analyses controlling for potential confounders. Gender, coronary heart disease history, region of the country and insurance type were not associated with diet counseling. CONCLUSION: In conclusion, health care providers primarily use diet counseling as a treatment for diet-related illnesses, rather than as primary prevention, where it was noted that fewer than 1 in 5 patients were counseled about their diet during routine physicals. . This testing is typically with a sensitive activated partial thromboplastin time (aPTT) and dilute Russel viper venom time (dRVVT), followed by mixing studies and confirmatory tests. With decision analysis, we investigated the optimal testing strategy for detecting LA in three clinical settings. METHODS: A decision-tree was constructed with 12 strategies, using a combination of aPTT and dRVVT with confirmatory tests, tissue thromboplastin time (TTI), platelet neutralizing procedures, and mixing studies. Probabilities of adverse events, utilities of these events, and costs were obtained from a literature review. Sensitivity and specificity of each strategy was calculated by testing 90 healthy people and 77 patients, with true positives defined as per the algorithm recommended by the International Society on Thrombosis and Haemostasis. RESULTS: For healthy people with a prolonged aPTT, the optimal strategy is not to test for LA and assume that no patient has LA. For patients with systemic lupus erythematosus (SLE), it is cost saving to test with TTI alone if the cost of the test is less than $13 (base case -$9), or if the prevalence of LA in the population is > 33% (base case-35%). For patients with past thrombosis or fetal loss, it is cost saving to avoid testing and assume no patient has LA, or to use TTI alone if the expected prevalence of LA is > 33% (base case-9%). CONCLUSION: It is optimal in healthy people and in those with a low likelihood of LA to avoid testing and to assume that these patients do not have LA. However, for patients with SLE, or for patients with past thromboses and a high likelihood of LA, TTI is a cost saving test. These results suggest that the current strategy for detecting LA needs to be modified.\nPURPOSE: symptoms of hyperthyroidism and hypothyroidism include weight changes, fatigue which can be confused with symptoms of cancer.very few data exit about the prevalence of thyroid disease in cancer patients. This was a prospective study testing thyroid functions in patients with various diagnosis of cancer in order to assess the prevalence of thyroid disease in those patients. METHODS: cross sectional study was conducted at the Comprahensive cancer center of Saint Vincents Hospital in NYC. Patients were recruited to have thyroid function done at the same time other blood work was done. Later data was collected from the patients chart regarding their type of cancer, the stage of their disease, and type of therapy they were receiving(chemotherapy, radiotherapy, hormonal therapy, etc)Thyroid function were performed by the hospital laboratory using standard kits.TSH normal range 0.49 ± 4.67Free T4 normal range 0.17 ± 1.85 RESULTS: Total of 158 cases analyzed to date.44 cases of breast cancer,25 cases of gastrointestinal cancer, 7 cases of lung cancer, 60 cases of hematological malignancies, and 22 cases of other types of cancer (ovarian, prostate, ks, etc).The mean age of patient was 57 y/ o(range 28 ± 86)There were 85 females and 73 males in the study.17 patients had abnormally low TSH suggesting subclinical hyperthyroidism and 1 patient had high T4 suggesting Thyrotoxicosis. 6 patients had elevated TSH and 3 had low T4 suggesting hypothyroidism. In total 26 cases (16%) of abnormal thyroid function in the population studied. CONCLUSION: The prevalence of thyroid disease in cancer patients is significant, and since the symptoms mimic those of cancer, both internists and oncologists caring for the patients should be aware of that. METHODS: We surveyed 2,500 adult patients seen in the ambulatory clinics of a university hospital. We asked patients about their desire for information regarding the side effects of prescription drugs. We asked 190 physicians practicing in the same clinics about their perceptions of patients' expectations for this information. We also collected information on age, gender and level of education for patients and age, gender and specialty for physicians. We found significant differences in responses of patients and physicians. RESULTS: Please see the Researchers have suggested that patients may understand treatment benefits better when they are presented as numbers needed to treat (NNT) rather than as absolute or relative risk reductions. We sought to determine if NNT helps patients interpret treatment benefits better than absolute risk reduction (ARR), relative risk reduction (RRR) or a combination of all three of these risk reduction presentations (COMBO). METHODS: We surveyed 357 men and women, ages 50 to 80, who presented for care at a university internal medicine clinic. After answering three questions assessing their ability to handle numbers (numeracy skill), subjects were asked to (1) state which of two drug treatments for a hypothetical disease Y provided more benefit, and (2) calculate the effect of drug treatment on a patient with a given baseline risk of disease. Risk information was presented to each subject in one of four randomly allocated risk formats±NNT (100 versus 250 people just like you would need to be treated for 5 years for a benefit against disease Y to be seen in one of you), ARR (treatment reduces the chance that you will develop disease Y by 10 versus 4 out of 1000 over the next 5 years), RRR (treatment reduces the chance that you will develop disease Y by 25% versus 10% over the next 5 years), or COMBO. RESULTS: Subject ability to interpret treatment benefits varied significantly with the risk presentation format they received. Patients correctly interpreted the treatment benefit most often when it was presented in the RRR format: when asked to state which of two treatments provided more benefit, subjects who received treatment benefit information in the RRR format responded correctly 60% of the time, whereas subjects receiving the COMBO, ARR, and NNT presentation formats responded correctly only 43%, 42%, and 30% of the time, respectively (p < 0.01). Most patients were unable to calculate the exact effect of drug treatment on a patient with a given baseline risk of disease, although subjects receiving the RRR and ARR formats responded correctly slightly more often (21% and 17%, respectively, compared to 7% for COMBO and 6% for NNT, p < 0.01). Higher numeracy skills were associated with increased ability to interpret treatment benefits: 88% of subjects who answered all three numeracy questions correctly stated which of two treatments provided more benefit versus only 63% of subjects who answered two questions correctly and 35% of subjects who answered one or no questions correctly (p < 0.01). This association persisted when subjects were asked to calculate the effect of drug treatment on a patient with a given baseline risk of disease. CONCLUSION: Patients are best able to interpret the benefits of treatment when they are presented in a RRR format. NNT is often misinterpreted by patients and should not be used alone to communicate risk to patients. PURPOSE: Depression is very common, particularly in primary care. While depression in otherwise healthy young patients is rather straightforward, it is less clear how to manage depressed patients with multiple medical problems. We assessed the level of physical and mental health impairment in primary care patients who had recently had depressive symptoms. METHODS: We identified two samples of veterans from the Primary Care Clinic at the VA Sepulveda Ambulatory Care Center who had depressive symptoms during a 7-month period: 290 patients whose primary care encounter form had a depression-related diagnosis (PC group) and 130 patients seen in primary care by Consultation L group). These patients all were sent a letter from their attending physician, inviting them to participate in a study of assisted referral back to primary care for patients who were still depressed. 19 PC group patients (7%) and 23 C&L group patients (18%) declined to participate and over half never responded (65% PC, 52% C&L). Respondents (82 PC, 39 C&L) were contacted 6 months after their initial diagnosis of depressive symptoms. The interviewer-administered initial survey included the Veterans Short Form-12 (SF-12V) and three questions on where they received health care. Respondents not currently seeing a mental health specialist were asked to complete a more detailed survey, which included measures of depression, anxiety, alcohol use and sociodemographics. RESULTS: Completed interviews were available for 86 patients (53 PC, 33 C&L). 14 patients who agreed to participate could not be reached and 20 interviews were still pending. The average Mental Component Score for respondents was 36.4. While 40% had no mental health impairment, 17% had impaired mental health (MCS 31 ± 40) and 43% had severely impaired mental health (MCS 30 or less). The average Physical Component Score for respondents was 37.3. The breakdown was similar to that for mental health: 38% had no physical health impairment, 24% had impaired physical health (PCS 31 ± 40), and 37% had severely impaired physical health (PCS 30 or less). 20% of respondents were both physically and mentally impaired and another 14% were severely impaired physically and mentally. Of those with current mental health impairment, the prevalence of physical health impairment was similar between the group seeing psychiatry and those not currently seeing psychiatry. CONCLUSION: Six months after having depressive symptoms identified, most veterans still have profoundly impaired mental health. Of note, the degree of physical health impairment is equally profound. This suggests that for many depressed patients in primary care, treatment models need to account for severe disability both physically and mentally. We assessed whether or not this same relationship holds among veterans, who themselves tend to be sicker and have more health care utilization than the general public. METHODS: As part of the baseline patient survey for a multi-site trial of evidence-based quality improvement for smoking cessation, we interviewed veterans at 18 sites in the Southwestern U.S. The survey, conducted by computer-assisted telephone interview among patients with at least 3 primary care visits in the prior 18 months, covered smoking history, health habits, health status and demographics. Questions were adapted from previously validated sources, including the California Tobacco Survey, Medical Outcomes Study, CES-D (depression), AUDIT (alcohol abuse), and others. Outpatient utilization data for FY 1999 were extracted from the VA Outpatient File at Austin and grouped by type of visit. Inpatient utilization data were obtained from the Patient Treatment File at Austin. Comparisons were done using chi-squared tests and ANOVA. For this preliminary analysis, we also analyzed the utilization data splitting subjects into those under 65 (n=3745) and those 65 or older (n = 3961). RESULTS: Of the 7,706 subjects interviewed, current smokers were more likely than former smokers or those who never smoked to be younger, less active, and divorced (all p < 0.001). Current smokers were more likely than the other two groups to have severe mental health impairment (21% vs. 12% vs. 14%, p < 0.001) and severe physical health impairment (55% vs. 48% vs. 46%, p=0.026). Current smokers were less likely than former smokers or those who never smoked to report being in excellent or very good health (16% vs. 22% vs. 26%, p < 0.001). Among current smokers, approximately half reported they never drink alcohol, but 18% met AUDIT criteria for alcohol abuse. Forty-three percent of current smokers met CES-D criteria for depression at the time of the interview. Current smokers, former smokers, and those who never smoked did not differ in the total number of outpatient visits or for emergency room or general medicine visits. Current smokers did average more mental health and social work visits and fewer medical subspecialty visits than former smokers or those who never smoked (p < 0.001). The rate and length of acute care hospitalizations did not differ among the three groups, but smokers had more nursing home admissions. When we analyzed the data separately for subjects over 65 and under 65, the utilization differences tended to occur predominantly in the half of the sample under age 65. CONCLUSION: While smokers had higher outpatient mental health use, they did not have higher utilization for most other categories. They tended to have worse functional and health status than former smokers and those who never smoked.\nBENEFICIARIES WITH CANCER. L.R. Shugarman 1 , C. Bird 1 , J. Lynn 1 ; 1 RAND, Santa Monica, CA PURPOSE: Medical care at the end of life is quite costly; over one quarter of Medicare payments support care of beneficiaries who will die within a year. The aggregate costs and utilization patterns of men and women facing death may not be the same due to differences in their age, preferences, and cultural stereotypes. We analyzed Medicare services utilization and payments for men and women dying of cancer to determine whether differences occur within 5year age strata. We selected cancer because of its clear diagnosis, generally accepted standards of care and limited treatment options for which we do not expect gender differences. METHODS: We analyzed 1993 through 1998 Medicare claims and eligibility data for an overall 0.1% random sample (a 2% sample of an initial 5% sample) of beneficiaries who: were age 65 and over with a diagnosis of cancer but without a diagnosis of end stage renal disease, died in the target time interval and had one year of continuous data before death. Beneficiaries were identified as having a cancer diagnosis if cancer accounted for the plurality of physician spending in the year of death. Age strata were based on the beneficiary's age at death. RESULTS: Across all age strata, women were more likely to use skilled nursing facility care than men (35% vs. 27%, p < .10) and hospice care (42% vs. 37%, p < .10). With the exception of 80 ± 84 year-olds, Medicare total reimbursements for hospitalizations were higher for women than for men ($18,531 vs. $17,166, p < .05, overall) . The beneficiary's responsibility for Medicare costs (e.g., deductibles and co-payments) was higher for men aged 65 ± 69 ($5,188 vs. $4,863, p < .10) and for women in the 70 ± 74 age stratum ($5,163 vs. $4,403, p < .05) and in the 75 ± 79 age stratum ($4,501 vs. $4,298, p < .10). CONCLUSION: Observed gender differences in aggregate spending and utilization are attenuated by age stratification. The persisting differences in use of skilled nursing facilities and hospice and higher hospital costs represent both differences in care and coverage under Medicare. These findings require more study, such as analyses stratified by other primary diseases, type of cancer, and type of service. Our data support the contention that social setting, cultural setting, and patient preferences may combine to create modest but persistent differences in treatment patterns by gender. PURPOSE: Although cervical cancer deaths account for fewer than one percent of cancer deaths among women aged 65 and older, most elderly women report continuing to undergo periodic Pap smear screening. We sought to describe the burden of downstream testing following Pap smears in elderly women who undergo screening. METHODS: Using three years of Medicare Part B 5% Files (1995 ± 1997), we identified women over age 65 who underwent Pap smear screening during a 16-month period in 1996 and 1997. In order to exclude women undergoing follow-up Pap smears or surveillance smears (women with a previous procedure or diagnosis suggestive of cervical dysplasia or malignancy), we required a one-year observation period without such testing prior to the screening Pap smear for each woman. We measured downstream events including subsequent Pap smears, colposcopies, and other diagnostic and therapeutic surgical procedures (e.g. conization) during the 8 months following the screening Pap smear. RESULTS: In 1996, almost two and a half million female Medicare beneficiaries over age 65 underwent Pap smear screening. For every 100,000 women screened, 3020 had at least one additional Pap smear, 669 underwent colposcopy, and 183 had other surgical procedures during the eight-month period following the screening Pap smear. Rates of downstream interventions were similar for women aged 66 ± 75 and 76 ± 85, and declined only modestly in women age 86 and older. Overall, for every 100,000 women screened, 3497 experienced downstream interventions within 8 months of the initial Pap smear. For comparison, according to population-based surveillance data (SEER,1996) , 16 women per 100,000 aged 65 and older are diagnosed with cervical cancer each year, while nine are expected to die from the disease. CONCLUSION: Relative to the small number of expected cases of cervical cancer, women aged 65 and older continue to be screened in large numbers and undergo substantial follow-up testing in response to abnormal Pap smear results. Whether this testing reduces the subsequent risk of death from cervical cancer is unknown. There are no data, however, regarding the frequency at which women actually undergo screening. We sought to describe the frequency of cervical cancer screening in the United States. METHODS: We used the 1998 Behavioral Risk Factor Surveillance System of the Centers for Disease Control and Prevention (CDC), a cross-sectional population-based telephone survey conducted annually on a random sample of civilian non-institutionalized adults. We focused on female respondents aged 18 and older (n=86,715). For women who reported ever having had a Pap smear, we estimated screening intervals based on the timing of a woman's most recent Pap smear. We assumed that each woman either was not being regularly screening, or was being screened at one of three discrete screening intervals (every year, every 2 years, or every 3 years). RESULTS: The vast majority (93%) of American women report having had at least one Pap smear in their lifetime. Among women who have not had a hysterectomy and who have been screened at least once, 90% report having had a Pap smear within the past three years. Based on the reported time since the last Pap smear, we estimate that 59% of women undergo Pap smear screening annually, 18% have a two-year screening interval, 13% are screened every three years, and 10% are not being screened regularly. Even the very elderly report regular screening Ð an estimated 34% percent of women aged 75 ± 84 and 18% of women aged 85 and older undergo annual Pap smear screening. Extrapolating these data to the country as a whole, more than 45 million American women undergo Pap smear screening annually. CONCLUSION: The majority of American women are being screened for cervical cancer too frequently. Lengthening the screening interval would not only reduce the number of pelvic exams, but would also reduce follow-up testing for abnormal smears and the volume of specimens that cytotechnologists are required to read. We performed a study to determine the utility of routine screening of transaminase and CPK values in patients taking statin medications. METHODS: We performed a retrospective on-line medical record review of the primary care practice at Beth Israel Deaconess Medical Center. A computerized search identified all patients at our institution with a statin on their medication list, as well as their ALT, AST and CPK values for 1998. Patients were separated into four categories based on which laboratory data had been measured during the year: CPK only, transaminase only, both CPK and transaminase or neither. From each of these four categories, 45 patient records were randomly selected for review to verify that they were followed at this primary care practice and were on a statin. The random samples were used to identify the rate of physician monitoring of transaminase and CPK values within this practice. We reviewed the on-line medical records of all patients within this practice on statins who had significantly abnormal test results (defined in prior trials of statin therapy as greater than 3x the upper limit of normal for transaminases and greater than 10x the upper limit of normal for CPK) to determine the relationship to statin therapy and outcome. RESULTS: 4556 patients at our institution had a statin on their medication list. Of the 180 random charts reviewed, 26% (corresponding to 1189 of the total cohort) were receiving care from this primary care practice and were taking a statin. Among the patients whose charts were reviewed, 53% were female with a mean age of 61 years. These data also demonstrated that physicians within our practice monitored serum transaminase values (defined as measurement of either AST or ALT during 1998, or within six months of starting a statin) in 84% of patients; physicians monitored CPK values in 54% of patients. 29% of the patients within this practice were on a dose greater than the recommended starting dosage. Of all the patients in this practice who had been monitored, 12 (1.2%) had a significant elevation of AST or ALT and 6 (0.9%) had a significant elevation of CPK. However, after a detailed chart review, none of these abnormalities were attributable to statins. CONCLUSION: In this study of statin use in a primary care practice, no cases of significantly abnormal transaminase or CPK values attributable to statins were discovered through routine monitoring. This questions the necessity of routine measurement of transaminase and CPK in all patients taking statin medications. and had no risk of zoster during that time. Thereafter, vaccinated patients had half the usual zoster and postherpetic neuralgia (PHN) incidence rates (or post-boost effect=50%). Agespecific zoster incidence was that of Rochester, Minn. Half the zoster patients received antiviral therapy; 20 ± 40% of all zoster patients developed PHN, depending on their age. Multiple sensitivity analyses were performed. RESULTS: In the baseline analysis, vaccination gained 0.0004 QALY compared to no vaccination at a cost of $51.13 or $143,000/QALY gained. Vaccination is least expensive at age 66, costing $129,000/QALY gained. In 60-year-olds, costs/QALY gained is greater than $50,000 with wide variation of: immunologic boosting duration, post-boost effect, PHN probability or duration, herpes zoster duration or utility, or antiviral therapy or hospitalization parameters. Vaccinating 60-year-olds costs $103,000 or $61,500 per QALY when lifetime zoster risk (baseline 11.0%) increased to 1 in 7 (14.3%) or 1 in 5 (20%) respectively. If lifetime zoster risk is 20%, costs/QALY gained are less than $50,000 if post-boost effect is < 38%, median PHN duration is > 114 days (baseline 90), average PHN utility is < 0.76 (where 0=death and 1=perfect health), or vaccination cost < $54.21 (baseline $61.96, including $5 administration cost). CONCLUSION: Varicella vaccination of 60-year-olds is expensive compared to many accepted medical interventions in a model biased toward its use. Results are most sensitive to the lifetime risk of zoster, which varies significantly in different populations. 1994) is a valid and reliable assessment tool designed to enable rapid determination of current or past psychiatric disorders in primary care patients. The current analyses were conducted to assess whether or not smokers with current psychiatric diagnoses, as determined with the PRIME-MD, are at higher risk for early relapse to smoking after quitting. METHODS: The PRIME-MD was administered as part of the study screening procedures in an NIH-funded smoking cessation clinical trial that enrolled 677 smokers. Brief individual smoking cessation counseling was provided prior to quitting and on the quit day; nicotine patches were dispensed on the quit day. The outcome measure in the current analyses was biochemically-verified smoking status at one week post-cessation. PRIME-MD diagnoses included major depressive disorder (MDD), panic disorder (PD), generalized anxiety disorder (GAD), and probable alcohol abuse/dependence. RESULTS: A total of 182 smokers (27%) were diagnosed with at least one of the four disorders and 30 smokers had two or more diagnoses. 63% of the participants without any current diagnoses were abstinent at one week compared to 20% of participants with MDD (n=133), 5% with GAD (n=20), and 0% with PD (n=3). No statistically significant increase in risk of relapse was observed for 62 participants with alcohol abuse/dependence (50% abstinence rate). Only 7% of participants with two or more disorders were abstinent at one week post-quit. CONCLUSION: Smokers with current mood and anxiety disorders appear to be at significant risk for early relapse to smoking. The PRIME-MD can provide rapid assessment of these psychiatric diagnoses that are commonly seen in the primary care setting. Identification of smokers with significant depression and anxiety can aid in the development of smoking cessation treatment plans (e.g., including an anti-depressant such as bupropion SR for depressed patients) and can focus attention on the importance of aggressive relapse prevention in these individuals. Russian language interpreters who administered the survey to adults with a valid phone number, using a list provided by the resettlement agency. We report comparisons with the 1999 Behavioral Risk Factor and Surveillance System (BRFSS) data (www.cdc.gov). RESULTS: Of 426 available adults, completed interviews were obtained in 56% (n=241), with mean age 54.2 yrs (18 ± 94), 54% women, and subjects had resided in the county a mean of 7.5 yrs (1 ± 29) . 91% had some type of health care coverage 91% (n=220), comparable to reports for BRFSS. Self-rated health was rated poor/fair by 60% (n=241), compared with responses on the 1999 BRFSS of 13.1% National and 21.6% Kentucky. Preventive measures shown in Table [ To elucidate the impact of the individual and combined components of a physician's preventive``medical message'' on people's intention to adopt more healthy behaviors. The specific risk used in this study was that of developing manifestations of coronary artery disease. METHODS: 86 young people (ages 20 ± 30) and 64 older people (ages 60 ± 80) in France indicated their degree of intention to adopt a healthy behavior in response to a series of 64 hypothetical messages from their physician about their risk of developing a cardiac event. The messages were all possible combinations of 4 components: the type of cardiac event (angina or a heart attack), the probability of developing it (5, 10, 15, or 20%), the time frame (within 5, 10, 15, or 20 years), and the degree of the patient's control over developing it (``given your family history, it is probable that you could not do much to reduce this risk'' or``the reduction of this risk depends entirely on your behavior''). They were asked about adopting 1 of 3 behaviors: à`m edical treatment,'' a strict dietary regimen, or regular exercise. The results were examined graphically and by ANOVA. RESULTS: All components had a significant effect on the intention to change behavior, but these effects varied according to age. The young people were influenced most by the degree of controllability, the probability, and the nature of the cardiac event; the older people by the time horizon. Even without a promise of controllability, participants were inclined to adopt protective behavior. An increase in probability had less added effect as the probability got larger; nonetheless, the level of intent to change behavior associated with a large probability over a long time (e.g., 20% over 20 years) was higher than that associated with a smaller probability over a short time frame (e.g., 5% over 5 years). The type of protective action had no effect on intention. CONCLUSION: The preventive medical message should include the type of event, the probability of developing it, the time frame, and the degree to which the patient can prevent it. The physician should stress the time frame in talking with older people and the other factors with young people. TREATMENT, 1987 TO 1998 . R.S. Stafford 1 , E. Macdonald 1 , S. Finkelstein 2 ; 1 Massachusetts General Hospital, Boston, MA; 2 Massachusetts Institute of Technology, Cambridge, MA PURPOSE: Physicians' approach to the treatment of depression has changed drastically in the last 15 years with the availability of selective serotonin reuptake inhibitors (SSRIs) in 1988. We sought to closely examine trends in antidepressant use, as well as assess broader changes in treatment patterns. METHODS: Using data available from the National Disease and Therapeutic Index (NDTI), a physician survey conducted by IMS Health, we examined trends in antidepressant prescribing from 1987 ± 98. NDTI provides nationally representative diagnostic and prescribing information on patients treated by U.S. office-based physicians. We selected visits by patients reported to have depression. Annual sample sizes varied from 3,901 visits in 1987 to 6,639 in 1998. We examined annual prescribing data to measure: 1) the frequency of visits where depression was noted and 2) the likelihood of specific medications being reported as treatment. RESULTS: The NDTI data show a dramatic shift in the treatment of depression between 1987 and 1998. In 1987, tricyclic antidepressants (TCAs) were the predominant drug class prescribed to patients with depression (47% of patients). Among individual antidepressants, the most common were amitriptyline (14%), trazadone (12%), doxepin (8%), and desipramine (6%). In 1989, a year after its introduction, fluoxetine was prescribed to 21% of patients with depression. Despite the introduction and growing use of other SSRIs in the 1990s, fluoxetine has maintained a constant share of patients at around 25%. The introduction of sertraline in 1992, paroxetine in 1993 and more recent SSRIs led aggregate SSRI use to grow to 38% in 1992 38% in , 56% in 1994 38% in , 60% in 1996 38% in and 75% in 1998 38% in . In 1998 , sertraline (21%), paroxetine (17%) and bupropion (6%) were the leading antidepressants. TCAs accounted for only 8% of depression patients in 1998. The prescribing of non-TCA, non-SSRI antidepressants decreased from 13% (chiefly trazadone) in 1987 to 10% (chiefly buproprion) in 1998. The use of benzodiazepines in depression declined from 11% of patients in 1987 to 3% in 1998. The rate of patients with reported depression not receiving an antidepressant decreased from 30% in 1987 to 9% in 1998. The estimated national number of physician visits by patients with depression increased from 14.4 million visits in 1987 to 22.5 million in 1998. CONCLUSION: The increasing therapeutic dominance of SSRIs has been pivotal to changes in depression treatment. Perhaps because of their effectiveness and side-effect profile, increasing SSRI use also may have contributed to the declining use of benzodiazepines, increased aggregate antidepressant treatment rates, and the increasing frequency of physician visits where depression was reported.\nNATIONAL TRENDS IN RECOMMENDED CARDIAC MEDICATIONS. R. Stafford 1 , C. Chaisson 1 ; 1 Massachusetts General Hospital, Boston, MA PURPOSE: Previous studies suggest that recommended cardiac medications are underutilized. We evaluated recent national patterns of medication use in the ambulatory care setting for warfarin use in atrial fibrillation (AF), beta blocker (BB) and aspirin (ASA) use in coronary artery disease (CAD), and ACE inhibitor use (ACEI, including angiotensin receptor blockers) in congestive heart failure (CHF). METHODS: We used the 1989 ± 1998 National Ambulatory Medical Care Surveys to identify nationally representative samples consisting of patients with AF, CAD and CHF who lacked specific contraindications to therapy. For AF, 1,370 office visits were identified; for CAD, 8,773 visits (for BB) and 8,808 visits (for ASA); and for CHF, 3,310 visits. We examined time trends in the proportion of visits reporting the selected medications, weighted to reflect national patterns. Logistic regression was used to evaluate the independent predictors of cardiac medication use in 1995 ± 1998, including patient and physician characteristics. RESULTS: In patients with AF, warfarin use increased steadily from 13% of visits in 1989 to 38% in 1994 to 50% in 1998 (see Table) . In CAD patients, BB use increased from 16% in 1989 to 19% in 1994 to 33% in 1998 . ASA use in CAD was 11% in 1989 increasing to 22% in 1992 where it plateaued, reaching 25% in 1998. In CHF patients, ACEI use increased slowly from 22% in 1989 to 28% in 1994 to 32% in 1998. Patterns of medication use for 1995 ± 98 varied by patient and physician characteristics. Women were less likely to be taking warfarin for AF (OR=0.35 P=0.06) or ASA for CAD (OR=0.79 P=0.04). Nonwhite CAD patients were less likely to take BB (OR=0.68 P=0.03) or ASA (OR=0.69 P=0.05) than were whites. The oldest and youngest patients tended to be less likely to receive medications than those aged 60 ± 69; this was most dramatic for BB in CAD in those 80+ (OR=0.57 P < 0.001). Patients with CAD visiting cardiologists were more likely to receive recommended drugs. CONCLUSION: Recommended cardiac medication use increased from 1989 to 1998, but some increases have not continued into the late 1990's and have not been uniform across subpopulations. Further adoption of these effective therapies could result in additional benefit for patients with cardiac conditions. identified by ICD-9 codes. Our outcome measure was the pattern of medication use for these conditions. In comparing indemnity and managed care, we used logistic regression to adjust for case-mix: patient age, gender, number of physician visits, Diagnostic Care Group (DxCG) risk score, and a series of DxCG co-morbidity indicators. RESULTS: With few exceptions, the use of chronic disease medications was more likely among managed care patients. For patients with DM, managed care plans had greater use of sulfonylureas than indemnity (42% vs. 37%), metformin (26% vs. 17%) and troglitizone (9% vs. 6%). In multivariate analysis to adjust for case-mix, these differences remained significant (p < 0.001). Insulin use was no different in managed care and indemnity plans. For CHF patients, managed care patients had greater use of loop diuretics (44% vs. 39%), ACE inhibitors/ARBs (50% vs. 39%), and beta-blockers (25% vs. 16%); differences that were significant in multivariate analysis (p < 0.001, except loop diuretics [p=0.003]). No differences in digoxin use were observed. For asthma patients, managed care had greater use of inhaled corticosteroids (33% vs. 28%), systemic corticosteroids (18% vs. 15%), short-acting betaagonists (41% vs. 31%), long-acting beta agonists (10% vs. 8%), and leukotriene modifiers (5.3% vs. 3.9%); differences that were significant in multivariate analysis (p < 0.001, except for long-acting beta agonists [p=0.03] and leukotriene modifiers [p=0.04]). No differences were found for cromolyn or methylxanthine use. CONCLUSION: Chronic disease patients in managed care plans are more likely to receive a broad range of medications. Three of 4 exceptions to this pattern are older medications whose use has been partly supplanted by newer therapies. The general pattern of greater medication use in managed care could reflect patient selection, reduced out-of-pocket cost barriers, or a strategy to avoid other, more costly services. Managed care plans do not appear to withhold expensive, newer medications. The emphasis on screening and minimally invasive breast techniques has led to an increase in detection of both benign and malignant breast disease. Optimal management for benign breast disease remains uncertain, and population-based studies of surgical intervetions are lacking. This study uses population-based administrative data to examine use of excisional breast procedures across 5 states and 7 years, comparing benign to malignant breast diagnoses. METHODS: Data are from the Healthcare Cost and Utilization Project, a federal-stateindustry partership in administrative data. Ambulatory surgery(AS) and inpatient(IP) data are used from CO, CT, MD, NJ and NY for 1990 ± 1996. All women with a lumpectomy(ICD-9 85.21) or subtotal mastectomy(85.22, 85.23) were extracted. Lumpectomy(LUMP) and subtotal mastectomy(STMAS) were further classified as either benign disease or cancer using diagnoses codes. Overall IP and AS age-adjusted rates per 100,000 women for each state, year and procedure were calculated using direct standardization. RESULTS: The rates of breast conserving surgery(LUMP+STMAS)for breast cancer increased across all five states, from between 33 ± 71/100,000 women in 1990 to between 70 ± 140/100,000 women in 1996. The rate of LUMP is 3 to 4 fold higher for benign disease as compared to that for cancer in every state. For instance, in 1996, the rate of LUMP in NY for cancer was 91/ 100,000 women, while for benign disease, 271/100,000. However, in contrast to cancer, the rate of LUMP for benign disease is relatively stable across all of the states and years. The overall rate of STMAS is one-tenth that of LUMP in all the states. Similar to LUMP, the rate of STMAS for benign disease was stable across time. However, there was a great deal of variation among the states in use of STMAS for benign breast disease. The rate varied from a negligible rate in CO, to as high as 50/100,000 women in CT in 1996. In addition, for CT and NJ there were equal rates of STMAS for benign and cancer diagnoses(50 and 27/100,000 respectively), while the STMAS rate for cancer was higher in the other 3 states. CONCLUSION: This is the first population-based study comparing interventions for benign breast disease to cancer. This study demonstrates unexpectedly high rates of surgical interventions (LUMP and STMAS) for benign breast disease. These rates may be explained by women and physician fear of missed cancer diagnoses, lack of best practices, upcoding of procedures, or AS data not capturing cancer at discharge for short stays. However, the latter would indicate a lack of two-stage decision-making process for women. PURPOSE: Anecdotal reports have highlighted the stories of patients who skip doses or otherwise avoid using their medications because they cannot afford the expense. However, little is known about which elderly patients without prescription coverage are at highest risk of not taking their medications because of cost, and how prescription coverage modifies this risk. METHODS: We performed a cross-sectional study of 4896 subjects in the 1995 ± 1996 wave of the Survey of Asset and Health Dynamics Among the Oldest Old (AHEAD), a population-based survey of Americans age 70 years and older. Subjects were asked the extent of their prescription coverage, and whether they had taken less medicine than prescribed for them because of cost over the prior 2 years. We used bivariate and multivariate analyses to identify risk factors for restricting one's use of medications because of cost in subjects who lacked prescription coverage. Among subjects with these risk factors, we then examined the effect of prescription coverage on rates of medication restriction. RESULTS: Of 4896 seniors who regularly used prescription medications, 39% had no prescription coverage, 44% had partial coverage, and 17% had full coverage. Not taking medications because of cost was reported by 8%, 3%, and 2% of patients with no, partial, and full prescription coverage, respectively (p < .01 for trend). Among subjects with no prescription coverage, the strongest independent predictors of foregoing medications because of cost were ethnicity (prevalence of restriction 21% for minority subjects, 6% for white subjects, P < .01), income (16% for annual income < $10,000, 8% for $10,000 to $19,999, 4% for !$20,000, P < .01), and out-of-pocket prescription drug costs (13% for monthly cost > $100, 7% for $20 to $100, 3% for < $20, P < .01). Prescription coverage markedly reduced the rate of medication restriction in these high-risk groups. For example, rates of medication restriction in minority and low-income subjects were 21% and 16% among those with no coverage, 8% and 8% among those with partial coverage, and 4% and 2% among those with full coverage, respectively (p < .001). Almost half (43%) of minority patients with low income, high drug costs, and no prescription coverage reported restricting their use of medications, compared with 12% of patients with these three risk factors and partial prescription coverage. CONCLUSION: Restricting one's use of medications because of cost is common in vulnerable groups of seniors who lack prescription coverage. Among these high-risk groups, prescription coverage markedly reduces the rate of medication restriction. PURPOSE: Hospital contact isolation policies are designed to prevent the nosocomial transmission of infectious diseases, but may inadvertently promote the neglect of isolated patients. We tested whether the frequency and quality of recorded vital signs differed between isolated and non-isolated inpatients. METHODS: We identified consecutive adults admitted to a large Canadian teaching hospital between January 1, 1999 and January 1, 2000 who were placed in contact isolation during their hospital stay (n=81). Controls were selected by identifying the two patients who occupied each isolated patient's hospital bed immediately before and after their admission (n=162). Vital signs recorded in the medical records were compared for the two groups using t tests. Adjustments for age, gender, Charlson Comorbidity Score, living status (nursing home vs. home) and admitting service (medicine vs. surgery) were done using propensity score matching. RESULTS: We found no difference in the frequency of daily vital signs ordered for isolated and non-isolated patients (2.8 vitals/day vs. 3.2 vitals/day, p > 0.20). However, isolated patients had a significantly higher percentage of their vital signs incompletely recorded (missing at least one of heart rate, blood pressure, respiratory rate or temperature) as compared to non-isolated patients (17.3% vs. 12.7%, p=0.03). Both groups had surprisingly high percentages of their respiratory rates recorded as twenty (53.2% vs. 50.9%, p > 0.20). CONCLUSION: Patients placed in contact isolation appear to have their vital signs ordered as frequently though not recorded as completely as non-isolated patients. Differences in the recording of vital signs may reflect underlying differences in the quality of care received by isolated patients and warrant further investigation. OBJECTIVE: Several studies have previously reported that patients want their physicians to address spiritual issues as part of their health care. It has also been reported that patients feel their individual spiritual faith can help them recover from illness. Surveys of physicians have shown that they often do not routinely address these issues with patients. We sought to examine patient and physician perceptions regarding inclusion of religiosity and spirituality in the medical encounter. METHODS: We surveyed primary care physicians and patients in primary care clinics at 7 sites in 4 states (NC, FL, GA, VT). For the patient survey, a trained research assistant verbally administered a 112 item survey that included questions pertaining to demographics, health status, utilization of health care, physical function (SF-36), and a religious/spiritual assessment (Spiritual Well Being, or SWB) scale. Physician surveys were mailed to primary care faculty, residents, and recent graduates at the same 7 sites. Bivariate analyses were done to examine associations between patient/physician characteristics and questions regarding the impact of prayer on health outcomes. RESULTS: 299 patients and 444 physicians were surveyed. Patients' ages ranged from 19 ± 86 years. Fifty-six percent were male, 46% were African-American, 51% Caucasian, and 63% were Protestant. Fifty-three percent of the patients surveyed had annual incomes of $20,000 or less. Of the physicians, 45% were in practice, and 55% were residents/fellows. Forty-five percent were Protestant. Fifty-three percent work in an academic setting. Of the patients, 30% felt that praying with their doctor would improve their own health. Thirty-nine percent of patients vs. 19% of physicians agreed that their doctor praying for them would improve their health. In addition, 42% felt that praying for their doctor would improve their own health, while 32% of physicians felt that patient prayer for them would help them provide better health care. Nearly half of physicians felt that patients who prayed for themselves or had strong religious/spiritual beliefs would have improved health outcomes. Fifty-three percent of patients as compared to 19% of physicians agreed that faith alone can cure disease. Bivariate analyses in the patient sample showed that the perception that prayer improves health outcomes is associated with higher attendance of organized worship services, hospitalization within the past year, higher score on the SWB scale, and lower total household income. In the physician surveys, bivariate analyses associated the equivalent perceptions with attendance of organized worship services and higher SWB score. CONCLUSION: A significant percentage of patients perceive that prayer (whether it is done by their doctor, with their doctor, or for their doctor) is linked to improvements in health. Although there are a significant number of physicians who perceive that prayer is linked to improved health outcomes, there is still a large disparity in the prevalence of physicians compared to patients who share this perception. These data suggest that a more reliable quantification of the benefit of spiritual behavior in medical encounters might be needed to bridge this gap. The prevalence of soft tissue infections among injection drug users (IDUs) is estimated between 11% ± 32% with 40% of those presenting for care requiring hospitalization. Risk factors for hospitalization from these infections are not known. This study sought to identify factors associated with increased health care utilization. METHODS: This was a cross-sectional study of all IDUs seeking initial care for soft tissue infections at an urban, public hospital emergency department (ED) from November, 1999 through April, 2000. Demographics, infection prodrome, and clinical measures were extracted from medical records. Systemic infection was defined as temperature !388C or WBC !15,000 per milliliter, prolonged hospitalization as !2 days, and delay in seeking care as !5 days of symptoms before the ED visit. RESULTS: Two hundred forty-two patients sought initial care for IDU-related soft tissue infections. Most patients were male (64%), Caucasian (69.4%), unemployed (78%), and uninsured (52%). Soft tissue infections included abscesses (74.4%), cellulitides (31%), and infected ulcers (5%) and were mainly located on the lower arm (49.6%), deltoid (14.1%), leg (22.7%), and buttock (19.8%). Ninety-seven (40.1%) patients delayed seeking care. Systemic signs of infection were identified in 118 (48.8%) patients. Of the 97 (40.1%) patients hospitalized, 70 (72.2%) stayed 2 or more days. Ethnicity, gender, age, employment status, and homelessness were not associated with having systemic infection, with hospitalization or with prolonged hospitalization. Compared to those with other types of infection, patients with abscesses were much less likely (OR 0.13, 95% CI 0.02 ± 0.73) to be hospitalized while those with cellulitides were 3.2 times more likely (95% CI 1.47 ± 6.79) to be hospitalized and 3.8 times more likely (95% CI 1.70 ± 8.57) to be hospitalized !2 days. Among patients who delayed seeking care, those with systemic signs of infection were 5.36 times (95% CI 1.67 ± 17.19) more likely to have a prolonged hospitalization compared to those without systemic signs of infection. Prolonged hospitalization was not associated with systemic infection among those who did not delay seeking care. CONCLUSION: Among IDUs seeking ED care for soft tissue infections, systemic signs of infection after delay in seeking care, and cellulitides, were each associated with increased use of health services. Gender, ethnicity, employment status, insurance status, and homelessness did not alter the risk of hospitalization, of prolonged hospitalization, or of having systemic signs of infection. PURPOSE: Stroke is the third leading cause of death and a major cause of disability among postmenopausal women in developed countries. Although postmenopausal hormone replacement therapy (HRT) is one of the most widely prescribed drugs, it is associated with increased rates of thromboembolic events and therefore may be important etiologically in stroke. We conducted a systematic evidence review and meta-analysis for the third US Preventive Services Task Force (USPSTF) to investigate the relationship between HRT and stroke. METHODS: We searched the MEDLINE database from 1992 to 2000 for all published English language studies reporting the association between HRT and stroke. In addition, reference lists of key articles were reviewed for related studies, including those predating the search. Thirty-three observational studies met inclusion criteria and were reviewed; however, only those studies considered good or fair quality based on criteria developed by the USPSTF were included in the detailed review and meta-analyses. We identified no randomized controlled trials. We used the Bayesian data analysis framework for the meta-analysis. RESULTS: The pooled relative risk of stroke mortality in women who had ever used HRT was 0.83 (95% Cl 0.64 ± 1.05). Stroke incidence was significantly increased among ever users, with a pooled relative risk of 1. 15 (1.03 ± 1.29). On subanalyses, the risk of thromboembolic stroke was significantly elevated among women who had ever used HRT (RR 1.30 [1.10 ± 1.58]); however, not subarachnoid hemorrhage (RR 0.93 [0.69 ± 1.25]) or hemorrhagic stroke (RR 0.71 [0.25 ± 1.29] ). CONCLUSION: Studies evaluating the association between URT and stroke mortality suggest no effect. Our meta-analysis suggests an increase risk of the incidence of total stroke, largely due to thromboembolic stroke, among women with exposure to HRT. These results are consistent with preliminary findings from the Women's Health Initiative. The results are limited by the observational nature of the data and randomized controlled trials will be the most valid way of clarifying the association between stroke and HRT. METHODS: A community-based, in-person survey of Chinese women was conducted in Seattle, Washington during 1999. The total estimated response rate was 64% and the cooperation rate was 72%. Four hundred and thirty-two women in the 20 ± 79 age-group were included in this analysis. The main outcome measures were a history of at least one previous Pap smear and Pap testing within the last two years. RESULTS: Nineteen percent of the respondents had never received cervical cancer screening and 36% percent had not been screened in the previous two years. Eight characteristics were independently associated with a history of at least one Pap smear: being married, thinking Pap testing is necessary for sexually inactive women, having concerns about embarrassment or cancer being discovered, having received a physician or family recommendation, having obtained family planning services in North America, and having a female provider. The following characteristics were independently associated with recent screening: thinking Pap testing is necessary for sexually inactive women, having concern about embarrassment, having received a physician recommendation, having obtained obstetric services in North America, and having a female provider. CONCLUSION: Pap testing levels among the study respondents were well below the National Cancer Institute's Year 2000 goals. The findings suggest that cancer control interventions for ethnic Chinese women are more likely to be effective if they address multiple barriers and facilitators. Results also indicate that efforts to increase Pap testing rates among Chinese should target the health care providers who serve Asian American communities. We have randomly assigned 252 women with at least 3 months amenorrhea who experience > = 35 hot flashes per week to 3 months of daily Promensil(r) (80 mg total isoflavones), Rimostil(r) (50 mg total isoflavones), or an identical placebo. At randomization and close-out visits, participants health related quality of life is evaluated using the Medical Outcomes Study 36 Item Short Form (SF-36). Non-parametric statistical tests were used as most of the SF-36 scale scores were not normally distributed. RESULTS: Participants' mean (SD) age is 52.3 (3.1) years and they average 3.3 (4.5) years since menopause. Ten percent of the participants are African American and 84% are Caucasian. Forty-two percent had completed college and all but 2 participants had completed high school. At baseline, women in the study scored higher than age and gender equivalent normative data on the Physical Component Summary (PCS mean 51.2, median 52.9) and the Mental Component Summary (MCS mean 51.9, median 54.8). Among the 8 domains of the SF-36, the participants scored lower than population norms on only the bodily pain scale. The weekly hot flash count decreased 36% for the 232 women who have completed the 12 week protocol. The PCS was unchanged over 12 weeks, while the MCS increased 6%. Change in hot flash count was not correlated with change in any of the eight SF-36 domains or with changes in the summary components. CONCLUSION: Since the blind has not been broken, it is not possible to definitively assess the effects of these dietary supplements on health related quality of life. The lack of association between the change in hot flash count and the SF-36 scales suggests that the SF-36 may not be a sensitive measure of menopausal quality of life. PURPOSE: Chronic heart failure (CBF) is costly, morbid, and common. CHF care is often suboptimal because appropriate drug regimens are complex and should be modified according to patients' symptoms. Standard measures of CHF symptoms and health status would be a valuable tool in providing CHF care. METHODS: To assess a measure of CHF symptoms and compare it with CHF-specific and generic health status and satisfaction with care, we studied 497 veterans(80% of those eligible) who had an outpatient diagnosis of CHF, objective left ventricular systolic dysfunction, and being actively treated for CHF by their primary care physicians (PCPs). We administered the Kansas City Cardiomyopathy Questionnaire (KCCQ) (to assess CHF-specific symptoms) and the SF-36 [to obtain generic summary measures: Physical Composite Score (PCS) and Mental Composite Score (MCS)]. Symptoms were used to assign New York Heart Association (NYHA5 functional class. We also assessed patients' satisfaction with their PCP and the most recent prior outpatient PCP visit using locally validated measures; higher scores denote better status or greater satisfaction. RESULTS: Patients' mean age was 69 years; 98% were men and 86% Caucasian. Patients' had moderate to severe limitations (means: NYHA=2.6, PCS=34, MCS=54). As shown in the table, KCCQ symptoms and NYHA class were highly correlated with CHF-specific physical limitations, functional status, and PCS (p < .0001 for each but less so with MCS (p < .005). KCCQ physical limitations and functional status scores and the PCS were highly correlated, but less with the MCS (p < .005 for each). There was no correlation between satisfaction with the PCP and any symptom or health status measure. Satisfaction with the most recent primary care visit was correlated modestly with PCS and MCS (p < .0001 for each) but less strongly with symptoms (p < .05). CONCLUSION: Measures of CHF-specific and generic health status are highly correlated with each other and with severity of CHF symptoms, but they tap different aspects of patient status. But there was little impact on satisfaction with their PCPs or primary care among these veterans with CHF. These health status measures can provide important data to assess and improve the care of patients with CHF. To determine the level of interest in taking tamoxifen for the primary prevention of breast cancer among women over age 50. METHODS: We conducted a cross-sectional, one-time structured interview of women over 50 years old in outpatient geriatrics and women's health clinics at an inner-city academic medical center. Data on age, race, socioeconomic factors, comorbidities using the Charlston comorbidity index, and objective/subjective risk of breast cancer and interest in taking a breast cancer chemopreventive agent were obtained. RESULTS: From the forty-eight (48) participants enrolled in this pilot study, 31 (65%) were black, 17 (35%) white, Hispanic or other, 35 (73%) were > 65 years old. A substantial proportion, 37.5%, of older women were interested in taking preventive medication for breast cancer. Interest in taking a cancer preventive agent appeared to be higher among white women (59% vs 39%), women who have had a breast biopsy (71% vs 35%), and those who believe their risk of cancer warrants taking an agent (93% vs 24%). Of women receiving Medicare, those with additional drug coverage were more willing to take a preventive agent than those without drug coverage (61% vs 38%). Interestingly, those women who paid the highest percentage of their monthly income for medications were also the most interested in taking a breast cancer preventive agent (87.5% vs 36%). No difference was observed by educational level, income, family history, number of comorbidities, objective breast cancer risk, or personal acquaintance with a person with breast cancer. CONCLUSION: A substantial number of women in our pilot study were interested in taking a chemopreventive agent for the primary prevention of breast cancer. Interest appeared to be associated with race, subjective cancer risk, breast biopsy history, and prescription drug coverage. Given our limited numbers, further study is warranted to test the robustness of these associations. To determine the level, pattern, and factors associated with preventive medication use among women over age 50. METHODS: We conducted a cross-sectional, one-time structured interview of women over 50 years old in outpatient geriatrics and women's health clinics at an inner-city academic medical center. Data on preventive medication use, age, race, socioeconomic factors, insurance status, and comorbidities using the Charlston comorbidity index were obtained. RESULTS: From the forty-eight (48) participants enrolled in this pilot study, 31 (65%) were black, 17 (35%) white, Hispanic or other, 35 (73%) were > 65 years old. All (100%) women in this study took at least one preventive agent every day (defined as a multivitamin, calcium, hormone replacement therapy, aspirin, cholesterol lowering drug or antihypertensive (to prevent myocardial infarction or stroke)). Over 55% took at least two preventive medications a day. The frequency of medications used were: antihypertensive (69%), aspirin (50%), multivitamin (50%), calcium (35%), cholesterol lowering agent (31%), and hormone replacement therapy (15%). Women with Medicare alone versus those with Medicare plus additional drug coverage were less likely to take: 1) a cholesterol lowering agent (23% vs 46%); 2) an aspirin (38% vs 61%); and 3) an antihypertensive (69% vs 84%). Lower income women were less likely to take calcium (25% vs 53%) or a multivitamin (43% vs 65%). Older women were less likely to take a cholesterol lowering agent than younger women (0% vs. 38%), even though the same older women were much more likely to be taking an antihypertensive agent (90% vs 62%). Women cited that physician recommendation was the most important factor in deciding to take a preventive medication. Conversely, women cited that cost and insurance coverage were the least important factors. CONCLUSION: There is a high level of interest in taking preventive medications among older women. However, low-cost, proven therapies such as aspirin and calcium are underutilized. Ability to pay appears to drive decisions about medication use despite the fact that women cited cost/insurance coverage as the least important decision-making factor. Women whose Medicare lacks supplemental drug coverage were less likely to take both expensive drugs (cholesterol lowering agents) and inexpensive drugs (aspirin). Lower income women were less likely to take over the counter preparations such as calcium and multivitamins. In addition, the pattern of use of some medications appeared to be inconsistent; the very elderly women in our study, at risk for coronary artery disease as evidenced by their high rate of antihypertensive use, were not taking cholesterol lowering agents. Additional study is needed to test the robustness of our findings and to help understand how women make decisions about their medication use so that inequalities due to age or ability to pay may be addressed. We performed independent duplicate review of each study for both inclusion and data extraction. Global improvement was abstracted as a dichotomous variable. Effect on pain, fatigue and sleep were abstracted as continuous variables at 4 time points (2, 4, 8 and 12 weeks) . Analysis was done using a random effects model. Quality was assessed using the methods of Jadad. RESULTS: Six randomized, placebo-controlled trials were identified of which all were appropriate for some data extraction. Overall the quality of the studies was fair (mean score: 4.7), range 0 ± 8. The odds ratio for improvement with therapy was 3.7 (95% CI: 2.2 ± 6.3). The pooled risk difference for these studies was 0.28 (95% CI: 0.16 ± 0.40), which calculates to 3.6 (95% CI 2.5 to 6.25) individuals needing treatment for one patient to experience symptom improvement. Patients experienced improvement in pain at 2 weeks (Standardized Mean Difference: 0.44, 95% CI: 0.18 ± 0.88) but none at 4, 8 or 12 weeks. There was no significant effect on patient fatigue at any time point. Patients experienced a statistically significant improvement in sleep at all time points with SMD's ranging from 0.35 ± 0.49. Eighty-five percent of patients given cyclobenzaprine reported at least one side effect.\nCONCLUSION: While patients were nearly 4 times as likely to improve if given cyclobenzaprine, the effect was modest, with no effect on fatigue, and improvement in pain only at two weeks. Patients treated with cyclobenzaprine did report modest improvement in sleep at all time points as well as more side effects.\nPREGNANCY AND FAMILY PLANNING SERVICES AMONG INCARCERATED WOMEN. I. Tong 1 , J.G. Clarke 2 ; 1 Brown University, Department of General Internal Medicine, Providence, RI; 2 Brown University, Providence, RI PURPOSE: Among incarcerated women, pregnancies are often unplanned and high-risk due to high rates of psychiatric disease and illicit drug use. Obtaining birth control can be costly and challenging for this underserved population given its lack of medical insurance and poor access to health care. Offering women an intramuscular injection of progesterone or a three-month supply of oral contraceptive pills prior to their release from prison could prevent or delay potentially high-risk and unwanted pregnancies. In addition to pregnancy prevention, birth control provision would also introduce control and stability into the lives of this disenfranchised population. The aims of this study are to obtain pilot data to determine 1) the number of pregnancies among incarcerated women in Rhode Island and 2) the number of pregnancies occurring within 12 weeks of a prior incarceration. METHODS: Charts of all patients with positive (+) serum beta-human chorionic gonadotropin (b-HCG) values from January 1998 to January 2000 were reviewed. Women who were postpartum or who had a recent termination of pregnancy were excluded from the study. Chi-square and t-tests were used where applicable. RESULTS: A total of 129 women and 140 pregnancies were included in the study. Of the 140 pregnancies, seventy-seven (55%) pregnancies occurred in women with prior incarcerations. Of these 77 pregnancies, 38 (27%) occurred within 12 weeks of a prior release. Women who conceived within 12 weeks of their prior release had a higher number of prior incarcerations (5.5 vs. 3.7, p = 0.0043) and a longer mean length of incarceration during their pregnancy (61.4 days vs. 26.7 days, p = 0.0016). Women who were incarcerated for greater than 30 days during their pregnancy had a higher number of incarcerations (5.3 vs. 3.7, p = 0.0043) and a longer stay during the prior incarceration (58.9 days vs. 21.4 days, p = 0.0082). CONCLUSION: Twenty-seven percent of conceptions among incarcerated women occurred within 3 months of a prior release, suggesting that initiating birth control prior to release from prison could prevent a significant number of pregnancies. Women with potentially preventable pregnancies had multiple prior incarcerations and longer lengths of stay, thus providing multiple opportunities for medical intervention and counseling. Family planning services may also provide cost savings as women with potentially preventable pregnancies have longer lengths of stay while pregnant, thus requiring more financial support for prenatal care. The establishment of a family planning program for incarcerated women would not only provide an important service to a medically underserved population, but it may also be a cost-effective measure for the Department of Corrections. Smoking is strongly related to most health outcomes. However the contribution of smoking behavior to mortality benefits in this group has not been investigated. PURPOSE: In a recent national study, women reported poorer adherence to ART than men but socioeconomic differences may explain this finding. We used a pharmacy-based measure to assess adherence in HIV-infected drug users enrolled in New York State (NYS) Medicaid, a population where men and women have similar socioeconomic backgrounds. METHODS: We analyzed longitudinal claims files for 9,557 non-pregnant HIV+ drug users enrolled in Medicaid > 10 months in each year of 1997 ± 98. In persons prescribed ARVs for > 6 months and on 2+ ARVs for > 2 months in each year, adherence was defined as > 90% days covered by 2+ ARVs from the start of combination therapy through the last prescribed ARV(s) in each year, with inpatient days judged adherent. Demographics and clinical conditions (i.e., HIV, substance abuse, and general medical) and health care factors were identified from eligibility/claims files in 1997. Outpatient care in 1997 was categorized as: regular medical care ( > 35% of visits to one provider); regular substance abuse treatment (6+ months with one provider); both; or neither. We created separate indicators for any care in 1997 from: a provider offering HIV-focused care under NYS Medicaid contract; an infectious diseases (ID) specialist; and a psychiatrist. Two logistic regression models were estimated for adherence to 2+ ARVs in 1997 and, to reduce possible confounding due to concurrent assessment of medical care patterns, adherence to 2+ ARVs in 1998. RESULTS: Of 9,557 persons on Medicaid in 1997 ± 8, 2+ ARVs were prescribed for 4,299 in 1997 4,299 in and 4,589 in 1998 4,299 in . In 1997 .4% of 1,564 women and 37.6% of 2,735 men were > 90% adherent while, in 1998, 34.6% of 1,730 women and 40.3% of 2,186 men were adherent. In both models, the adjusted odds of adherence were lower for women than men (adjusted odds ratio (AOR) 0.67 [CI 0.58, 0.77] Uncertainty about the diagnosis and fear of an episode taking place in a crowded or dangerous situation can influence social functioning. Few and only small studies on quality of life of patients with loss of consciousness have been performed. METHODS: As part of the Dutch Fainting Assessment Trial (FAST) we asked consecutive patients presenting to all departments of the AMC with loss of consciousness to participate in this study. All patients were asked to fill in the SF-36, a generic quality of life questionnaire, and the Syncope Dysfunction Scale (SDS), a previously validated disease-specific questionnaire which assesses specific areas of impairment due to syncope and fear or worry about syncopal spells. RESULTS: Of the first 144 patients 125 (87%) were included in analysis of quality of life. Disease specific impairment was moderate, with acknowledged impairment in 32% of listed activities (e.g. driving, physical activities and ability to work). Fear and worry due to syncope was also moderately high at 40 (SD 29) (possible range 0 ± 100, 0 = no fear or worry, 100 = terrified/ all I do is worry). CONCLUSION: Quality of life in patients with episodes of syncope is seriously affected. Knowledge of problems and fears which these patients encounter are vital knowledge for physicians that can improve patient-doctor understanding and guide (practical) medical advice to this group of patients. PURPOSE: African-Americans and Latinos are more likely to have diabetes and experience worse outcomes from diabetes than Whites. We compared rates of outpatient office visits, emergency ward (EW) visits, and hospitalizations for persons with diabetes by race and ethnicity. We studied Medicaid recipients in New Jersey, thus controlling roughly for income and financial access to care. METHODS: From the 1994 New Jersey State Medicaid Research Files, we identified persons with diabetes by ICD-9-CM diagnosis code or by prescriptions for sulfonylureas or insulin. The final database excluded patients who were: < 18 or > 64 years old; enrolled < 3 months on Medicaid; in Medicaid managed care or Medicare; or pregnant. We compared Black, Hispanic, and White patients, eliminating persons with other or unknown race/ ethnicity. We identified visits using CPT evaluation and management procedure codes for outpatient office visits and for EW services, verifying each by place of service documentation. Unique hospitalizations were identified based on date of admission. We used a Poisson regression multivariate model to examine the effect of race on service use, adjusting for age, sex, reason for Medicaid eligibility, selected co-morbidities, and duration of enrollment. RESULTS: Of 11,841 patients, 44% were Black, 15% were Hispanic, and 41% were White; 73% were women; mean age was 47years; and 66% had claims for sulfonylureas or insulin. Of Blacks 51% had no outpatient visits, compared to 47% of Whites and 42% of Hispanics; 35% of Blacks had at least one hospitalization, as did 31% of Whites and 19% of Hispanics. For outpatient visits, compared to Whites, Blacks had an adjusted relative risk (aRR) of 0.84 (95%CI, 0.79 ± 0.89), and Hispanics had an aRR of 1.00 (0.92 ± 1.08). For EW visits, compared to Whites, Blacks had an aRR of 0.95 (CI, 0.89 ± 1.02), and Hispanics had an aRR of 0.68 (CI, 0.61 ± .077). For hospital admissions, compared to Whites, Blacks had an aRR of 1.21 (CI, 1.14 ± 1.28), and Hispanics had an aRR of 0.91 (CI, 0.82 ± 1.01). Additionally aRR's for all three services differed significantly between Blacks and Hispanics. CONCLUSION: Among persons with diabetes, Blacks had significantly lower rates of outpatient visits and higher hospitalization rates than Whites despite similar incomes and identical insurance benefits. Hispanics had similar outpatient visits and admissions rates, but significantly fewer EW visits than Whites. These results suggest that factors other than income and insurance, such as availability of primary care services and physician-patient factors, contribute to differences in utilization by race and ethnicity. 18) . We defined South Asian as patients who state on registration forms that they speak Bengali, Gujarati, Hindi, Punjabi, Nepalese or Urdu in the home. We defined diabetic as any patient with a billing diagnosis of diabetes in two or more visits over the last two years. Europid controls are those who meet the same criteria for a diabetes diagnosis and state that they speak English in the home. We controlled for socioeconomic class by including only patients who use CHA Free Care or a Medicaid variant as their primary insurance. We compared mean HbA1c, mean LDL cholesterol, and prevalence of microalbuminuria between the two populations. We will continue the analysis with the comparison of BP and ophthalmologic changes, and will report statistical tests of significance for all variables. RESULTS: Sixty-eight of 937 (7.26%) South Asian patients were diabetic, compared with 1,698 of 49,206 (3.45%) Europid patients (RR: 2.10). Mean Hb A1c (8.71% vs. 8.47 %), mean LDL-C (124.17 mg/dl vs. 116.44 mg/dl) and prevalence of microalbuminuria (21% vs. 13%) were all higher in the South Asian patients than in the Europid patients CONCLUSION: The South Asian population is at higher risk of type 2 diabetes mellitus and its complications, and may exhibit poorer metabolic control as compared to the Europid population. Our findings warrant increased research into this population to determine the factors that influence observed differences, and to elaborate culturally appropriate interventions for this high-risk population.\nResults SF-36 (scores 0 ± 100; 100 = perfect health) PURPOSE: The complications of type 2 diabetes result in substantial morbidity and mortality. Racial disparities in clinical outcomes of diabetes care are well documented; however, racial variations in disability rates due to diabetes have not been studied. We sought to evaluate the impact of higher complication rates in minorities on health status and rates of disability. METHODS: We used data from the 1992 wave of the Health and Retirement Study (HRS), a national household sample of people age 50 or older in the United States, to provide estimates of prevalence of disability in patients with type 2 diabetes. Logistic regression models were constructed to evaluate predictors of disability. Self-reported disability was the dependent variable. Independent variables included race and other sociodemographic characteristics and the presence or absence of common diabetes complications including stroke, heart failure, coronary artery disease, visual loss, and renal failure. Costs of disability were calculated by applying the median reported income in the HRS to the lost work time associated with disability. Results were extrapolated to the US population using survey weights. RESULTS: In bivariate analyses, we found that African-Americans with diabetes were substantially more likely to be disabled than non-Hispanic whites with diabetes (unadjusted OR = 2.38; 95% CI 1.79, 3.16), as were Hispanic-Americans (OR = 1.54, 95% CI 1.05, 2.26). However, after adjustment for age, education, and gender, only African-Americans were at higher risk of disability (adjusted OR = 2.09; 95% CI 1.78, 2.45). These effects persisted when adjusting for the microvascular and macrovascular complications of diabetes (OR 2.74, 95% CI 1.89, 4.00). There were no significant interaction effects between race and other demographic or clinical variables. In the US population, the total lost income since the onset of disability for African-Americans with type 2 diabetes is $26 billion, which represents an incremental lost income of $12 billion compared to non-Hispanic whites with diabetes. CONCLUSION: African-Americans with diabetes have substantially elevated risks of disability and loss of income compared to non-Hispanic whites. However, the reasons for this disability remain unclear, and do not appear to be solely related to the increased risk of microvascular and cardiovascular complications. More research is needed to better define the causes and effects of increased rates of disability in minorities with type 2 diabetes. PURPOSE: The complications of type 2 diabetes, such as cardiovascular disease, stroke, visual loss, and renal failure, result in high rates of morbidity and mortality. Disability from these complications is substantial and leads to dramatic losses in income and productivity. We sought to quantify the rates, causes, and costs of disability in patients with type 2 diabetes in the United States, which have not been well delineated. METHODS: We used data from the 1992 wave of the Health and Retirement Study (HRS), a national household sample of people 50 and older in the United States, to estimate risks of disability in patients with type 2 diabetes. Self-reported disability was the primary outcome variable; duration of disability was a secondary outcome. Predictors of disability in the population of patients with type 2 diabetes (n=1330) were evaluated using logistic regression models. Independent variables included sociodemographic characteristics, health status, and presence or absence of common diabetes complications including stroke, heart failure, coronary artery disease, visual loss, and renal failure. Costs of disability were calculated by applying the median reported income in the HRS to the lost work time associated with disability; these were estimated for the entire US population using survey weights. RESULTS: 23.1% of patients with diabetes were disabled, compared to 7.7% of subjects without diabetes (unadjusted OR = 3.62; 95% CI 3.14 ± 4.18). Although more patients with diabetes were disabled, they were not disabled for longer than patients without diabetes (2550 days versus 2847 days; p=.1663). In multivariate analyses, the strongest predictors of disability in patients with diabetes were the presence of poor visual acuity (OR = 3.48; 95% CI 1.23, 9.81), congestive heart failure (OR = 3.47; 95% CI 1.69 ± 7.14), coronary artery disease (OR = 2.41; 95% CI 1.26,4.64), or renal failure (OR = 2.41, 95% CI 1.25, 4.62); stroke was not a significant independent predictor of disability. After adjustment for these outcomes, diabetes was still an independent predictor of disability (adjusted OR=2.29; 95% CI 2.61, 3.55). Based upon a median reported income of $26,000 in this sample, the lost income in the current United States cohort of patients with type 2 diabetes is approximately $14 billion annually; the total incremental lost income attributable to diabetes is $9.3 billion annually. To date, the total lost income over the lifetime of the current US diabetes population is nearly $100 billion. CONCLUSION: Type 2 Diabetes is associated with a substantially increased risk of disability, and diabetes-related disability leads to staggering losses in income and productivity. Some, but not all of this increased risk is explained by their rates of microvascular and cardiovascular diabetes complications. Both health care and societal costs should be considered when setting health care policy. 4) and a survey that asked about socio-demographic and clinical characteristics and health status. Hearing was tested with a hand-held audioscope during the study's physical examination. Hearing impairment was defined as better ear threshold at 40dB at the 1kHz or 2kHz frequencies. Logistic regression models were constructed to evaluate the influence of hearing impairment on depressive symptoms. All models were adjusted for patient socio-demographic and clinical characteristics, health status, and system of care. RESULTS: There were 484 study participants (response rate 68%). Mean age was 75+6 years; 47% were women; 52% were white, 20% Hispanic, 18% African-American, 6% Asian/Pacific Islander, and 5% Other/Multiracial; 48% had annual income under $20,000 per year; 9% received Medicaid; 24% had not graduated from high school; and 24% were enrolled in FFS. Depressive symptoms were reported by 25%. Hearing aids were worn by 9%, 17% had hearing loss at 1 kHz, and 23% demonstrated hearing loss at 2 kHz. In the multivariate analysis, hearing impairment was significantly associated with depression (OR, 1.8; P=0.05). Although there was an association between depression and hearing impairment at a frequency of 2kHz, it was not statistically significant (OR=0.67; p=.09). Other characteristics that were significantly associated with depression were higher comorbidity score (OR=1.2; p=.005); worse physical well-being (OR=1.04; p=.009); low income (OR=1.8; p=.02); and male gender (OR=1.6; p=.05). CONCLUSION: Hearing impairment is significantly associated with symptoms of depression among older persons with diabetes, a group that is already at high risk for sensory impairment due to visual loss and diabetic neuropathy, and points to a potentially correctable cause of depression. BACKGROUND: Although traditional teaching has insisted the sensitivity and specificity of a diagnostic test should not change as the pre-test probability of disease changes, several authors have suggested this may not be true in practice as patients are referred from a setting of undifferentiated problems with lower pre-test probabilities in primary care to tentative diagnoses with higher pre-test probabilities in secondary and tertiary care. The purpose of this paper is to demonstrate how and understand why sensitivity and specificity change as a patient population is divided by the physician's decision whether to refer. METHODS: Using MEDLINE, a collection of published studies with various inclusion criteria was assembled{216}. Studies were included when a) the study provided enough information about the studied population to determine its position along the referral algorithm b) the article provided history or physical examination component data that could be combined with data from other articles, and c) the article provided enough information to construct 2  2 tables to calculate sensitivity and specificity for those components of the clinical examination. The studies were divided into two groups, depending on whether the patient population was studied before or after referral for surgery. Group A consists of those studies evaluating all patients in whom appendicitis was considered as a diagnosis, regardless of whether they were referred for surgery. Group B consists of those studies evaluating all patients who had been referred to the operating suite for suspected appendicitis. RESULTS: In the metanalysis of the two-by-two table data, there were several trends observed as the population studied changed from group A to group B. Sensitivity tended to rise and specificity tended to fall across most of the exam components reported. These changes were found to be statistically significant for the specificity of migration and rebound pain. When all components of the examination were combined, the fall in specificity was statistically significant for the aggregate data. CONCLUSION: This study provides the first evidence that sensitivity and specificity change in populations of patients evaluated at different positions along the referral spectrum. These changes in the operating characteristics of the finding on clinical examination are important to recognize. These changes have implications for teaching the clinical examination as well as interpreting their presence or absence in varying patient populations. For example, a finding that is felt to be non-specific in tertiary care settings may be quite specific in primary care settings. To determine the rate of hip fracture and the risk factors associated with hip fractures in disabled elderly persons who choose to live in the community rather than a nursing home. METHODS: We assessed potential risk factors in 5,187 persons who enrolled 1/90 ± 12/97 in 12 nationwide sites of the Program of All-Inclusive Care for the Elderly (PACE), which provides comprehensive care to nursing-home-eligible men and women living in the community (mean age = 79, 71% women, 49% white, 47% with dementia). Functional status on enrollment was assessed by each site's nursing staff and included degree of dependence in walking and 5 Activities of Daily Living (ADL): bathing, dressing, toileting, transferring and eating. Cognitive status was assessed using a 10-item mental status questionnaire (SPMSQ). Demographics and comorbidities were also recorded on enrollment. The main outcome measure was new hip fracture that resulted in hospital admission. Average follow-up was 2 years. RESULTS: A total of 238 hip fractures (4.6%) occurred during follow-up. The rate of hip fracture was 2.3% per person-year. Four independent predictors of hip fracture were identified using Cox proportional hazard analysis: age !75 years (adjusted hazard ratio (HR) = 2.0, 95% CI 1.4 ± 2.8); white ethnicity (HR= 2.1, 95% CI 1.6 ± 2.8); ability to transfer independently from bed to chair (HR= 3.0, 95% CI 1.2 ± 7.2); and SPMSQ errors !5 (HR= 1.6, 95% CI 1.3 ± 2.1). Several risk factors, including gender and history of stroke, were not independently associated with hip fracture after adjustment for the above risk factors. PURPOSE: Missed clinic visits interfere with good medical care and have adverse effects on clinic efficiency. Explanations for patient non-compliance in keeping appointments generally emphasize practical physical issues such as finance, transportation and socioeconomic ones. We evaluated other factors, as well, in a community hospital's medical clinic in order to define ways to improve compliance with appointments. METHODS: A 10-item questionnaire covering demographic, medical conditions, socioeconomic and support conditions, missed visits, and reasons for missed visits, were filled out by 24 clinic visitors. All charts were separately analyzed for kept and missed appointments in the previous year. 72 patients were compliant ( < 20% appointments missed) and 36 were noncompliant ( > 30% appointment missed; range=30.7 ± 85.7% median=42.3%). RESULTS: Contrary to expectations non-compliant and compliant patients did not differ by medical insurance, availability of social support, transportation, education, age, or ethnicity. The sole significant difference in medical conditions was the presence of depression. The odds ration for depression was 3.18 (95% confidence interval(1.7 ± 8.7); p=.016) times greater among noncompliers, 39% of whom admitted to depression, than compliers. The non-compliers were aware of their noncompliance. The most common reason cited for missed appointments were forgetting (n=13), inadequate money for transportation (n=12), personal problems (n=9), and too long a wait (n=9). However, these did not differ significantly from the reasons cited by the others. CONCLUSION: The most striking, and we believe important, finding in our survey was the significant association of depression with noncompliance far outstripped all other characteristics, including financial and social ones. This observation suggests a very important problem, as well as a potential solution to the problem of noncompliance. We conclude that it is essential to address the possibility of depression actively in such patients as both a therapeutic goal in itself, and a potential means to improve compliance with medical care. PURPOSE: Although intentional weight(wt) loss is recommended to many patients, whether it improves survival, particularly among men, is unclear. We examined the association between intentional wt loss and mortality in a national sample of men. METHODS: We used data from the 1989 National Health Interview Survey where 20,131 adults (91% response rate) were asked about sociodemographic and basic health information including self-reported health and smoking status, health care utilization, height, current and maximum(max) wt, and whether they were trying to lose wt. Date of death was obtained by linking to the National Death Index through 12/31/95. We determined wt loss by subtracting wt in 1989 from max wt. We classified wt loss as intentional if respondents reported that they were trying to lose wt in 1989; otherwise, it was considered of unclear intention. We used Cox models to examine if intentional wt loss was associated with time to death. We limited our analyses to men only to avoid misclassification and confounding due to pregnancy. We weighted all percentages to reflect population estimates and adjusted standard errors to account for the complex sampling. RESULTS: Among 8395 eligible men, 604 had died by the end of follow-up. Among men who lost wtintentionally,thosewholost5 ± 10lbshadthelowestunadjustedmortality(2.3%)whencompared to those who lost up to 5 lbs (5.2%), p=.004. After adjusting for age, max. BMI, race, education, smoking, health status, number of doctor visits, hospitalized days, and days in bed, and intent to lose wt, intentional wt loss was associated with improved survival among men who lost 5 ± 10 lbs (see Table) . The benefits of weight loss were most consistent among men with BMIs of 25 ± 29.9 kg/m 2 . CONCLUSIONS: Modest intentional weight loss appears to be associated with mortality benefit among men. The benefit of greater weight loss is less clear.\nACUTE PANCREATITIS. Z. Weikang 1 ; 1 Union Hospital, Wuhan, Hubei PURPOSE: Glycoprotein ulinastatin is a newly discovered proteinase inhibitor extracted from human urine that can restrict the activity of pancreatin. This study was designed to evaluate its clinical effect on patients suffering from acute pancreatitis.\nMETHODS: 257 patients were divided into two groups. The patients in the experimental group (132 patients) received ulinastatin (300,000U/day for two weeks). The patients in the control group (125 patients) were treated with routine protocol (anisodamine 20mg/d). Both groups received similar supportive treatments. Observations of clinical symptoms and signs were made on daily basis and measurements of serum amylase, hepatic/renal function, and TNF-I à A  were performed at the 3-day interval for three weeks. RESULTS: The results showed that the clinical symptoms and signs such as fever, abdominal distention and pain, belch, and constipation of the experimental improved faster than those of the control group. Analyses of serum amylase, glutamic pyruvic transaminase (GPT), creatinine, and TNF-I à showed that all these indexes were significantly lower in the treatment group than those of the control group (p < 0.01) at the 6th, 9th and 12th day, even though no such difference was observed at the 3rd day. CONCLUSION: Acute pancreatitis patients treated with ulinastatin recovered significantly faster than those patients treated by routing protocol. Thus, ulinastatin is an effective medicine for treating patients with acute pancreatitis. PURPOSE: Pharmacists may improve patient outcomes by engaging in pharmaceutical care (PC) activities (e.g., monitoring symptoms, counseling about medications, helping resolve drugrelated problems). We conducted a randomized trial in 36 community drug stores to evaluate the effectiveness of PC for patients with asthma or chronic obstructive pulmonary disease (COPD). METHODS: 36 drug stores were divided into 12 clusters of 3 stores matched on key criteria. Within each cluster, stores were randomized to PC or to 1 of 2 control groups.\nPharmacists in the PC group were provided with: recent patient-specific data [e.g., peak expiratory flow rates (PEFR), acute exacerbations resulting in hospitalization or emergency department visit] displayed on a computer in their stores; materials to facilitate implementation of PC; and training on the PC program. Customers of these stores were eligible if they: filled a prescription for a breathing medication during the past 3 months; reported having asthma or COPD; were 18 years or older; filled !70% of their prescriptions in a single study store; were able to be interviewed; and resided in the community. Primary outcomes (PEFR, health-related quality of life, acute exacerbations) were assessed at 6 and 12 months. We present 6-month interim data; differences at the p < 0.05 level are considered significant. RESULTS: Asthma subjects (N=660) had a mean age of 45 years, 80% were Caucasian, and 82% female. COPD subjects (N=453) had a mean age of 62 years, 86% were Caucasian, and 66% female. At the 6-month interim analysis, the PC group did not have significantly better health-related quality of life or PEFR than either control group. The proportion of patients in the PC group with an acute exacerbation (11.4%) was not significantly different from either control group (9.4%, 9.1%). Neither was their a difference in months until the first acute exacerbation between the PC group (2.91.8) and either control group (3.11.6 and 2.31.7). CONCLUSION: Our interim (6-month) analyses found that an intensive PC program in community drug stores had no significant effect on our primary outcomes. We are currently examining long-term (12-month) outcomes, secondary outcomes (e.g., patient satisfaction), and dose-response effects. Recent studies report inconsistent findings on the changes in the incidence of hospitalizations for ischemic heart disease. These reports have relied primarily on hospital discharge data. Preliminary data suggest that a significant percentage of patients suffering acute myocardial infarction in rural communities are transferred to urban centers for care. Patients transferred to a second hospital may be counted twice for one episode of ischemic heart disease. The purpose of this study is to describe the impact of double counting and transfer bias on the estimation of incidence rates and outcomes of ischemic heart disease, specifically acute myocardial infarction, in the U.S. METHODS: Methods: Analysis of state hospital discharge data from Kansas, Colorado, Nebraska, Arizona, New Jersey, Michigan, Pennsylvania, and Illinois for the years 1995 ± 1997 for patients reported to have suffered ischemic heart disease (ICD9 codes 410 ± 414, 786.5). We developed a matching algorithm for hospital discharges to determine patients counted twice for one episode of ischemic heart disease. We validate the matching algorithm with a sensitivity analysis and chart audit. RESULTS: We found that double count rates range from 10 ± 15% for all states and have increased over the past 3 years. Moderate sized rural counties had the highest estimated double count rates at 15 ± 20% with a few counties having estimated double count rates a high as 35 ± 50%. Older patients and females were less likely to be double counted (p < .05). CONCLUSION: Conclusions: Double counting patients has resulted in a significant overestimation in the incidence rate for hospitalization for acute myocardial infarction. Correcting for this double counting reveals a significantly lower incidence rate and a higher inhospital mortality rate for acute myocardial infarction. Transferred patients differ significantly from non-transferred patients, introducing significant bias into myocardial infarction outcome studies. Double counting and transfer bias should be considered when conducting and interpreting research on ischemic heart disease, particularly in rural regions. Jan 1, 1991 and June 30, 1996 for either PE or DVT were analyzed. For each record, the first hospitalization with a principal diagnosis of DVT (N= 51,233) or PE (N = 20,017) was considered the index hospitalization. Demographic and clinical characteristics (ICD-9 codes) were compared, as were the 6-month incidences of re-hospitalization for recurrent PE or DVT, bleeding and death. Surgery defined as operation within 61 days. RESULTS:\nAll differences p < 0.001 except: bleed Ð 6 month follow-up p= 0.05; death Ð 6 months p=0.52, NS. In multivariate logistic modeling, odds ratios (ORs) for recurrent VTE in 6 months: All differences significant with p < 0.001. Each race compared to others combined.\nAll differences p < 0.001 except: bleed Ð 6 month follow-up p= 0.05; death Ð 6 months p=0.52, NS. In multivariate logistic modeling, odds ratios (ORs) for recurrent VTE in 6 months: CONCLUSION: Patients with PE (versus DVT) were less likely to be Latino, more likely to be African-American, and more likely to have cardiopulmonary disease or recent surgery. Recurrent VTE manifested as PE was strongly associated with an initial diagnosis of PE, and conversely, recurrent DVT was strongly associated with an initial diagnosis of DVT. Coupled with recent data indicating that the prevalence of factor V-Leiden among patients with PE is significantly lower compared to patients with DVT, our findings suggest that there are undefined genetic or acquired factors that effect the phenotypic expression of VTE. complications after specific kinds of surgeries has been intensely studied, few studies have compared the 3-month incidence of VTE after different surgeries. METHODS: We analyzed the California linked discharge records of 2,159,747 patients hospitalized between Jan 1, 1991 and Oct 1, 1996 who underwent one of 75 selected surgical procedures on the first or second hospital day, and determined the 3 month incidence of rehospitalization for VTE. Data were stratified by age ( > 65 and < 65) and malignancy (present within 6 months or absent). A multivariate model was developed to compare the 3 month incidence of VTE using inguinal hernia repair (ICD-9-CM = 53.0) as the referent surgery, adjusting for age, sex, presence of malignancy, Charlson Index > 1, race (Asian vs. non-Asian), and prior VTE ( < 6 mo.). RESULTS: The table below shows the VTE incidence in a analysis restricted to age > 65 yrs, no cancer, > 10,000 procedures. In a logistic model that included all surgeries, parameters significantly associated with VTE included age > 65 (OR = 1.5, CI 1.4 ± 1.6), malignancy (OR = 2. (83%) returned questionnaires. Mean patient age was 42 years, 17% were female, 71% were white, and 66% were gay. In bivariate tests, general communication (r = 0.12, p = 0.003), ADH dialogue (r = 0.19, p < 0.001), overall satisfaction (r = 0.10, p = 0.01), willingness to recommend (r = 0.12, p = 0.006) and MD trust (r = 0.10, p = 0.01) were significantly associated with ADH. MCS (r= 0.226, p < 0.001), PCS (r = 0.160, p < 0.001), and age (r= 0.16, p = 0.0003) were significantly associated with ADH. Blacks reported worse ADH than whites (p < 0.05). In multivariable models, after controlling for MCS, age, and race, ADH dialogue was the only variable significantly associated with ADH (R2 = 0.12, p = 0.001). CONCLUSION: Only ADH dialogue was independently associated with adherence. Optimizing patients' ADH probably requires detailed and specific information gathering and problem solving directed at specific dosing times and/or specific medications. These data do not appear to be an automatic biproduct of good general MD-PT communication. The Surgeon General's Report on Oral Health highlights the inadequate oral health care provided to the vulnerable population of homebound older adults. Cost of dental care and transportation challenges serve as major barriers to dental care in this population. We sought to examine the impact of removing both cost and transportation barriers on dental care utilization in urban homebound older adults. METHODS: 225 older adults participating in a home and community based waiver program and/or a home-delivered meals program were invited to receive free nutritional and oral health assessments. Individuals with both poor oral health and nutritional needs were offered free dental care including free transportation. A dental hygienist and nurse coordinator made appointments on behalf of patients, provided appointment reminders and arranged transportation. RESULTS: Mean age of participants was 81.6. 81% were female, 53% were African American. Of 159 subjects who completed both nutritional and oral health assessments, one-third were eligible for free dental intervention and transportation. Of those offered free dental care, 80% agreed to receive care. Of those who agreed to receive care, 14% did not keep any dental appointments, 19% kept some of their dental appointments but did not complete their dental intervention, 67% fully completed their dental interventions. The only significant barrier to completing the recommended dental intervention was depression score (p=.03). Even when functional status and Mini Mental State Examination scores were adjusted in the model, depression remained an independent predictor for not completing the recommended dental care visits. CONCLUSION: When cost and transportation difficulties are removed as barriers to dental care, it is possible to complete needed dental interventions in a significant number homebound adults. Public policy changes would be needed to provide an infrastructure to facilitate this process. Depression remains an independent risk factor for incomplete interventions. PURPOSE: To avoid erroneous results in the estimation of the sensitivity and specificity of diagnostic tests, it has been recommended that a broad spectrum of disease be represented in the study population when evaluating the efficacy of a test. However, the actual effect of severity of disease on these indexes has not been evaluated on a clinically relevant population. The objective of this study was to assess the effect of the severity of disease on the diagnostic performance of a positive PPD and upper lobe disease on chest radiograph (defined as infiltrates and/or consolidation above the third rib posteriorly) for the diagnosis of pulmonary tuberculosis (TB). METHODS: Seventy five patients with positive sputum culture for TB and 75 subjects who were considered at risk of TB but whose sputum tested negative for TB were identified from the hospital's TB registry. Patients were divided into two groups, severe disease and non-severe disease according to the presence or absence of specific criteria that are known to be associated with worse clinical outcomes. Patients with TB were classified as having severe disease if the sputum smear tested positive for acid-fast bacilli. Patients without TB (most of which had pneumonia) were classified as having severe disease if they were older than 65 years of age, had a temperature greater than 408C, or had pleural effusion on chest radiograph. The sensitivity and specificity of a positive PPD and upper lobe disease were determined independently among the group of patients with severe and non-severe disease. RESULTS: The sensitivity of upper lobe disease for the diagnosis of TB was 41.6% and 21.6% among those with severe and non-severe disease respectively (P = 0.03). The specificity of this radiologic finding increased from 77.2% among patients with severe disease to 94.7% among those with non-severe disease (P = 0.05). Conversely, the sensitivity of PPD was lower among those with severe disease (22.2%) compared to those with nonsevere disease (42.0%, P = 0.05). The specificity of PPD was not statistically significantly different among this two groups (100.0% vs 89.0%, severe vs non severe disease respectively; P = 0.10). CONCLUSION: The spectrum of disease significantly modified the sensitivity and specificity of a positive PPD and upper lobe disease on chest radiograph for the diagnosis of TB. Interestingly, the effect of the severity of disease was not in the same direction in the two tests evaluated. This potential source of error in measuring test performance should be considered when translating the results of research studies to the clinical practice. Variables (and beta weights) negatively (p05) associated: age in decades (À0.9), being female (À2), being employed, retired, homemaker or student (À0.89) compared to being unemployed, having friends who provide encouragement when faced with a difficult situation (À.76), and frequency of physical problems interfering with social activities (À.26). Family CAGE, race, marital status, education level, having a close friend, losing a friend or relative, frequency of contact with those closest to you, self-report number of visits to a doctor in the past year were not significantly associated with AUDIT score. CONCLUSION: The associations presented do not necessarily suggest causality. As expected, being male, younger, having friends who are problem drinkers, and higher CAGE scores had the highest association with AUDIT scores. Counter to our a priori predictions, frequency of participating in spiritual activities and having a friend willing to help were associated with higher AUDIT scores. Interestingly, frequency of emotional problems is positively associated with AUDIT scores while frequency of physical problems is inversely associated. Also unexpected was the lack of association between Family CAGE and AUDIT score. Further investigation may help explain these unexpected findings. Patients were asked about their preference for first seeing their PCP or a specialist for 3 clinical scenarios and the health problem for which they sought medical care on the day on which they were surveyed. To identify the independent predictors of preferring to see a specialist, we combined patients' responses to the 3 scenarios and health problem and performed a multiple logistic regression, adjusted for clustering at the patient level using the Huber-White method. We also performed analyses separately for each of the 3 scenarios and the presenting health problem. RESULTS: The proportion of patients who preferred to see a specialist first for the 3 scenarios was 13% for chest pain, 15% for knee pain, 11% for a rash, and was 8% for the presenting health problem. In the multivariable analysis, blacks were less likely than whites to prefer a specialist (OR=0.45, p < 0.0001). Compared to those with fee-for-service insurance, those with VA coverage and Medicaid insurance were less likely to prefer a specialist (OR=0.55, p=0.04 and OR=0.42, p=0.01, respectively). Those who preferred a specialist had less trust in their PCP (OR=1.6, p < 0.0001), had less confidence in their PCP's ability to diagnose and treat their problem (OR=2.6, p < 0.0001), were more certain about their diagnosis (OR = 1.4, p < 0.0001), and perceived the health problem as being more serious (OR = 1.2, p = 0.02). Non-significant predictors included age, gender, employment, education, income, managed care insurance, health status and several attitudes including perceived susceptibility to illness, entitlement and assertiveness. These results were similar across the 3 scenarios and the presenting health problem. CONCLUSION: Controlling for other sociodemographic characteristics, health beliefs and attitudes, blacks were much less likely to prefer care from a specialist. Though blacks have generally had worse access to care, lower quality of medical care, and worse health outcomes, their satisfaction with medical care has not consistently been lower than that of whites. Our results support the hypothesis that differences in expectations for care might explain this incongruity and raise the question about whether differences in expectations might contribute to disparities in health care quality and outcomes. PURPOSE: Most providers say that they withhold protease inhibitors (PIs) from HIV-infected patients whom they believe will be non-adherent. We hypothesized that this prescribing attitude might influence patients' access to PIs and sought to determine whether this prescribing attitude explained why African Americans tend to receive PIs later than whites. METHODS: In the HIV Cost and Services Utilization Study (HCSUS), we prospectively studied a nationally representative sample of HIV-infected patients (n=2864) and their providers in the U.S. We examined data for those with completed provider and patient surveys (398 providers taking care of 1892 patients (66% of original patient cohort)). We asked providers whether they prescribe PIs to patients whom they believe will be non-adherent. Patients were followed over a 2-year period and were asked at baseline and both follow-up surveys whether and when they began using PIs. Using parametric survival models, we determined the impact of providers' PI prescribing attitude on time to first PI use and on racial differences in time to first PI use. RESULTS: Contrary to our hypothesis, having a provider who considers adherence before prescribing PIs (More Restrictive) predicted earlier PI use, adjusting for provider characteristics only (e.g. HIV knowledge and type of specialty) (p = 0.01). However, providers' PI prescribing attitude was not associated with time to PI use after additionally adjusting for patient CD4 count, symptoms, age, gender, HIV risk factor, education, income, insurance and perceived access to care. After adjusting for patient and provider characteristics, the median time to first PI use was 74.2 days later for African Americans than for whites (p = 0.001). This racial difference in time to PI use was similar for an individual with a More or Less Restrictive provider (adjusted racial difference in time to PI use was 74.7 and 73.3 days respectively, p > 0.20). CONCLUSION: Providers' PI prescribing attitude was not associated with patients' access to PIs. African Americans received PIs later than whites, but providers' PI prescribing attitude did not explain this racial disparity. Future studies will need to examine whether other patient or provider characteristics better explain why African Americans receive PIs later than whites. PURPOSE: Though blacks have greater mortality rates than whites, it is not known which diseases contribute most to this disparity. We examined mortality rates as well as years of potential life lost (YPLL), which is a more sensitive measure of loss of life due to premature death. METHODS: To examine nationally representative samples of adults in the U.S., we used data from the 1986 , 1988 and 1990 for participants greater than age 25 with corresponding National Death Index data on each cohort through 1995. YPLL was calculated as the difference between the age at death and the maximum years that a person could have lived, which we set at age 85. Mortality rates and YPLL due to all and specific causes of death were calculated for blacks and whites and standardized to the age, sex, and educational distribution of the total U.S. population. We then calculated the percent contribution of racial differences in YPLL from specific causes of death to racial differences in YPLL from all-cause mortality. RESULTS: Adjusted for age and sex only, YPLL due to all-cause mortality was 0.62 years/ person greater for blacks than for whites, averaging across the 3 NHIS cohorts. However, this disparity disappeared after adjusting for education in addition to age and sex (black-white difference was À0.08 years/person). Age and sex-adjusted YPLL was greater among blacks compared to whites for all of the specific causes of death that we examined and for all 3 NHIS cohorts. The contribution of specific causes of death to the racial disparity in YPLL was 25.6% for infections, 14.0% for cancer, 6.1% for diabetes, 4.6% for stroke, 4.0% for ischemic heart disease, 3.3% for renal disease, 3.1% for lung disease, and 4.0% for accidents, homicides, and suicides combined. An analysis of mortality rates revealed similar results, with the exception that cancer contributed most to the racial disparities in mortality (22.6%) followed by infectious diseases (14.6%). For deaths due to infections, the black-white mortality rate difference was more pronounced in those younger than 60 years old compared to those 60 years or older (6.0 vs. 0.6 deaths/10,000 person-years, respectively). In contrast, the racial disparity for deaths due to cancer was much greater in those age60 years than in those age < 60 years (black-white mortality difference=20.2 vs. 3.5 deaths/10,000 person-years, respectively). CONCLUSION: YPLL was greater among blacks than whites, though differences in socioeconomic status (using education as a proxy) appear to account for this disparity. Mortality rates and YPLL yielded slightly different results because YPLL accounts for the age at death and racial disparities in death due to infections were more pronounced at younger ages as compared to deaths from cancer. Future efforts to reduce the racial disparity in mortality may have the greatest impact if focused on infectious diseases and cancer, which we found contributed the most to the difference in YPLL and mortality rates between blacks and whites. OBJECTIVES: Multiple studies indicate that patient compliance with cardiovascular medications is poor. One possible explanation is that patients perceive medication use as difficult. The goal of this study was to assess characteristics of patients who found medication use difficult, including the physician-patient relationship, health beliefs, and socioeconomic factors. METHODS: We prospectively surveyed 273 White and African American patients who underwent nuclear imaging studies graded as positive for cardiac ischemia at 5 VA hospitals (Houston, Atlanta, St. Louis, Pittsburgh, and Durham). Of these, 33 (12.1%) patients rated medication use as difficult. Difficulty was assessed by asking patients``How bothersome is it for you to take your pills for chest pain, chest tightness, or angina as prescribed?'' RESULTS: Patients who found medication use difficult were less likely than those who did not find it difficult to agree that they were satisfied with their current treatment (59.4% vs. 77.0% respectively, P = 0.03) and that their doctors used words they could understand (68.8% vs. 88.3%, P=0.003). Among patients who found medication use difficult, there were also trends towards having less trust in their doctor's judgement about their medical care (78.8% vs. 87.5%, P = 0.17) and feeling that they were less able to get to know their doctor (59.4% vs. 70.6%, P = 0.20). There were no differences between patients who found medication use difficult and those who did not in the perception that their doctors were well qualified to manage their medical problems ( (SD 10.4) ]. In addition, 35/50 faculty and residents that provide DM care in the clinic completed physician surveys. Variables were coded as pt, provider or system barriers if 30% or > of respondents endorsed an item as a problem some or all of the time. Provider and system barriers were also identified from chart review when 30% or > of data was missing. RESULTS: Thirty-five (35) variables and 63 barriers to DM care were identified. Pt barriers (25.3%)included adherence with medication, glucose monitoring, diet and exercise. Noncompliance with appointments was affected by transportation, childcare, and work-related issues. Costs of care, pt literacy, lack of family support and other pt priorities were also cited as pt barriers. Provider barriers (45.2%)included knowledge of/agreement with ADA guidelines, reimbursement issues, prescribing uncertainty, and provider perceptions of pts with DM. System barriers (33.3%) included lack of insurance, DM care costs, reimbursement problems, lack of DM educators/nutritionists, poor record-keeping, difficulty obtaining lab data and provider communication issues. Chart review documented: HBA1c -67%, UA -52%, urine microalbumin -20%, total cholesteral -23.5%, LDL -11.8%, foot exam -25%,funduscopic exam -19%, influenza and pneumonia vaccine -18% and 7%, respectively. CONCLUSION: We found that provider and system barriers outweighed patient barriers to DM care in this high-risk population. In addition to pt empowerment, DM improvement programs must address provider and system obstacles that impede optimal DM management. Further clarification of specific barriers and their impact on diabetes care is needed, particulary in AA, underinsured and/or high-risk populations. Adult patients who had been cared for by the same physicians for more than 3 months and who had at least 4 visits during this period, were selected for this study. The patient trust of physician ratings were obtained by self-administered questionnaires, the Trust in Physician Scale ( modified by the Stanford Trust Study Group). In addition to patient trust, we obtained a General Trust Scale (Yamagishi, Japan), patients' demographic data, the lengths of the patientphysician relationships, the reasons for the patients' choice of physicians. 275 patients answered the questionnaire to rate 11 different physicians during their visits. Multiple regression analysis was used for this study. RESULTS: The Trust in Physician scale (5 Likert scales: 1 = totally disagree to 5 = totally agree) were transformed to a 0 to 100 scale, and it showed high internal consistency (Cronbach's alpha = .82) for Japanese patients. Mean scores of male and female were 86.3 (SD: 10.3) and 85.9 (SD: 10.5) respectively. The Trust in Physician scores were strongly associated with the General Trust scores (p = .017), the reasons for the patients' choices (p = .037) and the satisfaction with care (p < .001) after adjusted by patient characteristics. However,the Trust in Physician scores were not statistically associated with gender, age, education levels and the lengths of the relationships. CONCLUSION: The patient trust, a measure of the health care seeking behaviors, was associated with the societal trust in general in our study. In addition, the patient trust was a useful concept to assess the relationship between patient and physician, especially the satisfaction with care. Normal range (18.5 ± 24.9 kg/m 2 ) was the referent category in the analyses. RESULTS: Participants had a mean age of 54.6 years (SD=5.6); 69% were white, 29% were black and 94% were male. The distribution of BMI was: < 18.5 kg/m 2 (n=16, 1.3%), 18.5 ± 24.9 kg/m 2 (n = 254, 20%), 25 ± 29.9 kg/m 2 (n = 531, 42%), 30 ± 34.9 kg/m 2 (n = 308, 25%), 35 ± 39.9 kg/m 2 (n = 112, 8.9%) and > = 40 kg/m 2 (n = 32, 2.6%). Mean SF-36 subscale scores for the sample were lower than U.S. norms by a mean of À18.7 points (range: À32.9 on Role-Physical to À3.5 on Mental Health). After adjusting for covariates, individuals with BMI > = 40 kg/m 2 had significantly lower scores compared with normal weight individuals on the Role-Physical ( = À18.8, p=0.008), Vitality ( = À12.5, p=0.001) and Social Functioning ( = À9.3, p=0.017) subscales ( represents the deviation in mean score from the referent BMI group (3.9 vs. 2.8) than their male counterparts. Of the 652 calls during the study, female patients called more frequently than male patients (p < .001), and female patients generated more total calls than male patients (p < .001). Also, female physicians had significantly more calls and callers than male physicians did (p < .001). Physician age, specialty, and years in practice did not affect calling rate. CONCLUSION: Compared to male physicians, female physicians experience more demand for on-call service. In addition to slower advancement, factors intrinsic to their practice may contribute to higher reported job stress among female physicians. Physician gender may be an important factor when considering severity adjustments to determine reimbursement schedules.`W E'RE JINXED!'' -ARE RESIDENT'S FEARS FOUNDED? A.C. Ahn 1 , B. Nallamothu 2 , S.K. Saint 2 ; 1 Massachusetts General Hospital, Jamaica Plain, MA; 2 University of Michigan, Ann Arbor, MI PURPOSE: Many house officers believe that there is bad kharma bestowed on them when supportive remarks are expressed during call days. Remarks such as``Hope you have a great call!'' or``Hope you have no more admissions!'' are interpreted as ill-fortuned greetings that may do more harm than good. The purpose of this study is to determine whether this fear of being``jinxed'' is based on objective findings or mere psychological superstition. METHODS: We performed a randomized controlled trial using senior medical residents at 2 university-affiliated hospitals. On the morning of a call day, each resident was randomized by selecting either a Jinx (``You will have a great call day!'') or No Jinx message (i.e. blank form) from a collection of unlabeled envelopes. Primary outcomes measured included the number of total admissions and hours of sleep for each resident, and the subjective level of difficulty of the resident's call day (on a scale from 1 to 5 with 1 being easiest and 5 the most difficult). Some residents were randomized more than once during a call month. Student t-tests assessed for differences in outcomes between the Jinx and No Jinx groups, with robust variance estimates used to adjust for the effects of clustering at the resident level. RESULTS: Thirty-three Jinxes and 36 No Jinxes were performed on 30 residents. No statistically significant differences were seen between the 2 groups in important baseline characteristics such as a priori level of superstition or admitting service (i.e., general medical service, cardiology service). Relative to those in the No Jinx group, residents who were jinxed had significantly fewer admissions (5.1 versus 6.2;P=0.021), more hours of sleep (3.8 versus 2.9; P=0.034), and a lower subjective level of difficulty (2.9 versus 2.5;P=0.040) during their call day. CONCLUSION: There is no evidence that offering encouraging remarks to residents on a call day``jinxes'' them. Therefore, such remarks may be given safely to residents without fear of causing more admissions or less sleep, or worsening a call day's level of difficulty.\nGAMING THE SYSTEM -PUBLIC PREFERENCES THAT DOCTORS PLAY? G. Alexander 1 , R. Werner 1 , A. Fagerlin 2 , P. Ubel 2 ; 1 University of Pennsylvania, Philadelphia, PA; 2 Ann Arbor Veterans Affairs Medical Center, Ann Arbor, MI PURPOSE: Efforts to contain rising costs have lead third party payers to restrict access to some healthcare services. Previous research suggests that the``hassle'' of the appeals process influences physicians' sanctioning of deception to obtain medically necessary services. However, little is known about whether the public supports such deception, and if so, whether this support is sensitive to the burden of the appeals process. METHODS: A clinical vignette was administered to 700 jurors and to a randomly selected national sample of 1617 physicians. Vignettes differed by condition severity (severe angina vs. moderate low-back pain), likelihood of successful appeal (95% vs. 50%), and length of appeals process (5-10 minutes vs. 1 hour). Participants were asked whether in response to a restriction on medically necessary healthcare the doctor in the vignette should (1) appeal the restriction, (2) accept the restriction, or (3) misrepresent the facts in order to obtain the desired service. RESULTS: Overall, the public was more likely than physicians to sanction deception (26% vs. 11% respectively, p < .001), and less likely to support accepting (4% vs. 12%, p < .001) or appealing (70% vs. 77%, p < .001) the restriction. Sanctioning of deception was greater among the public than physicians regardless of the vignette used. The public's support of misrepresenting (vs. appealing) was greater with a lower likelihood of successful appeal (Odds ratio [OR] 1.67, 95% confidence intervals [CI] 1.18, 2.37) but was not significantly related to the length of appeals process (OR 1.10, 95% CI .78, 1.55). Among physicians, the likelihood of sanctioning deception was significantly greater with both a lower likelihood of successful appeal and a longer length of the appeals process. CONCLUSION: Sanctioning of physician deception is even greater among the general public than among physicians, and unlike physicians, the public's support of deception is not sensitive to the length of the appeals process. Patients' preferences regarding deception of insurance companies may further pressure physicians who struggle to balance the tension between patient advocacy and honesty. There was no difference in the two groups in the percentage of patients discharged to Skilled Nursing facilities or to home with home health. However, for the patients needing SNF placement there was increased LOS for Friday admissions (mean LOS: Fri 4.7, Mon 3.7; p = 0.03). Similar trend was present for patients needing HH but did not reach statistical significance (mean LOS: Fri 4.4, Mon 4.0, p = 0.20) . There was no difference in mortality (% Expired: Fri 1.22%, Mon 1.88%; p = 0.45). Analysis of the yearly trend revealed decreasing cost and LOS through the four years, but the Mon-Fri difference was maintained. CONCLUSION: Patients admitted on Fridays have higher costs and LOS, despite similar case mix. They have more labs and diagnostic radiology testing done. They are admitted under`O bservation'' status less frequently and spend more time in observation. They wait longer for placement. Despite recent successes in decreasing cost and LOS, weekends continue to play a significant role in the cost of healthcare. Further reduction in cost will require attention to the factors we have identified including reduction in diagnostic testing and more efficient discharge planning over the weekend. this study was to determine the correlation between risk-adjusted hospital rates and rankings for postoperative mortality, postoperative pneumonia (POP), and respiratory failure (RF). METHODS: Cases were selected from those who underwent major noncardiac surgery at 44 VA hospitals participating in Phase I and II of the National VA Surgical Quality Improvement Program. Postoperative mortality was defined as death within 30 days postoperatively. POP was defined as a positive sputum culture with antibiotic treatment or an infiltrate on chest x-ray diagnosed as pneumonia or pneumonitis following surgery. RF was defined as mechanical ventilation greater than 48 hours postoperatively and/or reintubation and mechanical ventilation subsequent to postoperative extubation. Separate logistic regression models predicting mortality, POP, and RF were developed using Phase I cases (10/91 ± 12/93) and validated using Phase II cases (1/94 ± 8/95 PURPOSE: Due to a number of factors, many clinicians have experienced a decline in their income over the past few years. To compensate for this decline, some physicians have chosen to supplement their income through means not associated with their primary practice of medicine (e.g. drug trials, speaking engagements, malpractice case review, product sales). This is the first study to qualify and quantify the extent of participation in such supplemental income-generating activities (SIGAs). METHODS: We conducted a cross-sectional mailed survey of 1000 Maryland members of the American College of Physicians to determine the frequency of participation in various SIGAs. Members were sampled sequentially with a random start. The survey consisted of 24 items designed to collect information on demographics, practice patterns, income variables, and participation in nine different SIGAs. We used Chi-square analysis to determine whether relationships existed between personal and professional characteristics and involvement in SIGAs. RESULTS: After the first two mailings, our response rate was 40%. Sixty-three percent of those surveyed reported engaging in at least one SIGA. The three most common activities noted by these physicians are shown in the table below. Statistical analysis suggested a significant association between participation in SIGAs and male gender, married status, age between 41 and 50, having dependents, dissatisfaction with income, urban or suburban practice setting, and medical subspecialization. CONCLUSION: Physician participation in income-generating activities outside of their primary medical practices is widespread, perhaps reflecting an increase in financial pressures caused by decreasing reimbursements and managed care limitations. The impact of time diverted from the primary practice of medicine on continuity of care, as well as the ethical issues surrounding the enrollment of patients in clinical trials, the sale of medical and/or non-medical products from the office, and physician relationships with the pharmaceutical industry needs to be further examined. PURPOSE: Transthoracic echocardiograms (TTE) are frequently ordered by generalists to assess a heart murmur or screen for valvular heart disease. Little is known about interobserver agreement (IOA) in diagnosing aortic insufficiency (AI)or stenosis (AS)by TTE. The limited studies done have suffered from one or more of the following: 1) verification bias, 2) small sample size, 3) limited generalizability as observers were given a specific interpretation protocol and 4) possible Hawthorne effect. Despite these features fostering IOA, some studies have reported significant lack of IOA, particularly for AI. Our submission avoids the above deficiencies and appears to be the largest analysis of IOA in the diagnosis of AI and AS by TTE. METHODS: 143 patients undergoing TTE for any indication were prospectively enrolled in a blinded study assessing components of the physical exam for diagnosing AI. The TTE, incorporating color and continuous flow Doppler analysis, were performed by an experienced technician and officially read by one of three staff cardiologists. These studies were also interpreted by a blinded study cardiologist (SRB) for the presence of AI or AS. All observers were experienced in TTE interpretation and none (including SRB) were aware of each other's findings or the planned IOA analysis. No interpretation protocol was specified. SRB's findings were compared to the official readings. AI/AS was graded 0 (absent) to 3 (severe respectively. Data were not linked by respondent. The single item burnout measure used in this survey has previously been validated in this and other settings. RESULTS: Self-reported burnout increased in prevalence from 19% of the 439 respondents in 1991 to 41% of the 482 respondents in 1999 (p = .001). Between 1991 and 1999, the number of female and part time physicians increased as did the average age and organizational tenure. Burnout was higher in physicians in the middle range of tenure (6 ± 15 years) and in 1999 among physicians working part-time but was not associated with physician specialty, age, or gender. Burnout was associated with workload factors such as stress from long hours, inadequate time for patient visits, and the perception that the visit rate was too high to do a good job (p=.001).\nNegative attitudes regarding relationships with colleagues, dissatisfaction or stressful relations with patients, and negative attitudes about organizational control of practice were also associated with burnout (p = .001). From 1991 to 1999, the number of physicians reporting great stress from long hours increased from 15 to 41%, physicians reporting great stress from inadequate time for patient visits increased from 37 to 48%, and the number of physicians reporting that the visit rate was too high to do a good job increased from 31 to 54% (p = .001 for all). The quality of professional relations was perceived to be worsening by 20% of physicians in 1991 but by 40% in 1999 (p = .001). Although satisfaction with patient relations and continuity of care remained stable, physicians experienced higher levels of stress from demanding patients in 1999 than in 1991 (p = .004). Measures of perceived organizational control of practice were unchanged. CONCLUSION: Key correlates of the increased burnout rates noted in 1999 appear to be stress from workload factors, stress from demanding patients, and perceptions of declining quality of professional relations. Further research needs to elucidate organizational and structural changes that can prevent burnout by modifying perceptions of physician workload and improving relationships with colleagues and patients. RESULTS: Most clinic encounters were satisfying to physicians and patients. The mean physician satisfaction score was 4.01, SD .92 (5-point scale) and the mean patient satisfaction score was 9.46, SD 1.13 (10-point scale). Continuity of care was associated with patient satisfaction. Patient satisfaction was 9.65 if they had seen the physician previously; whereas, it was 9.24 if they had not seen the physician previously (t = 3.61, p = .0004). However, physician satisfaction was not influenced by previously seeing the patient. Physician satisfaction for an encounter was greatest if the patient was the first of the clinic day compared to all others (4.15 vs. 3.96, t = 3.05, p = .002) . Similarly, patient satisfaction was greatest if they were the first patient of the clinic day (9.69 vs. 9.39, t = 3.13, p = .001). Physician satisfaction with the immediately preceding patient encounter also appears to influence satisfaction within a clinic visit. Physician satisfaction with a clinic visit is lower if the physician was dissatisfied with the patient from the immediately preceding clinic visit (p = .001). However, physician satisfaction with the preceding clinic visit does not influence patient satisfaction. CONCLUSION: The majority of clinic visits are satisfying to physicians and patients. However, clinic continuity, the order in which patients are seen and even the previous patient can influence satisfaction within a clinic visit. These insights can guide the development of educational strategies to improve the patient-physician interaction, which in turn should enhance the satisfaction of physicians and patients with clinical encounters. RESULTS: Overall unadjusted in-hospital mortality was 12.2%, and was similar during July ± September and October ± June in major (14.2% vs. 14.5%; p = .37) and minor (11.5% vs. 11.5%; p = .97) teaching hospitals, but differed in non-teaching hospitals (10.3% vs. 11.3%; p < .001).\nAdjusting for severity of illness using logistic regression, the odds of death was similar (p > .1) in July ± September admissions, relative to October ± June admissions, in major (OR 0.96; 95% CI, 0.89 ± 1.03), minor (OR 1.06; 95% CI, 0.96 ± 1.19), and non-(OR 0.95; 95% CI, 0.88 ± 1.01) teaching hospitals. Results were similar when admissions in July, August, and September were examined separately, as shown in the table below (data reflect OR (95% CI)).\nLikewise, the relative LOS of ICU admissions in July ± September was nearly identical to admissions in October ± June in major teaching hospitals (1.00; 95% CI, 0.98 ± 1.02). This was also true in minor teaching and non-teaching hospitals. CONCLUSION: We found no evidence to support the existence of a July phenomenon in ICU patients. Patients admitted to teaching hospital ICUs during July, August, and September, have similar adjusted mortality and LOS. These findings suggest that residency programs may compensate for the inexperience of new housestaff during the first 3 months of the academic year.\nOUTCOMES? RESULTS OF A REGIONAL ANALYSIS. W.A. Barry 1 , G.E. Rosenthal 1 ; 1 University of Iowa Hospitals and Clinics and Iowa City VAMC, Iowa City, IA PURPOSE: Discontinuity of care in teaching hospitals may occur when teams rotate on and off service. The purpose of this study was to determine relationships between the timing of admission (during the month) and in-hospital mortality and length of stay (LOS) in patients admitted to intensive care units (ICUs). METHODS: We conducted a retrospective cohort study of 156,136 consecutive admissions (mean age 63.0 years, 51.7% male) to 38 ICUs in 28 hospitals in a large mid-western metropolitan area during 1991 ± 1997. Hospitals included 5 major (n = 48,853), 6 minor (n = 29,995), and 17 non-teaching (n = 77,288) facilities. Data were collected from patients' medical records. Admission severity of illness was measured using the APACHE III methodology. Multivariable analyses compared mortality for admissions on Days 1 ± 5, 6 ± 10, 11 ± 15, 16 ± 20, 21 ± 25 , and 26 ± 31 of each month, adjusting for severity of illness and month and weekday of admission. RESULTS: In-hospital mortality was higher in patients admitted on Days 1 ± 5, 6 ± 10, and 26 ± 31, compared to admissions on Days 11 ± 25 in major teaching hospitals (14.7%, 14.7%, 14.5%, and 10.9%, respectively; p < .01). Adjusting for severity of illness and other factors using logistic regression, the odds of death was higher in patients admitted to major teaching hospitals ICUs on Days 1 ± 10 or 26 ± 31, relative to patients admitted on Days 11 ± 25 (OR 1.11; 95% CI, 1.04 ± 1.18; p = .01). Adjusted odds of death were not higher during these periods in minor teaching (OR 1.03; 95% CI, 0.94 ± 1.13; p = .59) or non-teaching (OR 0.95; 95% CI, 0.90 ± 1.00; p = .07) hospitals. Results were similar when admissions on Days 1 ± 5, 6 ± 10, and 26 ± 31 were examined separately, as shown in the table below (data reflect OR (95% CI)).\nIn linear regression analyses, LOS was nearly identical in major, minor, and non-teaching hospitals (relative LOS 1.01, 1.00, and 1.00 respectively). CONCLUSION: Patients admitted to ICUs in major teaching hospitals early or late in a month have a modestly higher risk of death, even after adjusting for admission severity of illness. A similar pattern was not observed in minor teaching and non-teaching hospitals. These findings may reflect the discontinuity of care that typically occurs when housestaff and attending teams rotate on and off service. PURPOSE: To explore the extent to which physicians agree that their main responsibility is to individual patients rather than to society, to understand which physician and practice characteristics may influence this attitude, and to examine how this attitude affects physicians' career satisfaction. METHODS: We surveyed 500 primary care providers from 80 outpatient clinics in 11 managed care organizations (MCOs) and received 414 completed surveys (response rate 83%). We used t-tests, chi-squared tests and logistic regression to compare the socio-demographic and practice characteristics of physicians who strongly agreed versus all other responses with the statement``The practitioners main responsibility is to each individual patient rather than to society'' (the individual-patient ethic). We then examined the relationship between the individual-patient ethic and career satisfaction using chi-squared tests and logistic regression with career satisfaction as the dependent variable. RESULTS: 28% of physicians strongly agreed, 42% somewhat agreed, 13% were neutral and 17% somewhat or strongly disagreed that their main responsibility was to each individual patient rather than society. Physicians with the individual-patient ethic were older (43% physicians > 50 years vs 21% physicians < 35 years, p = .009), spent more hours/week in direct patient care (36 vs 32 hours, p = .048), and tended to practice in network rather than staff model MCOs (33% vs 24%, p = .077). There was no difference in gender, race, specialty, or geographic region between physicians with or without the individual-patient ethic. In multivariate analyses, physician age remained the only significant predictor of the individualpatient ethic after controlling for all other physician and practice characteristics. When examining the relationship of the individual-patient ethic to career satisfaction, physicians with the individual-patient ethic were more satisfied with their ability to provide good quality care (38% vs 19% very satisfied, p = .000) and more satisfied with their ability to serve enrollee needs (28% vs 12% very satisfied, p = .000) but were not significantly more satisfied with their overall careers (20% vs 14%, ns). After controlling for physician age, gender, specialty, number hours/ week in direct care, type of MCO and geographic region, physicians with the individual-patient ethic were more likely to be very satisfied with their ability to provide good quality patient care (AOR 3.16, 95% CI 1.88 ± 7.75), to serve enrollee needs (AOR 3.81, 95% CI 1.87 ± 7.75) and more likely to be very satisfied with their overall career (AOR 2.13, 95% CI 1.04 ± 4.39). CONCLUSION: Physicians who strongly agree that their main responsibility is to each individual patient rather than society are older and more satisfied with their careers. The extent to which this attitude impacts patient care is unknown. PURPOSE: Fragmentation of health services may hamper women's ability to obtain comprehensive, coordinated healthcare. The Veterans Administration (VA) has encouraged its medical facilities to create specialized women's health clinics (WHC) to meet these needs. This study compares patient perceptions of the quality of primary care delivery in WHC versus traditional VA general medicine or primary care clinics (PCC). METHODS: We mailed an anonymous survey to women veterans from the 10 VA facilities in the Mid-Atlantic region. For the 8 facilities with a WHC code, we randomly sampled 170 veterans who used the WHC, and 80 who used a PCC from March 1, 1999 , to March 1, 2000 At the other 2 sites, we selected 250 women who visited a PCC. The initial mailing was followed at one week intervals by a reminder postcard then a repeat survey. Patient perceptions of primary care were measured using the original 19-item Components of Primary Care Index (CPCI) by Flocke (1997 . Additionally, other provider characteristics significantly influenced patient ratings. The presence of a regular VA provider was associated with higher ratings on all four subscales (OR 2.53, 1.53, 2.44, and 3.26 respectively, p < 0.001). Among the women who had a regular provider, those who used a female practitioner also rated their care significantly higher on all subscales (OR 1.67, 1.80, 2.16, and 1.83, p < 0.001) . CONCLUSION: Female veterans using specialized women's clinics perceive a higher quality of primary care delivery even after controlling for demographics, health status, and provider characteristics. Further details about model of care, type of provider, and health outcomes for women veterans need to be examined. Since WHC appear to differ from PCC on some factors, one may infer that selection bias accounts for the differences in patient ratings between clinics. Further analyses will account for selection bias through Heckman models or other methods. PURPOSE: Waiting lists for coronary revascularization are frequently managed without any explicit queuing criteria. Patients may thus not receive priority based on the severity of their clinical symptoms. This is of particular concern in Europe. We therefore convened a European expert panel to develop criteria for maximum acceptable waiting times for coronary revascularization. We assessed the applicability of this criteria and compared Dutch patients' waiting times with the panel's recommendations. METHODS: A panel of 13 cardiothoracic surgeons and cardiologists from Spain, the Netherlands, Sweden, Switzerland and the United Kingdom was convened to assess the appropriateness of, and priority for, a set of hypothetical scenarios for coronary revascularization. They rated the appropriateness of these scenarios using a modified delphi process and then assigned a maximum waiting time, on a scale of 7 time frames, for 200 indications that were not judged inappropriate. We then measured the waiting time for coronary revascularization (i.e. the number of days between when a recommendation was made that a patient should undergo revascularization and the time the procedure was performed) for 1713 chronic stable angina patients who were treated at one of 10 hospitals in the Netherlands. We also collected data on how the patient's clinical data was presented at the meeting where the recommendation was made for revascularization: (1) direct presentations occurred when the referring cardiologist attended the meeting; and (2) indirect presentations occurred when the patient's clinical data was provided by telephone, letter or facsimile. We assessed the proportion of patients who underwent revascularization within the maximum recommended time. RESULTS: There was significant variation in the maximum recommended waiting time among the panelists (mean maximum recommended waiting time 96 days; standard deviation 85 days). Angioplasty patients waited fewer days than bypass patients (48 vs. 83 days, p < 0.001). Thirtyseven percent of patients waited longer than the maximum recommended waiting time. The majority of patients with excess waiting times were referred for bypass surgery rather than angioplasty (e.g., 83% vs. 17%, respectively). Patients whose cases were discussed during a direct presentation waited fewer days than those patients whose cases were presented indirectly (61 vs. 74 days). CONCLUSION: One-third of patients referred for coronary revascularization waited for periods longer than those recommended by a multinational panel. Two contributing factors were the type of revascularization procedure the patient was referred for and how the patient's case was presented, factors not considered by the panel as they felt a patient's waiting time should be determined by clinical symptoms. was convened to assess the appropriateness of, and priority for, a set of hypothetical scenarios for coronary revascularization. They rated the appropriateness of these scenarios using a modified delphi process and then assigned a maximum waiting time, on a scale of 7 time frames, for all non-inappropriate indications. They also assessed the impact of social factors on a subset of 45 scenarios in which the patient was considered to have mild-moderate angina, moderate left ventricular function, and had a low-moderate operative risk. The scenarios varied in the patients ability to work, live independently or care for dependents which was categorized as being: (1) not threatened, but more difficult; (2) threatened, but not immediately; and (3) immediately threatened. The time frame was converted to a linear scale and multiple regression was performed on the 540 individual panelist's ratings to assess the impact of each factor on waiting time. RESULTS: Twelve of the 13 panelists responded. Large shifts in (p < 0.001) occurred in ratings of waiting time, with the order of priority being those whose ability to work, live independently or care for dependents was immediately threatened first, those whose activities were threatened but not immediately second, and those whose activities were not threatened last. The overall mean shift in maximum recommended waiting time due to a threat to ability to work, live independently or care for dependents (37 days longer wait for those who were not threatened vs. those immediately threatened) was less than the mean shift due to extent of coronary disease (e.g., 93 days longer wait for those with 1 or 2 vessel nonproximal left anterior descending artery disease compared to those with left main disease) or stress test results (62 days longer for those with a negative compared to very positive stess test). CONCLUSION: Cardiovascular specialists may place considerable weight on the threat of cardiovascular disease on a patient's ability to perform their usual social activities. However, the impact of this factor varies according to clinical presentation. The impact of these factors should be assessed in actual practice. PURPOSE: Teaching hospitals, due to their size, expertise, and location may find themselves in a position to treat alcohol and drug related diagnoses more often than their community counterparts. Previous studies have shown the existence, in some cases, of a volume-to-outcome relationship. Thus, though past research has shown that overall teaching hospitals may be less efficient, they may experience efficiencies in uniquely qualified or niche areas. This study examines the relationship of hospital resource use and the treatment of alcohol and drug diagnoses. Specifically the study tests the hypothesis that teaching hospitals will have greater efficiency than other community hospitals in the treatment of alcohol or drug related diagnoses. This study also examines the hypothesis that hospitals with more experience in treating alcohol and drug related diagnoses would experience greater efficiencies than hospitals with less experience. METHODS: This is a retrospective cross-sectional study that examines the association of hospital resource use and the treatment of alcohol and drug related diagnoses. The population for this study comes from the 1996 Healthcare Costs and Utilization Project's National Inpatient Sample. The final sample consisted of patients with the appropriate alcohol and drug related diagnosis (n = 9,528 patients). Data from the American Hospital Association, the Area Resource File, InterStudy, and the Health Care Financing Administration supplemented the patient level data from NIS. Teaching hospitals were identified using the Council of Teaching Hospital designation. Hospital experience was measured by the number alcohol and drug related admissions relative to the hospital's total admissions. Hospital resource use was measured by taking both the log of length of stay and total hospital charges. Least square regression was used to analyze hospital resource use while controlling for hospital, patient, and market characteristics. RESULTS: The average age of the sample was 39 with 68% of the patients being male and 66% of them white and 23% black. Twenty percent of the admissions were to teaching hospitals. Teaching hospitals had significantly lower charges (p = 0.029) than non-teaching hospitals and shorter lengths of stay (p = 0.042) than their community hospital counterparts. When examining the hospitals relative experience, hospitals with greater experience had significantly lower total charges (p < 0.001); however, contrary to our hypothesis they had significantly longer lengths of stay (p < 0.001). CONCLUSION: Though previous studies have shown teaching hospitals, as compared to non-teaching hospitals, are less efficient overall, the results of this study show that they are able to experience certain efficiencies in niche areas. Specifically, teaching hospitals are more efficient at treating alcohol and drug related diagnoses. As teaching hospitals continually find themselves competing for managed care contracts, finding and documenting niche areas in which they excel becomes increasingly important. PURPOSE: Routine screening for domestic violence (DV) is increasingly recommended in outpatient healthcare settings. However, little is known about the screening process. The purpose of this study was to document the screening process for women counseled for DV in a primary care setting. METHODS: We identified pregnant women who were counseled for DV between 8/98 ± 6/99 at a university obstetrics clinic. The DV protocol at this clinic, similar to national guidelines, recommends that women be asked about DV at every visit. Identified women are offered counseling using a specific form (usually performed by a social worker). Data were collected from the DV counseling form and from medical records for the previous 6 months. We assessed how the women were screened, who had counseled the women, and what types of abuse led to the counseling. We compared demographic and health characteristics of counseled women to a control group of pregnant women, matched on race, age and type of visit, who were not counseled for abuse. RESULTS: Of 63 pregnant women counseled for abuse, 53 had complete medical records available for review. Fourteen (26%) of the women were identified as victims at their first clinic visit. Thirty-three (62%) of women denied abuse in at least one previous screening attempt. Eighteen (67%) of the 27 women who were asked about abuse by their physician on the same day that they received counseling denied abuse to their physician. Of the 48 women (91%) referred by an MD/RN to social work for counseling, only 25 (52%) were referred for DV. Twenty-three (48%) were referred for non-DV reasons with the social worker subsequently identifying the abuse. Women referred for DV were more likely to be seen within a month of referral than women referred for other reasons (72% vs. 39%, p=0.01). The majority of abuse reported was physical (96%) and recent (83% with event within past month); 37% had been experienced abuse for more than 2 years. In the 40 cases we were able to match to controls, women counseled for abuse were more likely to be depressed (51% vs. 29%, p < 0.05), to be currently using drugs (20% vs. 6%), p < 0. Bivariate analysis revealed that physicians with a higher score on the SWB scale and more frequent church attendance were more likely to hold beliefs that health outcomes are improved with patient and physician prayer and that faith alone can cure disease. There were no associations between physician sex, age, race, or spirituality training and these attitudes. CONCLUSION: A large number of physicians believe religious and spiritual behaviors by patients will improve health outcomes. A smaller but significant number believe that physician prayer will improve patient outcomes. These findings suggest that there is a need for more rigorous examination of the connection between health outcomes and spirituality as many physicians seem willing to incorporate aspects of spiritual behavior into their practices. 1995) . Little is known about the impetus to develop a separate WHC, especially within the VA, where issues of access to care, privacy, and quality of care for women veterans have often been of concern. We examined organizational, provider, and patient-level determinants of developing a separate WHC for delivery of gender-specific and primary care services. METHODS: We used data from the 1999 VHA Primary Care Practices Survey, a national survey of senior VA primary care leaders at all VA health care facilities with > 4,000 unique patients and > 20,000 outpatient visits during fiscal year 1998 (response rate 93%, n = 219). Analysis of facility characteristics associated with a self-reported WHC for delivering primary care was performed using bivariate analysis and independent predictors were determined using logistic regression. RESULTS: Nationally, 62% of facilities reported having a separate WHC to provide primary care for women. WHC's tended to reside in urban locations (p < 0.05) and in more complex facilities (p < 0.05). Facilities with WHC's had more female patients (p < 0.001) with more visits (p < 0.001), but comparable visits-per-patient (7.1 vs. 7.4). Facilities with team-based primary care that integrated providers from multiple disciplines were more likely to have developed a WHC (p < 0.05). WHC's were more likely where primary care leadership was distinct from subspecialty care (p < 0.001) and a separate budget existed for primary care (p < 0.05). In addition, WHC's were more common in academic facilities (69% vs. 56%, p = 0.09) with higher numbers of internal medicine houseofficers (43.5 vs. 26.9, p < .05) and longer outpatient block rotations (p < .05). Having separate primary care leadership, and higher women veteran caseload remained significant predictors of WHC development after adjusting for other variables. CONCLUSION: Separate WHC's were present in larger, complex, more intensively academic medical centers with more established and authoritatively independent primary care programs. As a cross-sectional study, it is unclear whether WHC development occurred as a result of or resulted in a higher female patient volume. The implications of separate service delivery for women with respect to clinical quality, and patient satisfaction are still unclear and somewhat controversial; assessing WHC performance is a crucial next step. 1.14 ± 1.25), and for all encounters combined (RR 1.07; 1.04 ± 1.09). As compared to their use of Outpatient services, PDs were especially more likely than non-PDs to use high cost Emergency Department and Inpatient services. CONCLUSION: Using CAGE results and objective encounter data from continuity patients in a large group practice, problem drinking was common and associated with higher overall utilization as well as use of higher cost sites for specific alcohol-related conditions. These findings support initiatives to screen and intervene for problem drinking in primary care practices. Respondents were asked to rate how important it is for asymptomatic men to know each of the 17 facts about PSA screening using a 5-point Likert scale ranging from``not at all important'' to``extremely important.'' Because internists and family physicians had similar responses, they were combined as non-urologists and compared to urologists. We used logistic regression to compare differences between urologists and non-urologists and adjusted for the effects of age and gender. RESULTS: Urologists and non-urologists differed in rating how important it is for men to know 9 of the 17 facts. Urologists considered it extremely or very important for men to know that it is unclear whether regular PSA screening will reduce prostate cancer mortality, that done together the PSA and digital rectal exam (DRE) can screen for prostate cancer, and that PSA and DRE are most appropriate for men whose life expectancy is at least 10 years. Non-urologists considered it extremely or very important for men to know that PSA screening is controversial, that there are risks and benefits to PSA screening, that prostate cancer may grow slowly without symptoms, that it is unclear whether treatment of early, localized prostate cancer is helpful, that there are side effects to treatment, and that a man over age 70 is less likely to die from prostate cancer even though he has a higher risk of having it. RESULTS: Faculty and resident patients were similar with respect to sex, race, weight, years of diagnosis of diabetes mellitus and type of diabetes therapy. However, resident patients were slightly older than faculty patients were (mean age 63 vs. 57, p = 0.008). Residents and faculty were no different in frequency of adherence to the ADA Clinical Practice Guidelines for visits per year, HbgA1C testing, lipid profile testing, and foot exam. Faculty were significantly more adherent to urine evaluation (44% vs. 26%, p = 0.04) and referral for dilated eye exam (54% vs. 39%, p = 0.02). There were no differences between residents and faculty patients with respect to mean HbgA1C levels, mean control of blood pressure, use of ACE inhibitors and use of lipid lowering therapy. Resident patients were more likely than faculty patients to have LDL levels controlled to less than 100 (42% vs. 25%, p = 0.03). CONCLUSION: Residents and faculty provide primary care for similar groups of diabetic patients at this academic health center. Adherence to ADA Clinical Practice Recommendations was similar between faculty and residents for many, but not all areas. Outcomes such as HbgA1C levels and blood pressure control were similar for both groups, though more resident patients had better LDL cholesterol control. Though resident continuity practices are faculty supervised and often in the same setting as faculty practices it cannot be assumed that quality outcomes are the same for patients of these two groups. Interventions to improve rates of adherence to specific recommendations should be geared towards both faculty and residents at this academic health center. Younger generation for females and males were significantly less likely to receive primary care compared to older generation males and females (females p < .001 and males p < .006). Male relatives are significantly less likely to access primary care compared to females, 57% vs. 70% (p < .015) and are less likely to access the Charity system compared to females 20% vs. 34% (p < .014). Male relatives are more likely to obtain the majority of their health care in the ER and Walk-in Clinic compared with female relatives 20% vs. 8% (p < .001). There was no difference between work status, age distribution, and health care coverage between male and female relatives. CONCLUSION: The barriers to health care access in both males and females were Full/Part time-work status, younger generation, and lack of health care coverage. Our patients' male relatives are less likely to have a regular source of primary care compared to female relatives. Offering evening and weekend hours in our indigent continuity clinics may possibly reduce this barrier. Further studies are needed to better assess this inequality of primary care access between males and females. PURPOSE: Decreasing waiting times to next available appointment involves evaluation of the physian appropriateness and effectiveness of practice in the clinic system. Some studies have shown that patients receive``better'' care from a sub-specialist than those followed by a generalist. The appropriateness and variability of this care, however, has not been well scrutinized. The purpose of this study, was to identify if patients enrolled in the cardiology clinic belong there based on a set of criteria agreed between internal medicine and cardiology. Also, to assess the variation among cardiology fellow's discharge rates and revisit interval assignment. METHODS: Data was extracted from 194 cardiology clinic notes between 1/14/00 ± 2/27/00. Providers were cardiology fellows practicing at an urban VA. The main outcomes of interest were appropriate discharge from the cardiology clinic (per criteria established by the authors) and assigned revisit interval. Predictors of interest included provider data and patient level information such as a severity of illness score and ejection fraction. RESULTS: Overall, discharge rate was 24/194 (12.4%) . Of the 161 remaining in the clinic, 74 (46%) could have been discharged to their primary provider by the agreed criteria. In logistic regression, disease severity was correlated with discharge from clinic (r =0 .69). The more stable patients were more likely to be discharged from clinic. Overall, average clinic revisit interval was 3.8 months. There was a large amount of variability among providers. Individual provider average follow up time ranged from 2.2 ± 7.7 months. Individual discharge rates ranged from 0 ± 26%. Individual clinic appropriateness ranged from 0 ± 90%. There was no correlation of revisit intervals with severity of illness score (r = .021, p = 0.794) or with ejection fraction (r = .013, p = 0.91). At best, patient severity of disease accounted for only 4% of the variance. Individual providers saw patients back at a consistent length of time regardless of the needs of the individual patients for instance, provider #10 saw every patient back in 6 months and provider #31 saw every patient back in 3 months.\nCONCLUSION: There needs to be established criteria which patients must meet to remain in the clinic that will to be utilized by all providers in the clinic to reduce variability in care. There needs to be improved consistency and decreased variability in return to clinic times to improve access in the cardiology clinic setting. Return visit time should be based on severity and stablility of patient's illness and not on individual provider preference. Centers for Disease Control and Prevention, Atlanta, GA PURPOSE: Influenza vaccine is the primary method for preventing influenza. Delay in influenza vaccine distribution was expected for the 2000 ± 01 influenza season. We conducted a cross-sectional study to characterize practice adjustments and expectations of physicians regarding influenza vaccine delay. METHODS: We sent a two-page mail survey to a national random sample of 1606 internists (IM) and family physicians (FP). Participants answered questions regarding their usual influenza vaccine administration period, their practices' capacity for identifying high-risk patients, and modifications in their clinical practice for the 2000 ± 01 influenza season. The first mailing was conducted in September, 2000; non-respondents received another mailing in October, with a $2 incentive. Tests of significance were computed using likelihood ratio chisquare. RESULTS: Response rate was 60% after two mailings and did not differ significantly by respondent specialty. Of 952 respondents currently practicing, 756 (79%) typically administered influenza vaccine; family physicians were more likely than internists to administer influenza vaccine (82% vs. 76%; p < 0.05). Among physicians administering vaccine, 356 (47%) selfidentified as IM and the remainder as FP. At the time of survey completion, 13% of respondents had received their full order of vaccine; another 23% reported receipt of a partial order. Whereas 37% of physicians who had already received their full order had modified their practices this influenza season, 70% of physicians who had received either partial or no shipment reported practice changes (p < 0.001). The most commonly reported practice modification was to target high-risk patients for receipt of available doses. However, only one-quarter of physicians (28% IM vs. 24% FP; NS) had experience with mail or telephone reminder systems to contact high-risk patients. Of concern, 43% of providers were hesitant about administering vaccine after the onset of influenza season in their practice area. CONCLUSION: Facing delays in influenza vaccine distribution, many physicians reported an intention to vaccinate high-risk patients first. However, few physicians had experience using reminder systems, and nearly half of physicians were hesitant about administering vaccine after influenza season has begun. These findings raise concerns about the capacity for physicians to meet domestic vaccination goals during this influenza season and in the future, and the possibility of preventable morbidity and mortality attributable to influenza as a result. PURPOSE: Payers and institutions use chart abstraction to measure physician performance, despite its underestimation of the quality of care due to recording bias. We wondered if the medical record might also overestimate the quality of care through false, and potentially unethical, documentation by providers. To determine this, we compare the quality of care as documented in the medical record with the reports of actor patients. METHODS: Twenty physicians in the primary care clinics of two Veterans Affairs Medical Centers were randomly selected among consenting residents and faculty (97% agreed to participate). Data were collected from standardized (actor) patients, who served as the gold standard and presented undetected as patients to physician subjects, and from the medical record generated from these visits. Quality criteria were developed from national guidelines and a modified Delphi technique for four common medical conditions. These were then recorded by a standardized patient or abstracted from the medical record. Physician subjects completed 160 evaluations of standardized patients (8 cases  20 physicians). We determined the proportion of criteria reported in the medical record but not by the standardized patient (false positives). We also determined the distribution of false positives according to domain (history, physical exam, diagnosis, treatment), study site, physician subjects, and actor patients. False positive rates at the two study sites were compared by t-test.\nRESULTS: Compared to the gold standard of standardized patients, false positives were identified in the medical record for 6.4% of measured items overall. False positives were higher for physical examination (13.5%) and diagnosis (14.6%) than for history (3.8%) and treatment (3.4%) . The difference in false positive rates between site 1 (7.1%) and site 2 (5.7%) was not statistically different (p = 0.34). The proportion of false positives for individual physician subjects ranged from 2.2% to 13.0% and for actor patients from 1.4% to 11.6%. CONCLUSION: These results suggest that chart abstraction may overestimate the quality of care due to false positives. The clustering of false positives in the domains of physical examination and diagnosis suggests that these are not incidental occurrences or underreporting by actor patients. Though false positives in the physical examination could result from careless documentation by physician subjects, they may indicate intentional misrepresentation of the process of care, perhaps to up-code a visit or save time by adding an exam element not performed. Such fabrication would violate ethical standards essential to the integrity of clinical practice, potentially putting patients at risk by including misinformation in the medical record. By contrast, documentation of diagnosis in the medical record but not by the actor patient would represent an important lapse in communication of essential information. Improved evaluation methods are needed to detect such irregularities as well as guidelines to determine appropriate actions when potentially unethical conduct is identified in studies of quality. University of Washington, Seattle, WA PURPOSE: For many physicians, the environment in which they trained differs from the current practice environment of managed care, declining revenues, patients on the Internet, and insurance paperwork. Studies of physician career satisfaction have suggested that unmet expectations may play a role in declining satisfaction, but this area remains largely unexplored. We designed a survey to examine career expectations and satisfaction, as well as evaluating trends in satisfaction over time. METHODS: We mailed surveys to half the ACP-ASIM membership in Oregon (n=550) in Fall 2000. Survey categories were developed from previous literature on career satisfaction and physician self-care. We asked respondents to rate 16 features as``not important, important, or very important'' to their current view of their career. The features included: having an adequate income, being valued by others, intellectual challenge, control of professional life, helping others, collegial relationships, being part of a team, doctor-patient relationships, working on the cutting edge of science, providing quality health care, managing own business, and others. We then asked whether expectations about these features were``not met, matched, or exceeded'' in their career currently. RESULTS: We had a 75% response rate (n = 412). We removed surveys returned from retired physicians and residents, analyzing 345 (67% male, 33% female). The highest number of physicians ranked``providing a high standard of care'' as a``very important'' feature of their career (81%), with 93% stating that this expectation was either matched or exceeded in their current work.``Physician-patient relationships'' and``helping others'' were also ranked as very important (68% and 63% respectively) and were matched or exceeded (84% and 94% respectively). 98% of respondents reported``autonomy/control over professional life'' as an important or very important to their career, yet a high proportion of unmet expectations appeared in the following areas:``having control over my professional life'' (52% unmet), and`h aving autonomy in current position'' (38% unmet). There was also high rates of unmet expectations in``making as much money as anticipated'' (42% unmet) and``work is fun'' (33% unmet). 71% report being``somewhat-very satisfied'' with their current practice situation, while 83% selected these satisfaction levels for their situations 10 years ago. 61% would go to medical school again. CONCLUSION: Overall career satisfaction appears to be declining with high unmet expectations regarding professional autonomy and control while, in this sample, internists reported that expectations regarding quality of care were generally met. Improved understanding of expectations and practice changes should help educators and professional groups in their efforts to educate and promote professional development. Part B in 1998 and 1999 . We utilized Medicare claims to measure quality of care based on whether or not beneficiaries had received an influenza immunization in 1999, a pneumococcal immunization between 1991 and 1999, whether females had a mammogram in 1998 or 1999, and whether persons with diabetes had an eye examination or a lipid profile in 1998 or 1999 or a hemoglobin A1C in 1999. We linked patients to primary care providers based on office visit claims and merged providers into practices based on common provider numbers. We restricted our analyses to 141 practices with at least 50 patients in the denominator for each quality indicator. We identified benchmarks as the 90th percentile in performance on each indicator and compared benchmark performance with median performance and performance at the 10th percentile. RESULTS: We observed large discrepancies in performance among practices on all quality indicators (see table) . Performance on certain quality measures was highly correlated (i.e. practices performing well on one measure were more likely to perform well on other measures). CONCLUSION: There is substantial variation among physician's practices in their performance on commonly utilized quality markers. Further efforts are needed to describe the characteristics of practices that are performing at the highest levels. PURPOSE: Previous attempts to improve the triage of patients with suspected acute cardiac ischemia in the Emergency Department (ED) have been disappointing. We developed and prospectively evaluated a decision aid based on a validated clinical prediction rule to determine if physicians' triage behavior would change and whether any changes would be beneficial. METHODS: We constructed a decision aid from Goldman and colleagues' published clinical prediction rule, which predicted major cardiac complications (N Engl J Med 1996) . After achieving over 80% compliance with use of the decision aid in our ED, we prospectively collected data on 1011 consecutive patients admitted from the ED with suspected cardiac ischemia during late 1999. Clinical data collected in the ED allowed us to adjust for possible differences in case mix when comparing decisions and outcomes before and after the intervention. We compared triage decisions (CCU vs. inpatient telemetry vs. observation unit or ward) and their safety and efficiency with similar patient cohorts studied prospectively in our institution during 1997 (n = 207) and 1998 ± 99 (n = 1033). Triage safety was defined as the proportion of all patients who had major cardiac complications within 3 days who were admitted to either the CCU or inpatient telemetry unit. Triage efficiency was defined as the proportion of all patients who did not have major cardiac complications who were not admitted to either the CCU or the telemetry unit. RESULTS: Among the 1011 patients, 1008 were eligible, with clinical follow-up on over 98%. Major complications occurred within 3 days for 3.5% (35/1008). A greater proportion of patients were admitted to an observation unit in 1999 compared to 1997: 35% vs. 21%; P < 0.001. In 1999, a smaller proportion of admissions to the inpatient telemetry unit were very low risk patients compared to 1998 ± 99: 41% vs. 52%; P < 0.001. Triage safety was slightly higher in 1999 compared to 1997: 94% (33/35) vs. 89% (8/9); P = 0.3. Triage efficiency was much greater in 1999: 36% vs. 21%; difference: 15 percentage points (95% CI: 8% to 21%); P < 0.001. CONCLUSION: A new decision aid changed physicians' triage decisions and improved triage efficiency without compromising triage safety. We therefore surveyed internists about how they inform patients about bad news. METHODS: A survey instrument asked how frequently physicians perform different activities in giving bad news to patients, based on a four point Likert scale. Categories included previously recommended activities involving emotional support of the patient (11 items) and those involving the proper setting (9 items). The average amount of time spent with patients while giving bad news was assessed. The impact of demographic variables on the number of emotional support items, setting and time items, and number of minutes spent informing patients were analyzed via ANOVA. All significant variables for each category were entered into a multiple linear regression model. RESULTS: Of the 961 surveys which were received by subjects, 461 (48%) were completed and returned. A majority of physicians (52 ± 93%) always or frequently performed 10 of the 11 recommended emotional support items and 6 (60 ± 92%) of the 9 proper setting items while giving bad news to patients. The average time spent in giving bad news was 27 minutes. Although training in giving bad news had a significant impact on the emotional support items provided to patients (p < 0.05), only 25% of respondents had any previous training in this area. A number of demographic factors were also associated with an increased number of emotional support items provided to patients, including being single (p < 0.05), being female (p < 0.005), and having personally had a life-threatening illness ( p < 0.05). CONCLUSION: By their own report, internists generally inform patients of bad news in an appropriate fashion. Some deficiencies do exist, but training in giving bad news can improve this important type of communication. Educational opportunities about how to give bad news should be provided to practicing physicians; and curricula designed for medical students and residents.\nCARDS. P.D. Faris 1 , W.A. Ghali 1 , R.F. Brant 1 ; University of Calgary, Calgary, Alberta PURPOSE: In health outcome report cards comparing providers for binary outcomes such as mortality, a commonly used method of profiling providers is to use risk factor data from patients treated by the providers to construct logistic regression models. Such models are then used to obtain the expected number (E) of outcomes for each provider and the ratio of observed (O) to expected outcomes (O/E ratio) is used as a risk-adjusted measure of provider performance. To account for chance variation and to determine``outlier status'', confidence intervals (CI's) derived from standard deviations are placed around the O/E ratios. Here we compare two methods for calculating standard devations (SD's) and CI's. The typically used method is compared with a more complex method based on the propagation of errors (PE). METHODS: Typically, when calculating the variances and SD's of O/E ratios, only O is treated as a random variable, and variability in E is ignored. A variance estimate treating both O and E as random variables was derived using the propagation of errors (PE) method. The resulting PE-SD estimates were compared with typical SD estimates using a data set (N = 50,357) previously employed to profile CABG providers in Canada. O/E ratios were used to profile three levels of hospital patient volume (low, medium, high), 8 provinces, and 23 hospitals. For each O/E ratio, the ratio of the typical SD to the PE-SD was used to evaluate the relative sizes of the SD estimates. In addition, computer simulations based on a hypothetical data set with three providers were used to evaluate the SD estimates. Measures of performance included empirical coverage and bias. RESULTS: For the CABG data profiles, the ratios of the typical SD's to the PE-SD's were 1.04, 1.65, and 1.23 for the low, medium and high volume hospitals, respectively. For 7 of 8 provinces, the ratios of the SD's were only marginally larger than 1. However, for the province treating the largest proportion of patients (52%) the ratio was 1.40. For the 23 hospitals, the largest SD ratio was 1.09. The computer simulations confirmed that the typical SD's were less accurate than the PE-SD estimates, and the coverage probabilities indicated that confidence intervals based on the typical SD's are too wide. CONCLUSION: The typically used SD estimates and CI's were always larger than the PE-SD estimates that accounted for variability in E. The bias in variance and SD was greatest when one or more providers treated a large proportion of the patients. When only a few providers are being compared, or when some providers treat a large proportion of the patients, the use of typical SD's may lead to incorrect conclusions regarding the outlier status of providers. PURPOSE: Language concordance between physicians and patients is associated with greater patient satisfaction. However, it is not known what level of physician Spanish language competence is important to Spanish speaking patients. We used four measures to test the association between physician Spanish ability and the probability of these physicians having Spanish speaking patients in their practice. METHODS: Primary care physicians for Spanish and English speaking diabetics from eleven public health clinics completed a questionnaire containing four questions about Spanish language competence: 1) self-rated fluency 2) use of interpreters 3) confidence in conducting an H&P 4) confidence in discussing complex topics. We used chi-squared to test the association between measures of physicians' Spanish competence and the proportion of Spanish speaking patients in their practice. RESULTS: 105 MDs caring for 365 Spanish and 1023 English speaking diabetics participated. 17 (16%) MDs rated their Spanish fluency as excellent and they cared for 144 (40%) of monolingual Spanish speaking patients. Conversely 27 (26%) of MDs rated their fluency as poor or none and they cared for 26 (7%) of Spanish speaking patients. MDs whose self-rated Spanish was excellent, good, or fair were much more likely to treat Spanish speaking patients than those whose Spanish was self-rated as poor or none (OR 3.5, CI 2.3 ± 5.3) . Similarly, MDs who never, rarely, or occasionally use an interpreter were more likely to treat Spanish speaking patients than those who usually, or always use an interpreter (OR 3.8, CI 2.6 ± 5.4) . The two additional questions assessing clinican confidence in conducting a Spanish patient history or carrying out a complex discussion were less predicitive of treating Spanish speaking patients. CONCLUSION: While factors other than patient preference may affect the distribution of patients, our study demonstrates that a brief physician questionnaire can discriminate degrees of language competence that appear meaningful to patients in selecting their physician. University of California, San Francisco, CA PURPOSE: Prior research has shown that old age and chronic conditions such as congestive heart failure are associated with multiple admissions of patients to the medical wards of community hospitals. Risk factors associated with multiple admissions to public hospital wards are unknown, though poor access to primary care is known to be associated with preventable hospitalizations. We studied the demographic, medical, and utilization patterns of high utilizing patients of an urban public hospital to determine risk factors for multiple admissions. METHODS: Using an administrative data set, we obtained data on patient demographics, inand out-patient utilization, and in-patient and out-patient billing diagnosis on all patients discharged from a public hospital medicine/cardiology service between 7/97 ± 6/98. Patients with 3 or more discharges in a one-year interval were defined as high utilizers (HU). We used chi square to test differences between high utilizers and non-high utilizers (NHU) in utilization of emergency room and primary care services in the year following the index discharge, in diagnosis and in demographic characteristics. RESULTS: 3562 patients had 5573 discharges in the study period. 465 patients (13%) were HU who accounted for 35% of discharges, and 36% of hospital bed-days. Compared to NHU, HU were slightly more likely to be male (71% vs. 66% p = 0.02) and more likely to be African American (46% vs.30% p = 0.001) They were no different in age. HU had more visits to primary care clinics (mean 5.72 vs.2.5 p = 0.01), to the emergency department (mean 4.38 vs.2.5 p = 0.01) and to any network clinical site (median 24 visits vs. 5 visits p = 0.001). HU had more congestive heart failure (21% vs.12% p = 0.01), AIDS (30% vs.14 p = 0.01) and COPD (21% vs. 13% p = 0.02). HU were much more likely to abuse alcohol (47% vs.25% p = 0.01) or drugs (58% vs 30% p = 0.01) or either alcohol or drugs (71% vs. 42% p = 0.01). CONCLUSION: High users of urban medical wards in a clinical system with good access to primary care have high rates of use of primary care services. Our data suggests that interventions to reduce multiple hospitalizations in urban public hospitals need to integrate substance abuse services to traditional disease management programs. Indicator Benchmark 50th Percentile 10th Percentile Influenza 64 50 29 Pneumococcal 55 43 30 Mammograms 80 68 51 Eye examinations 88 81 69 HgbA1c 96 88 61 Lipid profile 80 57 PURPOSE: Heart Failure (HF) is a leading cause of hospitalization and re-admission in most hospital systems. Multidisciplinary``Discharge Transition'' programs aimed specifically at the education and close follow-up of HF inpatients have been evaluated in a number of relatively small randomized controlled trials (RCTs). This is the first meta-analysis to evaluate the effectiveness of peri-discharge, multidisciplinary HF patient management programs. METHODS: Electronic database searches were conducted on MEDLINE, HealthSTAR and EMBASE. Reference lists of identified articles and experts' opinions were also reviewed. All potentially relevant articles were obtained. Study selection criteria were: (1) RCTs of adult inpatients hospitalized for HF enrolled at the peri-discharge transition period; (2) HF-specific patient education intervention coupled with a post-discharge follow up assesment; (3) Primary outcome of unplanned all-cause readmission secondary outcomes of mortality, compliance and quality of life. Study inclusion and quality assessment using a modified Jadad Scale were independently assessed by all four authors. Agreement was rated by a weighted Kappa and final decision agreed upon by consensus. RESULTS: A total of 529 citation titles were identified: 199 from MEDLINE; 148 from HealthSTAR; 162 from EMBASE and 20 from personal files, reference lists, or communication with experts. Of these, 94 were deemed potentially eligible for pre-test selection. Two research trials in progress were identified and interim data obtained from the principal investigators. The four reviewers selected 10 papers and two studies``in progress'' for inclusion in the overview. The Kappa agreement statistic for multiple reviewers was 0.73 (SE 0.09). One study was subsequently excluded because it represented an earlier publication of the same trial, and another study was excluded because of substantial heterogeneity in methods, patient population and results. PURPOSE: Depression typically presents in a medical rather than mental health setting. VA and non-VA settings may differ in physician characteristics, training opportunities, and organizational factors. We sought to assess VA and non-VA medical physicians' recognition and treatment of depression. METHODS: We selected a random sample of VA and non-VA primary care physicians and medical specialists practicing in the Northeastern United States. Physicians viewed a 5 minute, professionally produced video of a patient with somatic symptoms, who described 5 symptoms meeting clinical criteria for major depression. After viewing the video, physicians answered interviewer-administered questions about differential diagnosis and management recommendations. Because of the experimental design, every physician reviewed identical clinical data, permitting direct comparisons of physicians' diagnostic reasoning and management approaches. Physicians also viewed two``control'' scenarios (a patient with chest pain and a patient with polymyalgia rheumatica), further masking the key study purpose.\nRESULTS: 81 VA and 129 non-VA physicians participated. Regarding diagnosis, 85% of VA versus 95% of non-VA physicians listed depression as a possible diagnosis (p = .02), and 47% versus 62% assigned depression a probability of greater than 50% (p = .04). Regarding management recommendations, 15% of VA physicians versus 2% of non-VA physicians said they would recommend a mental health referral (p = .001), and 9% versus 13% would recommend an antidepressant (p = NS). 35% of VA physicians versus 53% of non-VA physicians would see the patient portrayed in the videotape for a return visit within 2 weeks (p = .01). CONCLUSION: Non-VA physicians were more likely to recognize depression, although many VA physicians also considered it. Neither group followed Agency for Healthcare Research and Quality guidelines for follow-up and for initiation of pharmaco-or psychotherapy, although non-VA physicians were more likely to see the patient back within 2 weeks and VA physicians were more likely to refer to mental health. Differences in management by VA versus non-VA physicians may point to systems issues underlying non-adherence to depression management guidelines.\nDO HOSPITALISTS FOLLOW EFFICIENCY GUIDELINES BETTER THAN NON-HOSPITALISTS? S. Freer 1 , J. Cotter 1 , C. Pugh 1 , W. Smith 1 ; 1 Virginia Commonwealth University, Richmond, VA PURPOSE: The hospitalist model of inpatient care on general medicine wards offers the potential for a more efficient use of hospital resources, but limited formal evaluation has been conducted. The ability to effectively move patients out of the hospital on day of discharge is an important indicator of the improved use of hospital resources. This is the first study we know of to examine if hospitalists, when compared to non-hospitalists, could more efficiently discharge patients. Physicians have control over the initiation but not the completion of this process. METHODS: We studied discharge orders written by 11am and completion of discharge by noon as a measure for evaluating efficiency. These efficiency guidelines were established by the institution. We evaluated compliance with these guidelines through a retrospective analysis of clinical and utilization data on 3,040 patients admitted to general medicine wards of the Medical College of Virginia Hospital at Virginia Commonwealth University. We analyzed the number and percentage of discharge orders written before 11am and the number of patients discharged by noon by hospitalist and non-hospitalist physicians using chi-square to compare proportions. RESULTS: In FYOO the percentage of discharge orders written before 11am for the hospitalist teams (44.3%) was greater than that for the non-hospitalist teams (28.2%; p < 0.0 1). Very low percentages of patients were discharged by noon from either general medicine service. CONCLUSION: Hospitalist physicians performed significantly better than did non-hospitalist physicians on entry of discharge orders by 11am, a process measure of efficiency over which they have control. There was no difference between the groups on the completion of discharge, a process measure of efficiency over which physicians have less control. METHODS: Pharmacy records for all outpatient diabetes prescriptions during 1997 were obtained at four VA hospitals. Primary care providers (PCP) were defined as writing > 50% of prescriptions, and coded as staff attendings (AT), medicine house officers (HO), nurse practitioners (NP), endocrinologists (EN), or no PCP. Patients were grouped based on whether they received oral agents only, insulin only or both. ANOVA was performed to evaluate medication costs, glucose monitoring supply costs, total costs and glycemic control by site, provider type and treatment classification, controlling for demographics. Supply costs were calculated only across patients receiving supplies; but all patients were included in total monthly costs. Chart reviews were done on a subset from 2 sites to evaluate for differences in DM severity and comorbid conditions. RESULTS: 4544 patients were identified with complete information for cost analyses ( METHODS: In December 1998 we randomized 4 of 8 outpatient health centers that participated in a local HMO to have access to an ACS. The multi-disciplinary, telephone-based ACS evaluated the appropriateness of warfarin therapy, dosed the warfarin therapy, educated the patients, and monitored their international normalized ratios (INRs). At the control sites, physicians and their staff performed these tasks. We assessed the rate of hospitalization for a hemorrhagic or thrombotic adverse event by analyzing hospital claims submitted to the HMO. Using two-sided tests of the Poisson approximation, we compared the relative rates of adverse events between control and ACS sites using an intent-to-treat approach. RESULTS: Of the approximately 1100 patients taking warfarin at the 8 centers, approximately half (N = 503) were eligible and enrolled in the ACS. The remaining patients had their warfarin monitored and dosed by their physician. Using hospital claims data through July 30, 2000, the relative risk for an adverse event was 0.71 among patients who had access to the ACS, corresponding to a significant (p < 0.05) relative risk reduction of 29% compared with patients whose warfarin was managed by their physician. CONCLUSION: The telephone-based ACS was associated with a significantly decreased rate of hospitalization for warfarin-related adverse events. This finding suggests that multidisciplinary, disease-state management programs that empower nurses and pharmacists can improve medical care. RESULTS: Baseline patient satisfaction with their doctor was quite high. The 1,270 patients interviewed gave their physician a median rating of 9 on a scale of 0 (worst doctor possible) to 10 (best doctor possible). 47% of patients rated their doctor as a``10.'' Physicians also scored highly using a validated 10-item scale to designed assess communication skills and humanistic qualities; the median score was 46 of a possible 50 points, with 35% of patients rating their doctor as`e xcellent'' on all 10 items. Patients' ratings of physicians' communication skills were a powerful independent predictor of overall patient satisfaction (p < .001). However, analysis of variance showed that attending the workshop had no significant effect on patients' rating of their doctor's communication and humanistic skills (p = 0.32) or patients' overall rating of their doctor (p = 0.33). Physicians with baseline satisfaction ratings in the lowest quartile were no more likely to show improvement in their ratings than were physicians with higher baseline satisfaction ratings. PURPOSE: In the inpatient setting, physician order entry has been shown to significantly reduce serious medication errors (MEs). However little is known about the impact of computerized prescribing systems in the ambulatory setting on MEs and adverse drug events (ADEs). We compared the frequency of these events in outpatient clinics using handwritten versus computerized prescribing. METHODS: We prospectively studied 2 sites with handwritten prescribing and 2 with basic electronic prescribing in the Boston area. The computerized sites had printed prescriptions and required fields, but no defaults and optional or non-existent checks for allergies and drug interactions. We collected copies of prescriptions written by 24 primary care providers from Sept 1999 to March 2000 (6 weeks per clinic). Prescription copies were reviewed by a pharmacist to screen for MEs and potential ADEs. In addition, patients who received prescriptions were telephoned 2 weeks after their visit to ask about problems with their medications (response rate 59%), which were then classified as ADEs or not by 2 MD reviewers. RESULTS: Of 1173 prescriptions screened during this time period, 202 (17%) were rule violations (orders that violate strict standards but are generally understood and generate no additional work), 44 (4%) were MEs, and 59 (5%) were potential ADEs. Sites with computerized prescribing were significantly less likely to have rule violations (p < .02) and ME's (p < .01), but potential ADE rates were not significantly different. Of 661 patients surveyed, 178 (27%) reported a total of 206 ADEs, of which 74 (36%) were preventable. There was no significant difference between computerized and non-computerized sites in ADE rates and preventable ADE rates (37%, 35%). Of preventable ADEs (n = 74), 45 (62%) occurred in the ordering stage, and 26 (36%) in the patient administering stage. The main types of physician error were failure to act on results of monitoring or tests (50%) and inappropriate drug choice (24%). Improved computer ordering checks would have prevented only 20% of physician errors. CONCLUSION: Errors in the drug process were common in the outpatient setting. However, while basic computerized prescribing systems were associated with lower rates of rule violations and MEs, serious error rates (potential and preventable ADEs) were similar. Monitoring for ADEs was unexpectedly important. Prescribing systems with advanced decision-support, including monitoring and communication features as well as allergy/interaction checking, may be required to substantially reduce the frequency of serious errors. PURPOSE: Despite mandates for physicians to screen for and intervene with domestic violence as they should do for HIV/STD risk, alcohol abuse, and smoking, studies show that physicians face many barriers to doing so. We wanted to know how asking and intervening with domestic violence compares to asking and intervening with patients in these other three health-risk areas. METHODS: In November 2000, we mailed a questionnaire on physicians' screening and intervention behaviors for domestic violence, HIV/STD risk, alcohol abuse, and tobacco use to a national random sample of 1200 physicians (internal medicine and family practice). Our response rate to date, prior to our final mailing, is 49%. Chi-square tests with 3 degrees of freedom (p) and Bonferroni-Holm adjusted pairwise comparisons among topics (p * ) were used to test results. RESULTS: Sixty-six percent of our physician sample reported that intervening when domestic violence is identified is an essential part of their role as a physician. In contrast, 79% reported that intervening with patients identified for HIV/STD risk is an essential part of their role; 82% with alcohol abuse; and 85% with tobacco use (Chi-square=13.8, p=.003; p * < .032 for domestic violence vs. tobacco and vs. alcohol). Regarding screening, only 10% of physicians reported that they always ask new patients about domestic violence, compared to 22% for HIV/STD risk, 72% for alcohol use, and 84% for tobacco use (Chi-square=188, p < .001; p * < .035 for all 6 pairwise comparisons). When asked about their knowledge of and confidence in screening in these areas, only 19% strongly agreed that they knew how to assess patients for the risk of domestic violence, whereas 47%, 59%, and 70% strongly agreed that they knew how to assess patients for HIV/STD risk, alcohol abuse, and tobacco use respectively (Chi-square=68.3, p < .001; p * < .001 for all 3 pairwise comparisons with domestic violence and HIV/STD vs. tobacco). When asked if they would rather refer domestic violence victims to outside resources than provide counseling themselves, 79% agreed or strongly agreed that they would. This compares with 42%, 51%, and 23% who agreed or strongly agreed that they would rather refer patients identified with HIV/STD, alcohol, and tobacco risks respectively (Chi-square = 74.7, p < .001; p * < .006 for all pairwise comparisons except HIV/STD vs. alcohol). CONCLUSION: These data suggest that, despite physicians' view that screening for domestic violence is part of their role, physicians are not prepared or able to overcome barriers to ask their patients about abuse. It is even more difficult for physicians to screen their patients for the health risk of domestic violence than to screen for the other health risks, including the risk for HIV/ STD, which is also considered a sensitive and stigmatized topic. One solution may be simplifying the physicians' role and augmenting patient care with other experts and assessment tools.\nTHE COMPUTERIZED PATIENT RECORD SYSTEM: A TRADE-OFF BETWEEN PHYSICIAN TIME AND PATIENT BENEFIT. P.A. Glassman 1 , P. Belperio 2 , B. Simon 2 , K. Lim 1 , J. Sayers 2 ; 1 VA Greater Los Angeles, Los Angeles, CA; 2 VA Greater Los Angeles, LA, CA PURPOSE: Improving patient safety is a critical concern. As part of a larger evaluation study designed to reduce adverse drug events, we conducted a baseline survey on clinicians' attitudes towards and knowledge of the Computerized Patient Record System (CPRS) and associated drug interaction alerts.\nWe surveyed 319 clinicians at 12 sites within an integrated VA Healthcare System located in Southern California. Clinicians included attending-level physicians, nurse practitioners and physician assistants. The questionnaire, developed and tested locally, assessed extent of and comfort with CPRS. Questions assessed how CPRS in general, and clinician order entry and drug interaction alerts in particular, have affected clinician efficiency and patient care. Additionally, we assessed general knowledge about 21 common drug-drug and drug-disease interactions. This abstract reports the results of the first 63 (20%) respondents in this abstract. The survey period is expected to end in January 2001, with an anticipated response rate of 65% to 70%. RESULTS: In this preliminary analysis respondents were 71% male, 87% full-time employees and 72% Internists. Participants averaged 48 years of age, practiced 2.5 days per week in outpatient clinics and wrote 53 prescriptions per week. A majority of clinicians (range: 70% to 86%) preferred using CPRS to conventional written methods for entering patient notes, requesting consults, ordering radiological procedures, ordering laboratory tests, and prescribing medications. Overall, clinicians reported that CPRS improved the safety and quality of patient care but reduced clinician efficiency. For example, 81% of clinicians felt that drug interaction alerts increased the potential for prescribing safely but 70% perceived that clinician order entry increased the time required to write prescriptions. Also, while 71% felt that clinician order entry reduced errors in ordering laboratory tests, 68% reported that doing so required extra time. Clinicians recognized a median of 53% (range: 17% to 89%) of 21 common drug interactions. Eighty five percent (85%) of clinicians reported that they would have felt more confident about their answers had they had drug alerts to identify these interactions. In practice, however, respondents reported that they would be more likely to change a patient's medication based on personal interaction with a pharmacist (58%) rather than depend on a CPRS drug interaction alert (4%). Thirty-eight percent were equally likely to change medications based on an alert from either source. Clinicians reported the greatest barriers to effective use of drug alerts included non-relevant alerts (72%), system slowdowns and shutdowns (65%), and lack of time to review alerts (57%). CONCLUSION: Early analysis of an on-going survey suggests that clinicians perceive that CPRS improves the quality and safety of patient care but decreases their efficiency. Clinicians favor the CPRS drug alert system as a means to improve their recognition of drug interactions, an issue that needs to be further addressed based on our data, but note several implementation problems that impede effective utility. PURPOSE: Clinical practice guidelines (CPGs) are being implemented in many large health care systems; yet, little is known about clinician reaction. We evaluated clinician response to a hypertension (HTN) guideline implementation at a large, geographically-diverse VA medical center. METHODS: In the context of a facility-wide implementation of 10 CPGs, the HTN guideline was implemented with 36 attending physicians and nurse practitioners in a randomized trial comparing a general intervention (educational components and a list of the patient's antihypertensive drugs delivered to clinicians at each primary care clinic visit) with an individualized intervention (the general intervention plus patient-specific recommendations about drug therapy delivered to clinicians during primary care clinic visits). For the 4500 hypertensive patients of these study clinicians meeting study criteria, the individualized intervention improved guideline concordance of drug therapy (Med Dec Mkg 2000; 20:488) . At the end of the study period, two physicians, previously unaffiliated with the study, conducted structured interviews with 32 (89%) study clinicians. RESULTS: 31 (97%) clinicians were aware of CPG implementation at the facility and 28 (88%) were specifically aware of the HTN CPG. 29 (91%) and 28 (88%) clinicians, respectively, reported that they use CPGs and that CPGs help with patient care. 16 of 18 (89%) clinicians in the individualized group agreed with the recommendations, but only 3 (17%) reported that seeing the recommendations affected their management of patients. Barriers to following the recommendations included patient reluctance to change medication; concern about need for additional clinic visits and laboratory monitoring of recommended medications; previously demonstrated patient intolerance of the guideline drugs. Time constraints were mentioned frequently. CONCLUSION: Clinicians had a high level of awareness of the CPG implementation and a generally positive attitude toward CPGs. In the context of the randomized trial evidence of the impact of the guideline implementation, the small percent of clinicians who reported that the recommendations affected their management of patients suggests that clinicians may not be aware of the impact of guideline implementation. PURPOSE: Patient adherence to antiretroviral therapy (ART) is critical to effective treatment of HIV and may depend partly on health professionals' practices. However, little is known about providers' knowledge, practices, and barriers in promoting ART adherence. We sought to assess adherence counseling practices among the physicians (MD), pharmacists (PH), and case managers (CM) caring for HIV+ patients in NC and to identify barriers they faced in facilitating adherence. METHODS: In February 2000, using NC AIDS Drug Assistance Program records and commercial prescription records, we identified and surveyed, by mail, the 1,301 MDs, PHs, and CMs caring for HIV+ patients in NC. After 4 mailings, 440 (77%) of the PHs, 94 (85%) of the CMs, and 380 (63%) of the MDs responded. Among the responding MDs, only the 190 reporting having prescribed a protease inhibitor in the last year were included in the analytic sample. RESULTS: 31% of the MDs were general internists, 24% family practitioners, and 22% infectious disease specialists; half cared for < 10 HIV+ patients. 66% of PHs worked in chain pharmacies, 28% in independents and only 2% in hospitals; half cared for < 4 HIV+ patients. On average, CMs cared for 34 HIV+ patients. Regarding the average number of minutes counseling a patient on a new 3-drug ART regimen, PHs spent 7, MDs 13 and CMs 21. MDs seeing a higher volume of HIV+ patients spent no more time counseling but were more likely to: advise patients of side effects (SE), tailor the regimen, explain intake requirements, and provide a pill box (p < .01 for each). Over 90% of MDs explained dosing, asked for patients' questions and discussed drug resistance most or all of the time; < 60% discussed SE management, handling missed doses, ways to remember doses, or planning dose times. Over 70% of PHs explained dosing instructions and asked for questions but < 50% discussed SE management, handling missed doses, drug interactions or storage requirements. The most common CM adherence counseling behaviors were: praising adherent clients (84%), discussing nonadherence repercussions (74%), asking about clients' treatment concerns (74%) side effects (65%), and if medications were taken on time (71%). The vast majority of MDs, PHs, and CMs reported strong interest in doing adherence counseling and had positive attitudes toward ART, ART counseling, and HIV+ patients. The primary barriers reported were lack of space, time, and reimbursement for counseling. MDs viewed CMs' doing adherence counseling as acceptable but were dissatisfied with HIV CM availability. Both MDs and CMs indicated a high interest in improving collaborations with each other in the care of HIV+ patients. CONCLUSION: Innovative practice arrangements between all three provider groups may be needed and feasible to facilitate adherence counseling and address the multifaceted problem of antiretroviral adherence. Practice guidelines for sore throat, nasal congestion and cough illness developed by the Colorado Clinical Guidelines Collaborative were mailed to all primary care physicians belonging to the Colorado Medical Society in November 1999.`P rofiled physicians'' (having at least 10 office visit claims for adults with acute bronchitis in 1998) (n = 352) also received antibiotic prescribing profiles based on aggregated office visit and pharmacy claims from 7 HMOs participating in the Colorado Medical Society Joint Data Project; and``non-profiled'' physicians (n = 514) received summary data based on the entire cohort of patient visits. The office visit was the primary unit of analysis. Multivariate mixed effects models included patient age, physician specialty, HMO and time segments (baseline period = 1/98 ± 10/99; study period = 11/99 ± 2/00) as fixed effects, and unique physicians as random effects. Units of time were measured in months. RESULTS: There were 12,993 and 2,899 adult office visits for acute bronchitis in the baseline and study periods, respectively. The majority of patients were 18 ± 44 years old and treated by family physicians. Prescription rates for family physicians were about 5% greater than for internists (p = 0.006), and were positively associated with increased patient age. Adjusted monthly antibiotic prescription rates for acute bronchitis were stable during the baseline period for both physician groups (profiled: 65% to 63%, p = 0.10; non-profiled: 64% to 63%, p = 0.78), and declined significantly during the study period among profiled physicians (63% to 54%; p = 0.0005), but not among non-profiled physicians. The rate of decline in antibiotic prescription rates during the study period did not differ by patient age (p = 0.09) or physician specialty (p = 0.87). CONCLUSION: This study demonstrates that antibiotic treatment of adults diagnosed with acute bronchitis by physicians in private practice can be reduced using a combination of educational physician profiling and practice guideline dissemination. , 1994 ± 1998 . R. Gonzales 1 , J. Maselli 1 ; 1 University of Colorado Health Sciences Center, Denver, CO PURPOSE: Rising rates of S. pneumoniae resistant to penicillin and other antibiotics have been publicized in the US since 1994, and have led to numerous calls to limit excess antibiotic use in ambulatory practice. This study evaluates whether changes in antibiotic treatment of acute respiratory infections (ARIs) between 1994 and 1998 have occurred, and whether these changes are associated with specific patient and physician characteristics. METHODS: The National Ambulatory Medical Care Survey data files for years 1994 ± 98 were obtained from the National Center for Health Statistics web site. Office visits with a principal diagnosis of otitis media, sinusitis, pharyngitis, bronchitis and upper respiratory tract infections (URIs)/common cold were identified, and further limited to visits to a general or family practitioner (GFP), internist (IM) or pediatrician (PED). Antimicrobial treatment was assigned to the visit only if it was entered as the principal medication entry (out of 6 possible). Multivariate logistic regression analysis was used to evaluate independent associations between antibiotic treatment for ARIs and time (year); patient age, race and diagnosis; and physician specialty and practice location. To determine if a change in antibiotic prescription rates during this period were associated with one of the other variables, interaction terms were utilized. RESULTS: Overall, annual antibiotic prescription rates for ARIs declined between 1994 and 1998 (65%, 64%, 60%, 62% and 55%, respectively; p = 0.0001). This equates to 7 million fewer antibiotic prescriptions for ARIs dispensed by primary care physicians in 1998 compared to 1995 (the peak year). Independent of specific diagnosis and time, antibiotic treatment was less likely for patients age > 45 years (vs. children age < 5 years) (OR = 0.62, 95% CI = 0.51 ± 0.76), and of non-white race (OR = 0.75, 95% CI 0.65 ± 0.88). Because of a significant interaction between time and physician specialty, we stratified further analyses by specialty. Between 1994 and 1998, time was associated with a decrease in adjusted annual antibiotic prescription rates among PED and IM, but not among GFP (relative rate change = À8.9%; p = 0.0001; À9.3%; p = 0.043; and À4.5%; p = 0.17, respectively). Antibiotic prescription rates for specific diagnoses declined variably across specialties: sinusitis: no declines for any specialty; otitis media: PED; pharyngitis: PED; bronchitis: GFP; URIs/colds: IM decline > PED > GFP. CONCLUSION: Primary care physicians are heeding the call to limit antibiotic prescribing in ambulatory practice, although there remains much room for improvement. Changes in prescribing need to be correlated with trends in antibiotic-resistance rates. PURPOSE: Antibiotic treatment of the common cold and upper respiratory tract infections (URIs) by US primary care physicians declined between 1994 and 1998. This study evaluates the amount of public and professional media exposure provided to the topics of antibiotic-resistance and overuse of antibiotics, and the correlation between media exposure and changes in antibiotic treament of colds and URIs during this period. METHODS: Treatment related to primary care physician (general and family practitioner, internist or pediatrician) office visits with a principal diagnosis of the common cold or URI were analyzed using the National Ambulatory Medical Care Survey (years 1994 ± 98) . Key word searches relating to excess antibiotic use and antibiotic resistance were performed on all databases, and final tallies confirmed by manual review. Lexis-Nexis was used to identify US news stories from large-circulation newspapers corresponding to the northeast (NY Times), midwest (Chicago Sun-Times), south (Atlanta Journal-Constitution) and west (LA Times), and one national newspaper (USA Today). The Vanderbilt Television News Archive was used to identify evening news stories on ABC, CBS, NBC, and CNN. Articles from medical journals represented in the Abridged Index Medicus were identified using MEDLINE.``Sentinel articles'' were defined as receiving evening network news coverage by at least 1 network within 3 days of publication. Segmented time series analysis (logistic regression) was used to compare the change in 60-day (the smallest time unit providing adequate sample size) antibiotic prescription rates between time periods corresponding to increases in media exposure. RESULTS: From 1994 ± 98, there were 207 newspaper stories, 31 major network evening news stories, and 65 journal articles identified. The number of newspaper stories reported in 1994 ± 1996 was stable, but more than doubled in 1997, and remained high in 1998. Evening news stories doubled in 1996 and 1997 compared to the previous years, but reverted to baseline levels in 1998. In contrast, journal articles have increased linearly between 1994 and 1998. There were 6 sentinal publications during this period: June 94, January 95, August 95, January 96, September 97 and March 98. Inspection of 60-day antibiotic prescription rates for colds and URIs reveals only 2 major trends: 1) relatively stable rates between 1994 and 1996 (p = 0.73); and 2) declining prescription rates from 1996 through 1998 (parameter estimate = (À)0.036; p = 0.06). CONCLUSION: The change in antibiotic prescribing for colds and URIs that occurred in 1996 appears to be more strongly correlated with an increase in public media exposure than with medical journal publications. However, one can not rule out an impact of other forms of physician education (eg. local or national conferences) on prescribing behavior. PURPOSE: Home glucose monitoring is a common practice in diabetes (DM) treatment, but adds significantly to costs of care. Several studies have questioned the benefit of glucose monitoring in patients with type II DM. We examined the impact of glucose test strips on glycemic control in outpatient DM patients at four VA facilities. METHODS: All patients receiving outpatient DM medications during 1997 were evaluated at four VA hospitals in Pennsylvania. Administrative data identified demographic variables (age, sex, race, marital status), glycosylated hemoglobin (HBA1C), all medications and DM supplies, and type of clinician (resident, nurse practitioner, staff internist, endocrinologist, and other). We examined differences among patients who recieved glucose test strips (strip+) and those that did not (strip-). Simple descriptive statistics was performed comparing strip+ and strip-patients, and analysis of variance was done, including main effects (medication type [oral agents, insulin, and both oral agents and insulin], site of care, and physician type), interaction terms, and demographic variables. Chart review of a subset of patients examined Charlson comorbidity scores, and presence of diabetes complications (neuropathy, retinopathy, and nephropathy). RESULTS: There were 2912 strip+ patients, and 2221 strip-patients. Strips added an average of $22.56 each month to cost of diabetes care. Average HBA1C for strip+ patients was 7.92; for strip-patients, 7.34. There were significant differences for use of strips in patients for site (ranging from 26% to 73% strip+), race (favoring white race), and marital status (favoring married). Differences in HBA1C persisted after adjusting for type of medication, site, and demographic covariates. There was no difference in Charlson comorbidity scores between groups in the substudy, but there were more diabetic complications in the strip+ group (15% more had at least one diabetic complication). CONCLUSION: Glucose control was poorer in patients who received test strips. Differences persisted after controlling for important confounders. Thus, our study does not suggest benefit from home glucose monoitoring in terms of improved glycemic control, although presence of more DM complications in the strip+ group suggests that strips may be used more often in difficult to control patients. Because home glucose monitoring is costly, further study should evaluate the cost-effectiveness and clinical benefit of its use. used to exclude subjects who are likely to be non-adherent with therapy or to experience side effects. While this approach may increase the chances of finding a treatment effect by eliminating non-compliers and subjects likely to experience adverse events, it diminishes the generalizability of the results of the trial, as patients are less likely to be as adherent and more likely to experience side effects. This is especially so if measures such as the``Number Needed to Treat'' (NNT) are used, as the``true'' NNT is likely to be higher than that obtained in the trial if an appreciable number of patients are excluded during the run-in period. As run-in periods would seem to increase the chance of finding a positive result, we hypothesized that there might be an association between the use of run-in periods in the design of randomized trials and the presence of commercial sponsorship, particularly by the pharmaceutical industry. METHODS: We searched all randomized trials published in The New England Journal of Medicine, The Journal of the American Medical Association, The Lancet, and The BMJ, from January 1, 1998 through December 31, 2000 . We included trials that had at least one arm consisting of either PO, dietary, intranasal, or subcutaneous therapy administered by the study subjects, of at least 4 weeks duration. We excluded studies of treatments not administered by subjects (e.g., intravenous or intramuscular treatments) and studies of treatment of acute events (e.g., myocardial infarction or COPD exacerbations), in which setting a run-in period would not be feasible. However, studies of treatments following acute events that did not require acute treatment were included. Trials of treatments during pregnancy were also excluded. For the purpose of our study, we defined a run-in period as follows: Any period preceding randomization during which time subjects took active treatment, placebo, or a dietary intervention to which they might subsequently be randomized. Pre-randomization periods used to assess subjects' symptoms were not considered run-in periods, even if the authors of the studies designated them as such. This is because these screening or``baseline'' periods serve a very different function from the above defined run-in periods. Drug``wash-out'' periods were also not considered run-in periods in our analysis, for similar reasons. Sponsorship was determined based on stated funding source at the conclusion of the article. We considered a study to be industry sponsored if at least one of the funding sources was from a commercial entity (e.g., a pharmaceutical company). We did not consider the provision of medication or placebo to constitute commercial support. All studies were evaluated separately by two reviewers, and determination of the presence or absence, as well as type of run-in period was made while blinded to the source of funding. Likewise, ascertainment of funding source was made blinded to presence or absence of a run in period. Any disagreement regarding presence or type of run-in period was resolved by consensus among the three authors, while blinded to the funding source. RESULTS: There were 214 eligible trials. Of these, 138, or 65%, were industry sponsored. Runin periods as defined above were found in 39 of the trials. Twenty-eight of these were placebo runin, 3 were placebo and diet, 2 were treatment, 2 were diet alone, 2 were treatment and placebo, and in 2 reports the type of run-in was not stated. In only 12, or 31%, of the 39 trials was the number of subjects excluded because of non-compliance or adverse events included in the result. Of the 39 trials with run-in periods, 36, or 92%, were commercially funded. The odds that a trial with a run-in period was commercially funded was 12:1, compared to 1.4:1 for trials without run-in periods (OR = 8.6). CONCLUSION: Run-in periods in clinical trials appear to serve a commercial, rather than a scientific, purpose. As their presence limits the generalizability of the results of these trials, we question their use in the design of randomized clinical trials done for scientific purposes. . Despite demonstrated efficacy of medical therapy in clinical trials, effectiveness of CVD risk reduction in actual practice may be sub-optimal. Our goals were to: 1) determine patterns of hyperglycemia (HG), hypertension (HTN) and hyperlipidemia (HL) management among patients with DM2, and 2) assess whether effective HG management was associated with effective HTN and HL management. METHODS: 601 confirmed DM2 patients attending our outpatient clinics between 3/96 ± 8/ 97 were randomly selected; chart abstraction data were linked to laboratory testing results. We defined 3 components of effective CVD risk reduction: 1) risk marker testing for HbA1c, systolic blood pressure (SBP), and LDL cholesterol, 2) initiation of medical therapy if above goal, and 3) use of high dose therapy if above goal. To assess the relationship between HG, HTN, and HL management, we compared risk marker levels, testing frequency, and medication dose intensity, using linear and logistic regression. RESULTS: Cohort characteristics included: mean age 64.7, 58.4% male, median DM2 duration 6 years, with 73% HTN and 50% HL prevalence. Sub-optimal management effectiveness was greatest for HL compared to HG or HTN. Fewer patients were tested for LDL than HbA1c (Fisher's exact test, p < 0.0001), and among tested patients, significantly fewer were at LDL goal than HbA1c goal (p = 0.045) [Table] . Levels of HbA1c, SBP, and LDL were not significantly correlated after adjusting for BMI. Achieving goal HbA1c did not predict meeting either SBP or LDL goals. There were no significant correlations between HG medication dose intensity (oral HG agent or insulin U/Kg dose) and either HMG CoA reductase (statin) dose or number of HTN medications. To explore factors associated with physicians' decisions to use warfarin in patients with non-valvular atrial fibrillation (NVAF). METHODS: We mailed a self-administered questionnaire to a random sample of general internists, selected from the AMA Masterfile. The instrument included questions on physicians' demographic characteristics, prior experiences and beliefs about the natural history and treatment outcomes for patients with NVAF. Case scenarios described patients with varying degrees of risk of thromboembolism or hemorrhage. Respondents who were in the upper quartile of warfarin use, as defined by the proportion of cases in which they recommended warfarin, were``high-users''. Results were summarized as proportions; chi-square was used for comparisons with a significance level of 0.05. RESULTS: Surveys were returned by 120 of 427 eligible respondents. The mean age was 46 years (79% male; 38% academic). The median perceived risk for TIA and ischemic stroke (CVA) in a NVAF patient who was 76 year old (otherwise healthy, not receiving therapy) were 10% (Interquartile Range -(IQR): 5 ± 20%) and 7.5% (IQR: 5 ± 16%) per year, respectively. For NVAF patients on warfarin, the median risk estimates for TIA and ischemic stroke were 2% for TIA (IQR: 1 ± 5%) and 2% for CVA (IQR: 1 ± 5%); the median annual risk estimate of intracranial hemorrhage (ICH) for these patients was 3% (IQR: 1 ± 5%). Respondents indicated they would recommend warfarin for a median of 9 of the 14 cases (IQR: 7 ± 11 cases). Physician age, gender, perceived risk of ischemic stroke and estimated stroke risk reduction attributable to warfarin were not associated with warfarin use. However, respondents whose estimated risk of warfarin-associated ICH was in the highest quartile were significantly less likely to be high-users than those who provided lower estimates (6.9% vs. 30.8%; p = 0.01). Use of warfarin was also inversely related to the belief that an ICH was a``worse outcome'' than an ischemic strok for a NVAF patient or a``more regrettable'' outcome for their physician. CONCLUSION: There is substantial variability in beliefs about the natural history of NVAF and the risks and benefits of warfarin. Warfarin use was impacted more by estimated risk of intracranial hemorrhage and by the perceived severity and``regrettability'' of this complication than by the potential benefits. However, little is known about the recruitment and evaluation of potential subjects in published trials. We therefore performed a review of published randomized controlled trials (RCT's) to assess the rigor with which enrollment experience was reported, and to analyze the available data. METHODS: We selected all RCT's that were published in the Annals of Internal Medicine, The New England Journal of Medicine, Lancet, or JAMA between April 1, 1999 and March 31, 2000 . From each article, we abstracted the number of patients that were screened by the investigators to determine if they were eligible, the number who were eligible, and the number who were enrolled. Results were summarized as proportions; chi-square tests were used to compare groups with a significance level of 0.05. RESULTS: A total of 172 RCT's were reviewed; the median number of participants was 260 (range 18 to 54,654). Of these, 90 (52%) papers included an estimate of the number of patients that were screened by the investigators for eligibility. The number of patients who were eligible for participation was reported in 74 (43%) studies. Multicenter trials were significantly less likely to report the number of eligible patients (33.9%) than single center trials (58.7%; p = 0.002). Only 54 (31.4%) papers included information about both the number of patients screened and the number eligible. In these studies, the median proportion of screened patients who were eligible for participation was 58.8% (range 1.4% ± 100%); and the median proportion of eligible patients who enrolled was 91.8% (range 35.1 ± 100%). CONCLUSION: Many RCT's published in major medical journals provided incomplete information about their patient recruitment experience. In the papers that did include this information, there was marked variability in the proportion of patients who were eligible and the proportion that enrolled. Clinician-scientists should be encouraged to collect and report this information in order to allow readers to consider the external validity of their work. we considered the RR's to be discrepant when they were not both less than or greater than 1.0.\nTo provide some perspective on the disease burden, we also calculated the ratio of DSM to ACM in the control groups for each cancer type. RESULTS: The RR's were discrepant in 5 of 14 trials. See table for details of RR and disease burden for each screening test. In addition, in one of the trials in which the RR's were both less than 1.0 (Edinburgh mammography trial), the observed difference in ACM was far greater than could be expected on the basis of decreased breast cancer mortality (5 times larger than the observed difference in DSM). CONCLUSION: ACM should be considered in randomized trials of cancer screening because it helps to identify threats to validity (such as the randomization flaws in the Edinburgh trial) and because it provides needed perspective on the magnitude of the benefit of screening. RR's based on ACM and DSM are often discrepant, raising questions about the validity of DSM and the potential for unrecognized harm from screening. For each study, we calculated the absolute difference in mortality rate between the highest and lowest volume strata reported and the number needed to treat (NNT) to prevent one death attributable to low volume. Two investigators independently abstracted each article using a standard form. RESULTS: Of 257 studies reviewed, 128 met inclusion criteria covering 27 procedures and clinical conditions. The methodological rigor of most studies was modest. Few studies used clinical data for risk-adjustment or examined hospital and physician volume effects simultaneously. Overall, 70% of all hospital volume and 74% of physician volume studies reported a significant association between higher volume and better outcomes. The strongest associations were for surgery on pancreatic cancer, esophageal cancer, abdominal aneurysm, and pediatric cardiac problems, and treatment of AIDS (median, 8 to 14 excess deaths per 100 cases attributed to low volume; NNT, 7 to 12). Although statistically significant, the magnitude of the volume-outcome relationships for CABG, PTCA, MI, carotid endarterectomy, other cancer surgery, and orthopedic procedures was much smaller (median, 0.2 to 3 deaths per 100; NNT, 35 to 500). CONCLUSION: High volume is associated with better outcomes across a wide range of procedures and conditions, but the magnitude of the association varies greatly. The clinical and policy significance of this finding is complicated by many methodological shortcomings. Differences in processes of care between high and low volume providers may explain much of the relationship between volume and outcome. (10):769) met all important quality criteria. In that study, 90% of total costs were attributable to drug costs alone, and the cost-effectiveness ratios were > $50,000/QALY for all 240 sub-groups analyzed. The statin dosage range used in that study is achieved at our institution by using half tablets of currently available preparations, and the actual VA costs for statins were 14% of those drug costs assumed in this study. When the VA costs were applied to this analysis, the cost-effectivneness of using statins for primary prevention was < $50,000/QALY for the majority of patient subgroups. CONCLUSION: In at least one large health care system, statins are available at acquisition costs considerably lower than assumed in published CEAs. These acquisition costs permit statin use for primary prevention of CAD to have an acceptable cost-effectiveness ratio. If these drug prices were widely available to consumers, this important category of medication would have great potential for improving the nation's health at reasonable cost. Mechanisms for creating a uniform pricing structure for these widely used medications should be explored. . Qualitative studies suggest that DCs give patients more information and more control over their treatment. Among primary care patients, information and control generally lead to greater satisfaction. We estimate the effects of provider type, explanation of treatment, and self-care advice on satisfaction in a randomized trial of DC vs. MD treatment for low back pain. METHODS: 681 adult low back pain patients were randomly assigned to treatment by either a DC or a MD and were surveyed at baseline, 2, and 4 weeks; follow-up was 99.7%. We used ordinary least squares linear regression to estimate the effects of provider type, explanation, selfcare advice, and other factors on satisfaction with provider. Explanation of treatment was reported as``yes'' or``no.'' Self-care advice was the number of items of advice (0 ± 10) the patient reported receiving. Satisfaction was measured on a 10-item scale (5 = low to 50 = high). RESULTS: The mean satisfaction score was 36.1 for patients in the DC group and 30.6 in the MD group; the crude difference was 5.5 (95% confidence interval [CI] = 4.5, 6.5). Adjustment for sociodemographic and baseline illness characteristics alone did not substantially change these estimates. The mean number of items of self-card advice was 2.3 in the DC group and 1.6 in the MD group (p < 0.001). Sixty-one percent of patients in the DC group vs. 16% in the MD group received an explanation of treatment (p < 0.001). In a regression model that included provider type, explanation, self-care advice, improvement in disability, prior experience with and confidence in treatment, baseline sociodemographic and illness characteristics, and interaction terms, the DC-MD difference in satisfaction was 4. PURPOSE: Although patient demand is high, physicians have not widely adopted use of e-mail with patients. Our purpose was to explore the experiences of physicians frequently using e-mail with their patients, as a window to the future. METHODS: To identify the rare``vanguard'' physicians who frequently use e-mail with their patients, we recruited 11,859 physicians using Physicians' Online, an Internet professional information portal, to participate in a survey. To identify frequent users of e-mail, we asked``In a typical day, please estimate the average number of e-mails you and your immediate staff receive from patients who are currently in your care.'' Additional questions assessed common clinical topics in e-mail, physician's adherence to published guidelines from the AMA and American Medical Informatics Association, and physician satisfaction, measured by``Would you recommend to a colleague use of e-mail with patients.'' RESULTS: Among the 1,325 physicians responding (including individuals from all 50 states), we identified 204 frequent users who received one or more e-mails from patients daily. The mean age was 49 years, 82% were male, and 35% were primary care providers. Participants were from a variety of specialties including adult medical subspecialties(9%), pediatrics(11%), surgery(11%), and psychiatry(7%). Common clinical topics, received frequently or sometimes by over 40% of physicians, include: non-urgent new symptoms, questions about lab results, and questions about information on the Internet. Although infrequent, 7% of our sample did report frequently or sometimes receiving e-mails about urgent issues such as chest pain. The most common practice adherent with published guidelines was including the e-mail in the medical record (38%), but they rarely incorporated other guideline issues such as educating patients about appropriate topics (10%). Despite daily use of e-mail with patients, 25% of the 204 were not satisfied. Physicians who were not satisfied more frequently (80%) reported``patient request'' as the most important reason for using e-mail, compared with those who were satisfied (39%). (p < 0.01). Compared with dissatisfied physicians, physicians who were satisfied with e-mailing patients more frequently noted a decrease in their amount of telephone medicine (29% versus 2%; p < 0.001), and were more likely to use the emails to educate their patients (25% versus 14% p < 0.01). CONCLUSION: Experiences of these``vanguard'' physicians were mixed. Although patient demand is high, physician satisfaction of e-mail with patients may also be important to sustained use of e-mail with patients. Our data suggest that increased dissemination/adherence of published guidelines and increased perception of the efficiency of e-mail communication may improve acceptance. PURPOSE: Continuity of care is a highly desirable characteristic that may be threatened by recent changes in the health care system. We sought to assess patients' perceptions of continuity of care and the relationship between continuity and satisfaction in a managed care organization. METHODS: Within a randomized controlled trial of a patient-provider matching intervention in a group model health maintenance organization (HMO), we evaluated whether subjects reported usually seeing their own primary care provider (PCP), and the association of this perception with satisfaction and trust. Between October 1999 and September 2000, 2501 (64%) subjects completed a mailed, self-administered questionnaire, one year after receiving a new PCP. RESULTS: Among patients who had seen their PCP at least once during the study period, the majority (76%) reported usually seeing their own PCP during their visits. These subjects tended to be older (58 v. 52 yo, p < 0.0001), white (75% v. 69%, p < 0.02), and had more visits during the past year to their PCP (3.6 v. 3.2 visits, p < 0.04) than subjects who reported usually seeing another provider, i.e. lacking continuity of care. Moreover, compared to subjects without continuity, subjects perceiving that they had continuity reported greater satisfaction with their PCP and the health system. For example, subjects reporting that they usually saw their PCP had significantly higher levels of satisfaction with their PCP (72% v. 38% reporting excellent or very good overall satisfaction, p < 0.0001). These subjects were more likely to recommend their PCP to others (82% v. 52%, p < 0.0001), reported greater trust in their PCP (p < 0.0001), and in the health system (p < 0.0001). In addition, these subjects also were less likely to perceive that the PCP created barriers to access, such as with specialist referrals (p < 0.0001). CONCLUSION: In short, the majority of subjects in this study perceived that they usually saw their own PCP during visits. Furthermore, the perception of continuity of care with one's PCP was associated strongly to satisfaction and trust in the PCP, and also in the health system. Further research is needed to assess the direction of this association, as well as to identify potential areas for organizational improvements. Despite a growing focus on this problem many health care organizations provide inadequate interpreter services. A principal reason is the concern that uncertain benefits do not justify the costs of adequate services. The objective of this study was to assess the impact of an interpreter service program on the utilization and cost of health care services at a staff model HMO. METHODS: We conducted a 2-year cohort study of continuously enrolled adult members of a staff model HMO where new comprehensive interpreter services for Spanish and Portuguesespeaking ambulatory patients were implemented in year 2 of the study. Two groups were studied: an interpreter service group (ISG, n = 380) consisting of members who used the new interpreter services and a comparison group (CG, n = 4119) consisting of a 10% random sample of all other members who received ambulatory care in year 2. We abstracted demographic information and utilization of primary health care services (preventive services and outpatient services) and hospital-based services (ED visits and hospitalizations) from the HMO's administrative database. We calculated the unit cost per interpretation based on the cost of the services (salaries, fringe benefits, supervision, and overhead) and reported volume of use of services. We calculated induced costs of interpreter services by multiplying the change in utilization for each health care service by its 1997 fee-for-service reimbursement rate from the Massachusetts' Division of Medical Assistance. RESULTS: Utilization of primary health care increased in both groups after implementation of interpreter services. The changes (yr2-yr1) in utilization of preventive services (p < 0.05), utilization of office visits (p < 0.01), prescriptions filled (p < 0.01) and prescriptions written (p < 0.01) were significantly greater in the ISG compared to the CG. Utilization of hospitalbased services remained the same for both groups, except for a reduction in ED use by the ISG. The change in rate of ED use (yr2-yr1) was not significant when compared to the CG. However, relationships between patient satisfaction and other performance measures are poorly studied. The goal of this study was to examine the relationship between patient satisfaction and severity-adjusted mortality rates. METHODS: The current study used data from a regional initiative to measure performance in 31 hospitals in Northeast Ohio during 1991 ± 1995. Satisfaction was assessed using the Patient Judgement System, a 41 item survey mailed to patients after discharge. Responses were obtained from 40,344 medical patients (response rate 48%). We utilized 5 scales (physician care, nursing care, coordination of care, information provided, and discharge instructions) and 1 single item assessment (overall quality). For each hospital, mean scores were determined, adjusting for self-reported health status, age, gender, education and other demographic factors. Severity-adjusted mortality was determined for patients with 6 medical diagnoses. For each hospital, observed mortality was compared to predicted mortality derived from validated disease-specific multivariable models that were based on data abstracted from medical records. Comparisons of observed and predicted mortality rates were summarized using the z-statistic. RESULTS: Mean satisfaction scores for the 5 scales and 1 single-item indicator ranged from 68.0 (information provided) to 72.6 (physician care). Z-statistics for mortality rates ranged from -7.6 to 4.6. In analyses including data for all 5 years (1991 ± 95), patient satisfaction scores, at a hospital level, were inversely correlated with mortality for each of the 6 measures of satisfaction. from each group who met these criteria: > half-time practice for the last 2 years, > 40% minority patients, and providing primary care of HTN. The 2-hour sessions with 2 ± 4 observers were audiotaped. A facilitator elicited comments and discussion on JNC6 recommendations, perceived barriers, efficacy of traditional guideline interventions, and recommended solutions for future HTN care. One investigator analyzed tapes for emerging themes and concepts. The resulting summaries were distributed to the research team to discuss alternative interpretations. All focus group observers did a final reading of the summary to confirm the validity of the themes and conclusions. RESULTS: Similar themes emerged in the two groups. Physicians felt the JNC6 recommendations were realistic but that physician behavior was not the problem with implementation. Physicians outlined the barriers to optimal control of HTN: poor office systems for tracking care, inability to address lifestyle modification in office visits, impracticality of self-monitoring, the disincentive of co-pays, lack of generic samples for cost-effective prescribing. There was no interest in traditional physician directed interventions such as seminars, academic detailing, incentives for chart review to change therapy, pocket or wall versions of the guidelines, or practice profiling. Physicians suggested making monitoring more practical, providing generic samples, and utilizing group appointments, pharmacies, the internet, and ethnically sophisticated mass-media campaigns for patient education. CONCLUSION: Physicians agreed with the goals and recommendations of the JNC6 HTN guidelines but felt that existing efforts to improve guideline compliance by changing their behavior are bothersome and ineffective. They suggest intervening with the patient to address barriers outside the control of their practices. Future research should examine patients' perceptions of the acceptability and efficacy of these interventions. METHODS: This retrospective cohort study evaluated consecutive ICU admissions to a VA hospital (n = 1,142) and 27 private sector hospitals (n = 51,249) serving the same health care market in 1994 ± 95. Mortality and ICU LOS were adjusted for severity of illness using APACHE III, a validated method that considers age, comorbidity, ICU admission diagnosis, admission source, and abnormalities in 17 physiologic variables during the first 24 hours of ICU admission. We used two multivariable statistical methods to estimate the risk of death in VA patients, relative to private sector patients. 1) Logistic regression provided the odds of inhospital death in VA patients. 2) Cox proportional hazards regression was used to account for potential differences in LOS and in the timing of death by censoring patients at the dime of discharge. RESULTS: Unadjusted in-hospital mortality was higher in VA patients (14.5% vs. 12.0%; p = .01), as was hospital (28.3 vs. 11.3 days; p < .001) and ICU (4.3 vs. 3 .9 days, p < .001) LOS. Using logistic regression to adjust for admission severity of illness, the odds of death was similar in VA patients, relative to private sector patients (OR 1.16, 95% C.I. 0.93 ± 1.44; p = .18). However, a higher proportion of VA deaths occurred after 21 hospital days (33% vs. 13%; p < .001). Using proportional hazards regression, the risk of death was actually lower in VA patients (hazard ratio, 0.70, 95% CI, 0.59 ± 0.82; p < .001). This result was consistent in stratified analyses of medical (hazard ratio, 0.80, p = .02) and surgical (hazard ratio, 0.46, p = < .001) patients. After adjusting for severity using linear regression, differences in ICU LOS were no longer significant (p = .19). CONCLUSION: Severity-adjusted mortality in ICU patients was lower in a VA hospital than in private sector hospitals in the same health care market, based on proportional hazards regression. This finding differed from logistic regression analysis, in which mortality was similar. This difference suggests that comparisons of hospital mortality between systems with different hospital utilization patterns may be biased if LOS is not considered. Moreover, if generalizable to other markets, our findings suggest that ICU outcomes are at least similar in VA hospitals compared to private sector hospitals. BACKGROUND: The evidence to support colorectal cancer (CRC) screening has become definitive though it is well known that most adults do not receive the screening that is recommended. METHODS: We merged patient self-report data from 1998 for respondents > =50 years (n=20,199) with surveys of medical directors of 53 medical organizations [29 medical groups (MG) and 24 Independent Practice Associations (IPAs)] from 3 west coast states to evaluate rates of CRC screening and patient and organizational predictors of those rates. We used logistic regression adjusted for intra-organization clustering of patients within medical organizations to estimate CRC screening rates as a function of patient demographic and comorbid characteristics, receipt of care within a MG or IPA, receipt of care within an organization that uses their corporate structure to implement guidelines, tracking, or feedback of CRC screening results, and the unique identity of each of the medical organizations. RESULTS: Overall, screening rates were modest [45% for sigmoidoscopy or colonoscopy during the last 5 years (SIG), 43% for fecal occult blood tests by the patient at home (FOBT), 64% for either SIG or FOBT, and 24% for both]. Screening rates varied by demographic characteristics. After adjustment for comorbidity, use of any CRC screen was significantly lower for females (OR 0.69 *** ), Asians (OR 0.73 *** ), Hispanics (OR 0.86 * ), and those from other nonwhite race (OR 0.76 * ); screening rates were higher for African Americans (OR 1.3 ** ). Respondents receiving care in IPAs reported less screening than those in MGs (OR 0.83 * ) after adjustment for patient characteristics as well as use of guidelines, tracking and feedback pertinent to CRC screening, and clustering of patients within organizations. In addition to the amount of variation in CRC screening explained by patient characteristics alone, receipt of care in a MG or IPA and corporate policy for use of guidelines, tracking or feedback for CRC screening explained 8% of the unique variation in CRC screening rates, while the unique identity of the 53 medical organizations explained 51% of the unique variation. CONCLUSION: Although there is no clinical reason demographic characteristics should influence rates of CRC screening, they do. In addition to demographics, the corporate structure of the medical organization is associated with rates of patients receipt of CRC screening such that screening occurs more frequently within MGs than IPAs. Even after adjustment for patient characteristics, the use of guidelines, feedback, and tracking of guideline performance, and whether the patient received care in a MG or IPA, most of the variation in screening is explained by the unique identity of their medical organization. PURPOSE: State medical associations may choose to participate in public debates surrounding physician-assisted suicide (PAS). However, physicians lack consensus on the ethics of PAS and its legalization. We examined physician attitudes about PAS and the prospects for consensus regarding its legalization. METHODS: Anonymous questionnaires were mailed to all (1456) members of the Connecticut Chapter, ACP-ASIM. The survey instrument measured attitudes toward PAS; consensus on health policy issues generally; participation in public discussions about PAS; prospects for consensus on its legalization; demographic data including age, gender, experience caring for terminally ill patients, religious affiliation, and religious service attendance. The proportion of foreign-born residents in the United States is at its highest point in over 100 years. Provision of interpreter services for non-English speaking patients is now a federal requirement and general internists will need to use interpreters to care for these patients. We surveyed clinicians in three academic generalist practices to describe how interpreters are being used. METHODS: We surveyed all of the primary care internists and nurse practitioners (n=194) in three academic outpatient settings in San Francisco regarding their most recent patient encounter which involved an interpreter. Questionnaires were self-administered and took about 10 minutes to complete. RESULTS: 158 questionnaires were completed (81% response rate). Among the respondents, 53% were women, 64% White, 66% residents, 78% spoke and understood a language other than English, and 60% had some prior training regarding working with interpreters. Reflection of the most recent patient encounter requiring an interpreter showed that 67% used professional interpreters, 49% felt that not enough time was allotted for the appointment, 90% felt satisfied that they had accomplished their own goals during the encounter, and 83% felt satisfied that the patient had accomplished his/her goals. Most respondents (78%) were very satisfied or satisfied with the medical care they provided, 85% felt satisfied with their ability to diagnose a disease and treat a disease, but only 45% were satisfied with their ability to empower the patient with knowledge about their disease, treatment or medication. Even though 71% felt they were able to make a personal connection with their patient, only 33% felt they had learned about another culture as a result of the encounter. Clinicians had trouble eliciting exact symptoms (70%), explaining treatments (57%), and eliciting treatment preferences (51%). Almost half (45%) felt that too much time was spent on translation. A majority of clinicians perceived that lack of knowledge of the patient's model of disease and lack of knowledge of the patient's culture hindered their ability to provide quality medical care. However, only 18% felt they were unable to establish trust or rapport. CONCLUSION: Although most clinicians feel good about their encounters with patients in which they utilize an interpreter, many of them have received no training regarding that use, and their ability to elicit information, convey information, understand the patient in his/her cultural context, and empower the patient with regard to his/her own healthcare appears to be compromised. We conducted a controlled trial of a guideline-derived intervention, which included 3 components: 1) training of clinic nurses and medical assistants to use a modified vital signs stamp in identifying and counseling smokers at each visit, 2) feedback to clinic staff on performance of AHRQ guideline-recommended activities, and 3) proactive telephone counseling by a smoking cessation counselor (2 sessions) and free nicotine replacement therapy (NRT) for all pts who indicated willingness to make a quit attempt within 30 d of their clinic visit. After a period of baseline data collection (8/99 ± 1/00), the intervention was implemented at 1 pilot family practice (FP) clinic (2/00 ± 3/00); patterns of usual care were observed concurrently at 5 control FP clinics. We obtained exit interviews of consecutive adult smokers who presented for routine, non-emergency care. Abstinence (no cigarettes over prior 7 d) and quit attempts were determined by telephone interview during 6 mo follow-up. Odd ratios (OR) were computed for each outcome. RESULTS: There were no significant differences in demographic or smoking variables between baseline and intervention pts, 87 and 85% of whom completed 6 month follow-up. Concordance with guideline recommendations at the clinic visit and quit status are shown below ( * p < 0.05). For comparison, the 6 mo quit rate for pts at control clinics during baseline and intervention periods was 12 and 10%, resp. 37% of intervention group pts received telephone counseling plus NRT. CONCLUSION: Effective reduction of tobacco use requires redesigning health care systems to increase the identification of smokers and the delivery of smoking cessation advice in a timeefficient manner. The 9% absolute difference in 6 mo quit rates associated with this nursebased, primary care intervention is comparable to that observed in clinical trials of NRT. Cleveland between 1991 and 1997. Patient level data were obtained through the Cleveland Health Quality Choice (CHQC) program; mortality was obtained by linking CHQC data with MEDPAR files. Pneumonia was identified from secondary ICD-9-CM codes. Cox regression analysis was used to estimate the effect of pneumonia on mortality up to 30 days from admission after adjusting for severity of illness using a model of 30-day predicted mortality from admission data (c-statistic = 0.78). Patients dying or having a``Do Not Resuscitate'' order within 3 days of hospitalization were removed a priori from the regression analysis to focus on those for whom avoiding pneumonia was most pertinent. RESULTS: Pneumonia occurred in 6.9% (n = 985) of all patients hospitalized for acute stroke. There was a significantly higher incidence in patients: with hemorrhagic stroke (11.5% vs 6.3%,p < .001), men (9.0% vs 5.4%, p < 0.001), from nursing homes (11.4 vs 6.4%,p = 0.001), aged > 74 (7.2% vs 6.3%, p = 0.032), and with greater severity of illness on admission (predicted 30-day mortality 24.8% vs 13.2%, p < 0.001). Average length of stay for patients with pneumonia was 16.2 0.44 days compared to 7.5 0.05 days in patients without pneumonia (p < 0.001); fewer patients with pneumonia were discharged to home (12.0% vs 41.4%, p = 0.001). Crude 30-day mortality rates were 37.4% for patients with pneumonia and 13.0% for those without (p < 0.001). There was a steady increase in the adjusted hazard ratio for death among stroke patients with pneumonia throughout the 30-day period; adjusting for admission severity, the hazard ratio for death within 30 days was 4.31 (95% C.I. 3.55 ± 5.23). CONCLUSION: In this largest community-wide study of stroke complications, pneumonia conferred significantly higher severity-adjusted hazard for mortality within 30days of admission and was associated with dramatically longer lengths of hospital stay and reduced chances of discharge to home. Measures to reduce the incidence of pneumonia following stroke are warranted to reduce morbidity, mortality, and cost. , and personal experience with BRCA. The dependent variable, a sum score of attitudes towards BRCAS, was calculated such that a higher score indicated a more favorable attitude towards screening. Univariate and multivariate analyses with backwards ± stepwise regression assessed the relationship between the dependent and independent variables. RESULTS: The response rate for the survey was 82% (N = 140 responses); 120 physicians provided complete data for analysis. Of these physicians, 62% were male, 60% were under 30 years old, 12% worked in a private office, 42% worked in a health center, 46% worked in an outpatient setting, 38% had some personal experience with BRCA, and 68% had some professional experience with BRCA. In both univariate and (multivariate) models professional experience p = 0.006 (0.03) was associated with a more favorable attitude concerning BRCAS, but age p = 0.10 (0.54), gender p = 0.54 (0.59), practice type p = 0.23 (0.62), and personal experience 0.54 (0.37) were not. CONCLUSION: Prior professional experience of the physicians, but not demographic or personal variables, was significantly related to a favorable attitude towards BRCAS. Our data differs from previous studies, which found that BRCAS may be influenced by physician characteristics like age, gender, practice type, and personal experience. Increasing awareness through the education of physicians has been a focus of many efforts to increase the incidence of screening for BRCA. If professional experience is associated with a favorable attitude towards BRCAS, then medical trainees should be exposed to environments where BRCAS is a high priority in an effort to increase BRCAS in women. (4.4 ± 29.4, 95%CI) . Most commonly cited missing elements that were needed included current notes types that were not online (33%), notes predating the system (20%), EKGs (16%), and vital signs (12%). On the same Likert scale, 75% agreed that they could give up the traditional paper chart in favor of the mobile computer. CONCLUSION: Overall, internal medicine residents responded positively to the implementation of the mobile CPRS system. Based on surveys and computerized order logs, all residents voluntarily used the devices to enter nearly half of orders over available desktop computers. Reported difficulties with the technologies did not appear to significantly affect the mobile computer users ordering pattern, suggesting that the technical problems were limited and the technology was robust. Finally, feedback from this study was sufficiently positive to justify implementation to all internal medicine teams. After one year, we assessed their impact on patient length of stay as well as resident education and service from the viewpoint of both medical residents and community physicians. METHODS: Two clinician educators were hired to provide inpatient attending medical care and resident education, including 1:1 intern mentoring. Length of stay data was calculated for Medicaid and self-pay patients based on DRG groups where > 50% of patients were admitted to the clinician educator service and was compared to data on the same patient mix from the preceding year. Categorical medicine residents (N=36) were given an anonymous survey assessing the clinician educators' impact on resident education and service, including resource utilization and resident roles. Community internists (N=150 on staff, 100 who actively use hospital) also received an anonymous survey assessing their perceptions of hospitalist medicine and the clinician educators' impact on housestaff behavior. RESULTS: Length of stay: LOS was reduced from 5.3 to 4.4 days comparing the last 6 months of the clinician educators' first year with the last six months of the previous year. Reductions in LOS > 1 day were found with GI, pulmonary, infectious disease, endocrine and neurology admissions and 1/3 of a day for cardiology admissions. Resident survey: Return rate was > 90% with all residents noting improvement in the quality of attending rounds, bedside teaching and the overall inpatient experience. Increased use of evidence based medicine resources was noted by 90%. Residents' roles as teachers and team leaders were increased or unchanged for over two-thirds and none felt they had been reduced to unacceptable levels. A potential influence on these behaviors is the availability of pharmaceutical samples. A prospective blinded study was designed to evaluate this influence in an academic clinic. METHODS: The antibiotics in the drug closet of our teaching clinic were manipulated over 20 consecutive weeks. The following changes were made at 4 week intervals: 1) removal of all antibiotics, 2) removal of macrolides, 3) removal of quinolones, 4) removal of cephalosporins, 5) removal of amoxicillin/clavulanate and nitrofurantoin. Upon completion of the study, a chart review was conducted to identify acute infectious illnesses for which an antibiotic was likely to be prescribed. Datapoints collected include: clinical diagnosis, antibiotic prescribed, postgraduate year (if a resident), date of service, and patient's gender and age. Fishers Exact 2-sided tests were performed to determine whether the presence of antibiotics in the closet influenced their frequency of use. RESULTS: 465 encounters fulfilled our inclusion criteria. 77% of these encounters led to an antibiotic prescription. Of these, 73% had an antibiotic prescribed that could be found in the drug closet. Defining the relative risk as the risk of being prescribed a specific antibiotic if it was available in the closet versus if it were not, the relative risk ratios (RR) with their associated 95% confidence intervals (CI) for each class of antibiotics are listed in the following table. CONCLUSION: The prescribing practices of clinicians in our academic clinic do not appear to be directly influenced by the availability of antibiotic samples in a drug closet. Although a direct relationship between the drug closet and the prescribing practices of clinicians was not observed, it is notable that 73% of all prescriptions were for antibiotics that could be found in the drug closet. This still suggests that pharmaceutical marketing exerts an influence on clinician antibiotic prescribing practices. is important that primary care physicians be aware of the safety of drugs in pregnancy given to nonpregnant women, as well as the effect of common medical problems in pregnancy. For the same reasons, it is also important that counseling and prevention of risk factors known to affect pregnancy outcome occur prior to conception. The purpose of this study was to assess the knowledge and practice of preconception care by primary care attending physicians. METHODS: All primary care attending physicians in general internal medicine and family practice at our institution were given a written survey regarding their knowledge and practice of preconception care. Knowledge questions assessed were 1) safety of medication use in pregnancy 2) risk associated with diabetes in pregnancy, and 3) the rate of unplanned pregnancies and estimated gestational age at presentation to a pregnancy provider. Practice questions included counseling of the nonpregnant patient regarding 1) the hazards of smoking, alcohol and illicit drug use 2) folic acid supplementation, 3) rubella screening, and 4) the effect of chronic medical disorders were pregnancy to occur. Surveys were mailed. Responses were kept anonymous and a number was used to identify participants. RESULTS: Of 25 surveys mailed, 21 were returned. Analysis was done of the returned surveys. 80% of responding attending primary care physicians were aware that 50% of women present to their pregnancy provider after organogenesis and that similar rates of pregnancies are unplanned. 45% counseled women with substance abuse of potential effects on pregnancy, 40% recommended folic acid use, and 10% screened for rubella. 55% counseled women with chronic disease of deleterious effects on pregnancy. Safety of medications in pregnancy were correctly identified by 35% for pain relievers, 45% for antibiotics, 25% for asthma medications, and 10% for antihypertensives. Only 45% of providers were aware of the risk associated with diabetes in the first trimester. No statistical difference using Chi Square analysis was found when the results were computed by the sex of the provider or by primary care specialty, possibly due to the small sample size. CONCLUSION: Despite the knowledge that women often do not plan pregnancy and do not present to a pregnancy provider early enough to prevent the toxic effect of medications, chronic medical disorders, or personal behaviors, the majority of primary care providers were not knowledgeable regarding preconception care and did not incorporate it into the continuing care of women of childbearing age. PURPOSE: The quality of care provided to patients hospitalized for heart failure has been shown to vary by physician, hospital and region. Hospitalists are physicians whose primary professional focus is the general medical care of hospitalized patients. While hospitalists appear to reduce costs and length of stay, their impact on quality of care is less certain. We sought to compare the quality of care provided to patients with heart failure by hospitalists and nonhospitalist physicians. METHODS: We conducted a retrospective medical record review at a 550 bed communitybased teaching hospital in Massachusetts that is home to 3 hospitalist groups as well as numerous small group practices that provide care for their own hospitalized patients. We identified all patients cared for by hospitalists and by non-hospitalist general internists who were discharged with an ICD-9-CM principal diagnosis of heart failure between 4/1/99 and 3/30/00. We evaluated quality of care by measuring adherence to a set of process measures derived from the JCAHO's``Core Measures Set'' and HCFA's Health Care Quality Improvement Initiative. PURPOSE: There has been an increasing amount of research in the area of religion and spirituality and its effect on patient care and health outcomes, but how this should be incorporated into physicians' practice has been an area of debate. We evaluated the extent to which general internal medicine residents and faculty acknowledge a personal belief in spirituality and religion; and to what degree internists feel it is appropriate to incorporate religion and spirituality into health care. METHODS: A twelve-item survey of the general internal medicine residents and faculty at Wake Forest University. RESULTS: 87/112 physicians responded (response rate 77% of residents and 82% of faculty), and an impressive majority (90%) of respondents professed a belief in God or other higher power. 82% considered themselves to be religious or spiritual. An overwhelming 92% felt that patients' beliefs can influence their health. 71% agreed that it would be helpful to inquire about patients' religious/spiritual concerns relating to their health. However, less than half (47%) of respondents felt there is a role for direct physician involvement in meeting patients' religious or spiritual needs, and even less (43%) had actually been directly involved in patients' spiritual care. CONCLUSION: A vast majority of respondents profess their own personal religious and spiritual beliefs, and feel that religion and spirituality can have a positive effect on health outcomes. Yet only a minority feels that doctors should actively pursue any action in this area, and even fewer have actually done this. This is a striking disconcordance between physician attitude and action. Given the growing amount of evidence that suggests religion and spiritual factors can affect health outcomes, efforts should be made to determine what barriers lie between these attitudes and actions, and improve physician education on how to approach these important patient-centered issues. PURPOSE: HMOs' financial incentives to restrict medical care have generated public concern about the quality of care they provide. Public disclosure of quality of care data might improve public accountability, enhance informed consumer decision making and promote quality\nimprovement. Yet, public disclosure of this data is voluntary. We sought to determine the association between HMO quality of care and willingness to publicly disclose quality of care scores. METHODS: We analyzed data from the National Committee for Quality Assurance's annual Quality Compass databases, including the Health Plan Employer Data and Information Set (HEDIS), a standardized set of quality of care measures (e.g. % of women > 50 who received biannual mammography). We determined how frequently HMOs that publicly disclosed HEDIS scores in 1997 or 1998 withdrew from public disclosure the subsequent year. We also assessed whether withdrawal from public disclosure was associated with being in the lowest (compared with the highest) tertile of plans ranked by HEDIS scores for 9 clinical quality measures, after adjustment for the method of data collection (administrative vs. medical record review). RESULTS: Of the 329 HMOs which publicly disclosed HEDIS scores in 1997, 161 (48%) withdrew from public disclosure in 1998. Of the 292 HMOs which publicly disclosed in 1998 (including newly disclosing plans), 67 (23%) withdrew from public disclosure in 1999. Quality varied greatly between plans with absolute differences in HEDIS scores between lowest and highest tertile plans ranging from 14.6 to 42.3 percentage points. When HEDIS measures were analyzed individually, HMOs ranked in the lowest (compared with the highest) tertile in 1997 were significantly more likely to withdraw from public disclosure in 1998 for 7 of the 9 measures (RR = 1.6 ± 2.7, p < 0.05 for all). In 1999, HMOs ranking in the lowest tertile in 1998 for 8 of the 9 measures were significantly more likely to withdraw from public disclosure (RR = 1.9 ± 7.0, p < 0.05 for all). When examined by average rank across all 9 measures, plans in the lowest tertile were significantly more likely to withdraw from public disclosure in 1998 ( Reprints and monographs were excluded. Clinical information was reviewed by two general internists and compared to information in Goodman s 9th edition textbook of pharmacology, in the pharmacology section of the UpToDate version 8.2, or in both. References cited were reviewed for correct citation and accessibility in any of the four major medical libraries in Buenos Aires. RESULTS: Of the 64 pieces of printed promotional materials collected, 30 were randomly selected and evaluated. In 21 (70%) of the thirty promotional printed materials evaluated the therapeutic effect promoted in the advertisement appeared in the references used. Only 18 (60%) had statements supported by cited references. From a total of 131 references cited in printed promotional materials, 60 (46%) were incorrectly listed according to the International Committee of Medical Journal Editors. These references were also inaccessible. Of the 71 references correctly cited, 49 (69%) were not available in any of the four major medical libraries in Buenos Aires and 8 (11%) were available in only two of the libraries. Twenty-two references were read, and in only 12 (54%) of these, did the objective of the research study concur with the statement of the printed promotional material. Adverse drug reactions, warnings about drug interactions and drug contraindications were absent from all printed promotional materials. CONCLUSION: The printed promotional materials distributed by pharmaceutical sales representatives in Buenos Aires are biased and provide misinformation more often than not. We recommend that practicing physicians should routinely disregard printed promotional materials as a source of clinical information. PURPOSE: Hospitalist physicians who specialize in inpatient care are rapidly increasing in number but there is limited evidence from randomized trials concerning their effects on resource utilization and outcomes or how they achieve their effects. This project aims to determine the effect of hospitalists on resource use and outcomes on a general medicine service in an academic medical center and the mechanism for their effects. METHODS: A longitudinal trial from July 1997 ± June 1999 with all patients admitted every fourth day assigned to teams led by hospitalist physicians (HPs) who care for inpatients 6 months per year versus teams led by non-hospitalist physicians (NHPs) who care for inpatients 1 or 2 months per year. Resource utilization was measured by length of stay and costs. Patient outcomes were measured by 30-, 60-, and 365-day mortality rates, readmission rates, reported physical function, and patient satisfaction. RESULTS: Of 6511 admissions to the general medicine service, 24.8% were to HPs and 75.2% to NHPs. Patients cared for by HPs and NHPs did not differ in age, race, gender, diagnosis mix, Charlson index, or payer mix. Average length of stay for the general medicine service was 4.7 days and average cost was $8517. In multiple regression analysis controlling for diagnosis with DRG weight and for comorbidity with Charlson index, HPs did not have different length of stay or costs than NHPs in year 1, but HPs had 0.5 day lower length of stay (p < 0.01) and $740 lower costs (p < 0.01) in year 2. There were no differences in mortality in year 1, but in year 2 HPs had lower mortality at 30 days (4.2% vs. 6.0% for NHP, p < 0.04), and 60 days (8.8% vs. 6.8%, p < 0.07). There were no statistically significant differences between HPs and NHPs in 365-day mortality rate, readmission rate, physical function, or overall patient satisfaction, but the trends favored HPs in all these measures. In analyses controlling for diagnosis-specific fixed effects, month of admission, total volume of patients seen by the physician to date, and total volume of patients with the same diagnosis seen by the physician to date, the effect of hospitalists on length of stay and costs, but not mortality, was explained by diagnosis-specific clinician volume to date. (1) preference rankings of reasons for selecting drugs in the treatment of patients with CAP, (2) knowledge and attitude questions regarding antimicrobial drug prescribing, and (3) measurement of thresholds for switching from hypothetical older drugs with increasing resistance to newer drugs with much less current resistance. Physicians were sampled from the AMA Masterfile and surveyed by mail with two follow-up mailings. Responses were calculated for the overall sample as well as stratified by clinical specialty. RESULTS: 833 out of 1582 physicians (53%) completed the survey. Response rates did not differ across specialty, gender, or US region. Overall, physicians ranked efficacy, severity of illness, and prior experience as the top reasons for selecting antimicrobial drugs, and this ranking was consistent across specialty. Concern over contributing to rising antimicrobial resistance was ranked low by both ID specialists and generalists. Although 88% agreed that`a ntibiotic resistance is a major health problem'' and 89% agreed that``over-prescribing antibiotics is a major cause of antibiotic resistance,'' only 54% agreed that``before prescribing an antibiotic, I weigh the potential benefit to the patient against the potential harm to society.'' In a hypothetical scenario for the treatment of an outpatient with CAP due to S. pneumoniae, 55% of generalists and 67% of ID specialists were unwilling to accept a 20% or greater level of resistance with an older drug before switching to a newer drug without resistance (p=.01). Physicians reported a higher threshold for switching to a newer antibiotic if they agreed that before prescribing an antibiotic, they weigh the potential benefits to patients against the potential harms to society. CONCLUSION: Many physicians report that societal issues created by antimicrobial drug resistance have at most a small impact on their prescribing decisions. These attitudes were generally consistent across different response tasks and between generalists and ID specialists. Since efforts to reduce antibiotic resistance focus on increasing provider knowledge about the societal risks of these drugs, these results suggest that such efforts alone will have limited success. PURPOSE: Primary care physicians have been the focus of efforts to improve the quality of preventive health care such as smoking cessation counseling. Many primary care practices have not implemented systematic protocols to identify patients who smoke or to encourage clinicians to provide smoking cessation counseling because such interventions are thought to be too time and effort intensive. With collaboration from Blue Cross Blue Shield of Massachusetts, we designed a project to assess the relative effectiveness, as compared with no intervention (usual care), of two brief primary care interventions on patient smoking cessation rates. We hypothesized that improved documentation would increase smoking cessation among patients. METHODS: We performed the study at the adult primary care practice of a large urban tertiary care center. We chose interventions that could be used routinely in a busy primary care practice, and which differed in the level of patient and physician involvement. Each intervention was implemented on a geographically separate``team'' consisting of 5 ± 6 attending physicians, 2 nurse practitioners, and 3 ± 5 residents. Three teams served as controls (usual care). Thè`m inimal'' intervention consisted of a smoking status``vital-sign'' stamp, which identified smokers and documented patient smoking status. The``enhanced'' intervention consisted of a simple, one page, 5-question form which identified smokers, assessed level of cessation readiness and motivation, and provided smoking cessation counseling prompts for clinicians. Outcomes were collected 8 ± 10 months after implementation of the interventions. Patients who had identified themselves as current smokers at the beginning of the study period and who had seen a clinician at least once during the study period were contacted and their smoking status assessed by self-report. Two individuals unaware of study design or purpose performed structured chart reviews to assess clinician documentation of patient smoking status and cessation counseling. RESULTS: Four hundred seven charts were reviewed for documentation assessment.\nSmoking status was documented at 84% of the visits on the minimal intervention team, 91% on the enhanced intervention team, and 49% on the control teams (p-value < 0.0005). Cessation counseling was documented at 36% of the visits on the minimal intervention team, 47% on the enhanced intervention team and 30% on the control teams (p-value = 0.015). The improvement in smoking status documentation was mainly noted 1) for routine and non-cardiac/pulmonary urgent care visits compared with new patient or cardiac/pulmonary urgent care visits, and 2) among nurse practitioners and a patient's primary care physician compared with residents or urgent care physicians. The trends were similar for cessation counseling documentation. Two hundred forty-five patients completed the smoking status assessment at the end of the study period: 58% were female, the mean age was 46 years, and 40% were evenly divided between the minimal and enhanced intervention teams. There were no significant differences between groups in mean length of follow-up, or median number of visits or cigarettes smoked. A total of 33 (13.5%) patients had stopped smoking by the end of the follow-up period: 4% in the minimal intervention team, 30% in the enhanced intervention team, and 11% in the control teams (p-value < 0.0005). The proportion of patients reporting increased awareness of the health risks associated with smoking or improved motivation to stop smoking was higher among the intervention (56 and 67%) than control teams (40 and 59%). CONCLUSION: An enhanced intervention that uses a short questionnaire to identify patients who smoke and assess their level of cessation readiness may significantly improve smoking cessation rates compared with minimal or no intervention. Both enhanced and minimal interventions can improve physician documentation of patient smoking status and cessation counseling. The rate of new applications to medical school in the United States has been declining over the past three years, possibly because of less interest in medicine as a career among young persons. It has been suggested, however, that even established physicians are leaving medicine for a host of reasons (e.g., financial issues, time factors). Our study attempts to qualify and describe this issue in relation to financial pressures of physicians. METHODS: We conducted a state-wide, randomized survey of 1000 Maryland chapter members of the American College of Physicians. Surveys were mailed, with a follow-up reminder letter and survey sent at one month to non-responders. The 24-item survey collected data on physician demographics, satisfaction with income, activities to generate income outside of medical practice, and plans to remain in medicine in 5 years. RESULTS: After initial mailings, the response rate was 40%. Mean age was 47.7 years, and 76.2% were male. Excluding the 11.9% of physicians who planned to retire, only 193 (62.1%) intended to remain in their current practice or position in 5 years. 11.6% felt they would still be in medicine, but in a different practice or setting, and another 6.5% expected to be in medical industry or management. A surprising 16.4% planned to be in a profession outside of medicine or were unsure about remaining in the medical field. In univariate analysis, factors associated with a desire to change positions were female gender, unmarried status, no dependents, generalist (vs. specialist), decrease in primary medical income over 5 years, and low level of satisfaction with primary medical income. If starting over, 25% of all responders would not have chosen medicine again as a career. CONCLUSION: In our survey of Maryland physicians, over 35% planned to be in a different practice or position, many not related to medicine. This documents further the growing dissatisfaction with medicine among physicians, much of which is related to financial pressures. The impact of these findings on continuity and quality of patient care deserves further study. The effects of maternal smoking on infant mortality and morbidity are well recognized. Brief physician advice has been shown to increase quit rates among pregnant smokers and to improve pregnancy outcomes. Frequent prenatal visits offer repeated opportunities for smoking cessation counseling. We evaluated how frequently physicians identify the smoking status of pregnant patients and how frequently physicians counsel pregnant smokers. METHODS: The National Ambulatory Medical Care Survey is an ongoing national survey of U.S. office based physicians. During the years 1991 through 1996, physicians answered questions both about patient smoking status and whether or not tobacco counseling was performed. We analyzed data from these six years to determine the frequency with which physicians identified the smoking status of pregnant patients and the frequency with which physicians counseled pregnant smokers. We compared smoking status identification and counseling rates for different specialties. RESULTS: 9807 physicians recorded data on 5622 visits by pregnant women. The Figure presents is not yet widely used. A wide variety of physician order entry systems currently exists making it important to determine which systems physicians are likely to accept. While studies have evaluated physician satisfaction with institution-specific order entry systems, few published studies have assessed commercial products, even though nationally these products will represent the majority of systems that will be implemented. METHODS: We distributed the Questionnaire for User-Interface Satisfaction (QUIS) to Internal Medicine physician housestaff who use both a commercially available order entry system and the Veterans Affairs Computerized Patient Record System (CPRS). The QUIS is a validated questionnaire that has been used to measure user satisfaction with the humancomputer interface with electronic medical records. It is a twenty-seven-item survey and questions are scored on a 0 (lowest) to 9 (highest) scale. RESULTS: 90 and 84 surveys were returned giving a response rate of 63% and 64% for the commercially available product and the CPRS surveys respectively. Overall, residents were dissatisfied with the commercial system giving it an overall mean score of 3.67 (95% confidence intervals 3.37,3.97). In contrast, the Veterans Affairs CPRS had a mean score of 7.21 (95% confidence interval 7.00, 7.43) indicating the residents were more satisfied with the CPRS (pvalue < 0.0001). Overall satisfaction was strongly correlated (& = 0.71) in both surveys with the ability to perform tasks in a``straight-forward'' manner. Overall satisfaction was also correlated with terminology consistency (&=0.60, &=0.71) and screen sequence (&=0.61, &=0.67) for the commercial product and the CPRS respectively. CONCLUSION: Satisfaction with the user interface for the two order entry systems differed dramatically between the two systems, suggesting that all physician order entry systems are not equally accepted. Satisfaction was highly correlated with the ability to perform given tasks in a direct and consistent manner. These data support prior studies that have found correlations between user satisfaction and efficiency with order entry systems. If maximal benefit is to be gained from the implementation of such systems further studies are needed to investigate what specific order entry features physicians find most useful and least disruptive to daily workflow. PURPOSE: Nearly half of all California physicians still practice in solo or self-employed group practices. The recent and well-publicized demise of independent practice associations (IPA) and practice management organizations in the state has underscored the tenuous financial status of these practices. As part of a larger study about the state of physician practice in California, we interviewed community-based physicians and asked them to describe various aspects of their practice lives. We describe here the variety of approaches that physicians are taking to enhance or maintain the viability of their practices. METHODS: We interviewed fifteen physicians throughout California who spend > 50% time providing care in office-based practices. Participants were identified using the snowball sampling method. Each individual was interviewed by telephone by a study physician. The average age of participants was 41 years (31 ± 55), 43% were specialists, 36% were female and 30% practiced in community health center or public clinics. Only 13% expressed any dissatisfaction with their practices. Transcribed interviews were thematically analyzed using ATLAS 4.1 software.4.1 software. RESULTS: Physicians in solo or small group practices were more apt to discuss financial issues related to their practices than those affiliated with larger groups or with health maintenance organizations. Specific methods for improving the financial viability of their practices included improving``customer service'' for patients and referring physicians, limiting contracts with low paying insurers and or patients when feasible, lengthening work hours, shortening duration of office visits, offering supplemental patient services for out-of-pocket pay, streamlining office processes and staffing, optimizing billing and using physician extenders. A few physicians described colleagues whose response was to leave practice for administrative positions or early retirement. CONCLUSION: Physicians are experiencing financial pressures that in California have been amplified by the financial insolvency of IPAs and other practice organizations. The predominant response to improve the financial viability of their practices has been more efficient and effective utilization of existing resources (including personnel). Less common approaches include providing supplemental services that patients are willing to pay for out-ofpocket and leaving clinical practice. These experiences, to some degree, mimic those experienced by other professions such as dentistry. The frequency with which the approaches are used and their implications for access, patient outcomes and physician workforce requires further study. Virginia has the greatest number of free clinics of any state. We evaluated the type and dollar value of medical services provided in 1999. METHODS: Survey completed by the directors of all 32 clinics registered in 1999 with the Virginia Association of Free Clinics. Dollar value of services provided was estimated to be thè`u sual and customary'' charges for the region of each clinic; prescription value was estimated to be $45, the average retail price of a prescription for Virginia in 1999. Operating expenses were expenditures for clinic administration, fund raising, and direct services, excluding capital expenses.\nRESULTS: See table. CONCLUSION: Free clinics in Virginia effectively leveraged $6.8 million operating expenses into $24.5 million worth of medical services. The average expense per visit, including prescriptions and health care provider services, compares favorably to private practice office benchmarks. Although many patients were served, they represented only the tip of the iceberg in Virginia in 1999, comprising only 4% of the approximately one million uninsured. While free clinics provide an outlet for patients who might not receive care otherwise, physicians and policy makers must continue to work for a more comprehensive solution.\nM. Nadkarni 1 , D. Hoffman 1 ; 3 University of Virginia, Charlottesville, VA PURPOSE: To ascertain how many patients in a medically underserved population receiving continuity care at a University Clinic actually qualify for additional health and social assistance benefit programs but are not yet enrolled. METHODS: A random sample of 200 medically indigent patients at University Medical Associates, a combined resident-faculty practice with 9000 active patients which provides care to a primarily underserved population in central Virginia, was selected for analysis of healthcare benefits they were eligible for but were not currently receiving. Many patients without health insurance are actually eligible for assistance programs and potentially could receive some form of healthcare insurance. A significant number of patients in underserved populations who are already covered by Medicare may still qualify for additional benefits but have not accessed them. Aggressive attempts to overcome barriers for enrollment in important assistance programs could substantially increase the welfare of many patients in indigent or uninsured populations. Interventions to increase enrollment of eligible patients and their families should be undertaken on national, state, and county level. Additionally, individual clinics and practices can help increase enrollment with relatively simple procedural measures.\nSURGEON VOLUME OF EARLY STAGE BREAST CANCER CASES. A. Nattinger 1 , R. Hoffmann 1 , R. Sparapani 1 ; 1 Medical College of Wisconsin, Milwaukee, WI PURPOSE: The percentage of U.S. women with early stage breast cancer who receive initial treatment in accordance with national guidelines has been declining. We are interested in whether physician volume of cases is related to processes or outcomes of care, and therefore determined for a population-based cohort the distribution of cases cared for by each surgeon operating on one or more cohort patients. METHODS: We used data from a population-based SEER-Medicare linked data base to study women aged 65 or older, who were diagnosed with a stage I or II breast cancer in 1993 ± 95, and underwent either breast-conserving surgery (BCS) or mastectomy. We determined the operating surgeons from the Medicare part B claims, and eliminated surgeons with an office zip code outside the SEER states. RESULTS: Over the three study years, 15,574 women were operated on by 3,265 different physicians. The median number of cases performed per physician per year was 1.0 with 1st/3rd quartiles of 0.0/4.0. Over three years, 35.3% of physician-years were zero-case years. We We developed a conceptual model for assessing the validity of existing guidelines using literature searches and expert guidance. Two physicians with expertise in literature retrieval and evaluation conducted limited literature searches for new evidence that could affect the validity of the guideline statements. We also searched for practice guidelines that had been published after the release of the AHCPR guidelines. Guideline statements were sent to original guideline panel members and other content experts, who were asked to evaluate the current validity of each statement and include evidence to support their assessment. The evidence collected was then used to determine the validity of the guidelines, which were classified as``still valid'' or``needs updating.'' Finally, we conducted a``survival analysis'' to estimate the rate at which guidelines become outdated using a Kaplan ± Meier survival curve and the Weibull parametric model. RESULTS: We reviewed 17 guidelines and surveyed 170 experts, 121 (71%) of whom returned their surveys. Our literature searches identified 6,994 titles, which were all reviewed.\nWe reviewed the abstracts of 610 of these articles and then narrowed our selection down to 173 full-text articles. We also reviewed 159 guidelines. There were 13 (76%) guidelines in need of an update, 3 (18%) guidelines that were still valid, and 1 (6%) guideline for which we could not make a determination due to insufficient information. The guidelines had an estimated``shelf-life'' of 3 years (90% of the guidelines were still valid) to 5.9 years (upper bounds of the 95% CI). CONCLUSION: Our study provides the first empirical evidence of the rate at which guidelines go out-of-date. More than 75% of the AHCPR guidelines are in need of updating. We recommend re-evaluation at a minimum every three years with modifications in this timeline based on whether advances in the field are rapidly or slowly evolving. Future guidelines could facilitate the updating process by including an estimate of the type of evidence necessary to prompt revisions. PURPOSE: Critics state that some of the cost reductions demonstrated by hospitalist services may be explained by``cost shifting'' to the outpatient setting. Timely and appropriate patient follow up is also of concern. The purpose of our study was 2 fold: 1)determine if a significant portion of the cost savings realized by our hospitalist service could be attributed to``cost shifting'' and 2)determine if follow up issues were addressed as an outpatient. METHODS: During the 1998 ± 99 academic year, WVU implemented a hospitalist service (H) which was compared with the two other medicine services, one staffed by General Internists (GIM) and the other by subspecialists (SS). Inpatient cost (COST) and length of stay (LOS) were the main outcomes. 956 charts (321 for H, 257 for GIM, 378 for SS) were retrospectively analyzed in a blinded fashion to determine the following for each group: 1) percentage of patients discharged with workups needing to be performed (% W/TJ) 2) percentage of patients with tests or procedures scheduled as outpatient (% TEST) 3) average costs of test/procedures specified at discharge (F/U COST)(elective surgeries excluded). Of patients who had follow up within our system, 25 charts in each group were reviewed to determine follow up rates (% F/U). RESULTS: See table. Among the 3 groups, there were no significant differences in the percentages of patients discharged with test procedures or workups pending (p > 0.6).Patients who had longer LOS or who were followed in our system tended to have more outpatient test and workups scheduled (p < 0.0 1). In terms of cost specified at discharge, there were no significant differences (p = 0.09). There were no significant differences in percentage of issues followed up, however the numbers were small (p = 0.2). CONCLUSION:``Cost shifting'' does not appear to explain the cost benefits realized within our hospitalist system. Most of the issues specified at discharge tend to be followed up in the outpatient setting. While alerts about life-threatening drug-drug interactions are integrated into many medical information systems, few data are available regarding whether these warnings affect prescribing behavior or prevent patient injury. METHODS: We examined all consecutive cases of alerts warning of life-threatening drug-drug interactions during a six month period at a 700-bed academic tertiary care hospital. The alerts examined were the 7 with the most severe consequences from a database of known drug-drug interactions. Data were automatically collected each time a physician entered an order that triggered an interaction alert. The hospital information system logged the drug pair involved in the interaction, whether the alert was overridden, and any subsequent orders to discontinue one of the drugs in the pair. Adverse events attributable to the drug interaction were ascertained by chart review. RESULTS: Over 6 months, there were 168 life-threatening drug-drug interaction alerts (see table) . After 65(43%) of the alerts, physicians immediately discontinued one of the medications. For the 85(57%) alerts where both medications were given despite the alert, there were 2 adverse drug events, both involving the drug cisapride, and neither resulted in subsequent disability. CONCLUSION: Physicians overrode alerts about life-threatening drug-drug interactions more than half the time. When they did so, few adverse drug events occurred. These data suggest that the severity level of some drug-drug interactions may be set too high. For the small number of drug pairs that should never be given jointly, stronger safety measures should be put in place. people. Some plans claim that providing higher quality care raises costs and lowers profits, and that plans withdraw because they cannot sustain high quality care under current payment levels.\nWe performed a study to assess whether higher quality performance by Medicare managed care plans was associated with withdrawal. METHODS: Taking each county where a contract was active as a unit of analysis, this was a cohort study of Medicare managed care plans active in 2310 contract-county combinations in 1997 and followed for three years. We measured quality using fourteen indicators from the Health Employer Data and Information Set which we collapsed into four summary measures. We used a separate Cox proportional hazards regression for each indicator and each summary measure to assess the association of high quality care with time-to-withdrawal from Medicare while adjusting for market, organizational, environmental, and demographic factors. RESULTS: Of 2310 managed care contract-county combinations, 877 (38%) withdrew. The withdrawal rate among plans with high scores on a summary clinical quality measure was onefifth that for low scorers (4.2% v. 20.5%). For the summary ambulatory care access measure, the corresponding ratio was two-fifths (12.8% v. 32.0%). Lower payment rates were associated with higher withdrawal risk, contrary to plan claims, with higher clinical and ambulatory care access quality performance. In separate multivariable analyses, both high summary clinical performance (HR 0.18, 95%CI 0.08 ± 0.42), and high summary ambulatory care access performance (HR 0.53, 95%CI 0.27 ± 1.07) were independently associated with lower withdrawal risk. There was no independent association between either physician qualification or primary care provider stability measures and withdrawal. CONCLUSION: Managed care plans that continue to provide care to Medicare beneficiaries have higher average performance on clinical and ambulatory care access measures than plans that withdrew. System (BI-RADS) category 4 assessments (suspicious for malignancy) or category 5 assessments (highly suggestive of malignancy) were considered positive and in need of biopsy. These mammograms were matched to a subsequent biopsy obtained by fine needle aspirate, core biopsy, or surgery. We calculated the proportion of patients who had biopsies performed within 30 days, had biopsies within 1 to 12 months, and not biopsied within 12 months after the positive mammogram. RESULTS: 5,713 mammograms met eligibility criteria. 57.8% of mammograms were followed by biopsy within 1 month, 18.5% within 1 month to 12 months, and 23.8% did not have a biopsy within one year. There was a significant variation (p < .001) in time to biopsy by facility with a range of 33.5% to 77.5% of patients biopsied within one month and 9.3% to 60.0 % not having had a biopsy within one year. CONCLUSION: Though a majority of women received an expedient biopsy after a positive mammogram, the large proportion of women having a delayed biopsy or apparent lack of biopsy is of concern.\nDELAY TO BIOPSY AFTER A POSITIVE MAMMOGRAM. R.G. Pinckney 1 , B.M. Geller 2 , M. Coahran 2 , P. Vacek 2 , B. Littenberg 1 ; 1 University of Vermont, Burlington, VT PURPOSE: Delay from detection to diagnosis has been associated with more advanced stage at diagnosis and decreased survival in women with breast cancer. We sought to determine the delay to biopsy after mammographic detection of suspicious lesions at a statewide level. METHODS: The Vermont Mammography Registry (VMR) collects information on all mammograms and breast pathology obtained in Vermont. Mammograms performed on Vermont women between 1/1/1994 and 8/31/1999 with Breast Imaging Reporting and Data System (BI-RADS) category 4 assessments (suspicious for malignancy) or category 5 assessments (highly suggestive of malignancy) were considered positive and in need of biopsy. These mammograms were matched to a subsequent biopsy obtained by fine needle aspirate, core biopsy, or surgery. We calculated the proportion of patients who had biopsies performed within 30 days, had biopsies within 1 to 12 months, and not biopsied within 12 months after the positive mammogram. RESULTS: 5,713 mammograms met eligibility criteria. 57.8% of mammograms were followed by biopsy within 1 month, 18.5% within 1 month to 12 months, and 23.8% did not have a biopsy within one year. There was a significant variation (p < .001) in time to biopsy by facility with a range of 33.5% to 77.5% of patients biopsied within one month and 9.3% to 60.0% not having had a biopsy within one year CONCLUSION: Though a majority of women received an expedient biopsy after a positive mammogram, the large proportion of women having a delayed biopsy or apparent lack of biopsy is of concern. The short-term follow-up of marginally abnormal mammograms presents a challenge to both physicians and patients. A variety of systems and individual factors may impact on the adherence to mammographers' recommendations. We sought to assess the extent of this problem and to examine the factors associated with the provision of appropriate follow-up care. METHODS: Among a sample of 579 women with abnormal screening mammograms or breast complaints, we analyzed the clinical data of 126 women who had marginally abnormal mammograms for which the mammographers recommended 6-month repeat follow-up mammograms. All women were contacted for detailed telephone surveys at the time of their index mammogram, and 7 to 8 months later. The medical records of these women were reviewed to determine the breast care they received before and during the follow-up period. Women who received follow-up mammograms as recommended, saw breast specialists, or underwent breast biopsies within the 7 ± 8 month follow-up period were considered to have received appropriate follow-up care. We then studied the differences between women who did and did not receive appropriate follow-up care April 1, 1996 and March 31, 1997 . Each discharge record contains up to 16 diagnoses, 16 corresponding diagnosis type indicators (value of`2' = complication), and up to 10 procedures. A clinically trained chart reviewer examined the corresponding medical charts for evidence of diagnoses and complications. A complication was defined as a new diagnosis arising after the start of hospitalization. We defined 12 common conditions using the ICD-9-CM coding system: stroke, cystitis/pyelonephritis, atrial fibrillation, respiratory failure, post-hemorrhage anemia, pneumonia, bowel obstruction, congestive heart failure, acute renal failure, myocardial infarction, atelectasis, and pleural effusion. We then selected records with presence of a condition in both administrative data and chart review data. Finally, we determined the extent to which the diagnosis type indicator in administrative data agreed with the chart reviewer's assessment (criterion standard) of whether a diagnosis was a complication or not.\nRESULTS: Observed agreement for flagging complications using diagnosis type indicators was generally high between the data sources (agreement ranging from 47 to 97%, and agreement > 80% for 8 of the 12 conditions studied). However, kappa varied greatly across conditions studied (range: -0.06 ± 0.87) and was low at < 0.20 for 6 conditions. Specificity was generally high (range: 68 ± 100%) and was > 90% for 8 conditions, suggesting that pre-existing conditions were usually appropriately coded as such in the administrative data. In contrast, sensitivity was lower (range: 0 ± 83%), with sensitivity < 50% for 7 conditions, indicating a tendency for complications to often be miscoded as baseline comorbidities. CONCLUSION: The validity of diagnosis type indicators in Canadian administrative discharge data appears to be sub-optimal for some types of complications. This is likely to be of greatest concern in studies that rely on diagnosis type indicators to define complications as outcomes. PURPOSE: Recent data suggest that there are substantial variations in the treatment of acute myocardial infarction (AMI) based on age, race, gender and socioeconomic status. Specifically, the use of proven pharmacologic interventions and lifestyle modification counseling have been shown to vary by patient characteristics. We evaluated the use of post-AMI therapies and counseling in patients with AMI treated in an academic referral center with special attention to those receiving medical management alone for AMI. METHODS: We reviewed the records of a convenience sample of 384 patients with AMI from 1997 ± 2000. Our outcomes of interest included use of post-AMI therapies such as aspirin (ASA), beta-blockers (BB), angiotensin converting enzyme inhibitors (ACEI) and other anti-coagulants (AC). We collected variables including use of primary angioplasty (PA), defined as a cardiac procedure within twenty-four hours of admission, demographics, comorbidities, do-notresuscitate (DNR) status, physician characteristics, and severity of illness using the ATLAS system. Univariate analyses were performed on the variables of interest, and subsequent logistic regression modeling incorporated all variables significant on univariate analyses. RESULTS: Unadjusted analyses of pharmacologic therapies revealed that severely ill patients were less likely to receive any post-AMI therapies (all p < .05). Older patients were significantly less likely to receive both ASA and BB post-AMI (p < .05), but were not less likely to receive ACEI or AC. Race and gender were not associated with differences in any medication use. Conversely, patients who had undergone PA were significantly more likely to receive all medications (p < .05). Unadjusted analyses of tobacco and cholesterol counseling, defined as any chart reference to tobacco use in current smokers and defined as any chart reference to serum cholesterol in all AMI patients, was significantly more likely to occur if one underwent PA, and significantly less likely to occur as severity of illness increased (p < .05). After adjustment for potential confounders, patients who underwent PA were significantly more likely to receive proven post-AMI therapies and counseling (p < .01), and this increased likelihood persisted across increasing levels of severity of illness. CONCLUSION: Our results indicate significant differences in the use of post-AMI pharmacologic therapies and counseling correlated with the use of primary angioplasty for AMI, despite evidence of proven benefit in AMI in patients undergoing medical management alone. These differences are not explained by severity of illness and suggest that standard therapies may be withheld from those who may benefit most. in the diagnosis and treatment of depression, PCC success in this area is problematic, and direct patient access to mental health care has been proposed as a solution. The purpose of these analyses is to learn how often PCCs in two large healthcare organizations that allow direct patient access to mental health services discuss depression diagnosis or treatment with depressed outpatients, and the relationship between PCC discussion and treatment rates. METHODS: We screened 19,793 consecutive patients attending one of nine primary care practices in one of two staff model managed care organizations in California: three practices based in a single academically-affiliated VA medical center and six in separate Kaiser Permanente (KP) medical centers. Of the 597 who met full criteria for current major depression by diagnostic interview and met other study inclusions criteria, 567 completed a 40-minute telephone-administered survey assessing their mental health care over the preceding six months.\nMeasures include PCC discussion of depression (the PCC asked about depression or gave the patient a treatment for depression or emotional problems at the last visit); recent depression treatment (the patient took an antidepressant or had at least 4 mental health specialty visits in the past 6 months); and high or low depression symptoms by CESD score. Significance is by Chi-Square. RESULTS: Of 567 patients with major depression, 414 (73%) had been recognized as depressed (64% HMO, 76% VA) as indicated either by PCC discussion of depression (333 or 59%) or by recent depression treatment in the absence of PCC discussion (81 or 14%). A total of 290 of the 567 (51%) reported recent depression treatment; treatment rates were significantly higher among the 333 patients who had discussed depression with their PCC than among the 233 who had not (63% vs 35%, p < .001). Excluding patients reporting recent treatment, PCCs who discussed depression were more likely to assess suicidality, mania, and alcoholism. Among the 567 patients, those whose PCC discussed depression were more likely to be treated with an appropriate antidepressant (35% vs. 18%, p < .001), and to visit mental health (51% vs 33%, p < .001). Results comparing treatment for patients with PCC discussion and those without remained similar and significant when analyzed separately for patients with high or low CESD scores. CONCLUSION: In two organizations with direct primary care patient access to mental health specialty, discussion with PCCs remains an important determinant of depression treatment quality. These findings support the importance of PCC recognition of depression, and the need for better integration of mental and physical health care. There are limited data about minority physicians' satisfaction with their professional lives. Previous studies suggest that among academic physicians, minorities may be less satisfied than whites. In this study, we describe, by race and ethnicity, satisfaction and job stress among a national sample of physicians. METHODS: We analyzed data from 2,326 respondents to a career satisfaction survey of physicians drawn from the AMA Physician Masterfile. Scales (from 1 ± 5) measuring overall job satisfaction, career satisfaction, and work-related stress were constructed from Likert-response items and tested using factor and reliability analyses. We examined the association between physician ethnicity and each of these scales. We also examined how the proportion of patients from ethnic backgrounds other than the physician's own affected satisfaction and job stress. RESULTS: Respondents included 56 black, 136 Hispanic, 409 Asian, and 1,651 white physicians. Black and Hispanic physicians, as compared with others, cared for higher proportions of minority, non-English speaking and uninsured patients. Scores for job and career satisfaction and stress by ethnicity are displayed in the table. Higher scores indicate higher satisfaction or stress, and p-values refer to comparisons of the highest and lowest scores. After adjusting for physician age, gender, practice setting, specialty, and patient panel characteristics (proportion of patients who were female, elderly, non-English speaking, medically complex, psychosocially complex, or substance-abusing), Hispanic physicians had higher job and career satisfaction than all other groups. The proportion of physicians' patients from ethnic groups other than their own was associated with lower job satisfaction and higher stress (p < .001). In adjusted analyses, these findings persisted but were most consistent among white and black physicians. CONCLUSION: Hispanic physicians were more satisfied with their jobs and careers than physicians from other ethnic groups. For white and black physicians, caring for ethnically dissimilar patients was associated with lower job satisfaction and higher stress. Future study should investigate how physician cultural factors and cross-cultural relationships with patients affect physician worklife and professional satisfaction. Family physicians were more likely (OR 1.254, 95% CI 1.253 ± 1.255) to see patients for acute problems unrelated to chronic medical conditions. Family physicians performed more pelvic exams (OR 1.447, 95% CI 1.446 ± 1.448) and Papanicolaou smears (OR 1.391, 95% CI 1.390 ± 1.392), and more frequently provided counseling on smoking cessation, mental health, injury prevention, family planning and sexually transmitted diseases (p < 0.001 for all). Family physicians were more likely to prescribe medication at patient visits (OR 1.229, 95% CI 1.228 ± 1.230) than internists, but internists more frequently ordered advanced radiologic tests such as CT, MRI, and ultrasound (p < 0.001 for all) even after adjustment for age, gender, and number of ICD-9 diagnoses. CONCLUSION: Significant differences exist between ambulatory practice patters of primary care physicians independent of patient age and gender.\nLAB-PHARMACY LINKS: LEGAL LABELING LANGUAGE OVERLOAD. G. Schiff 1 , G. Shah 1 , H. Lubin 1 , D. Klass 1 , D. Bates 2 ; 1 Cook County Hospital, Chicago, IL; 2 Brigham and Women's Hospital, Boston, MA PURPOSE: Lab and pharmacy are intimately related. However their departments, functions and information systems are often disconnected. The extent of these clinical interrelationships, and expectations upon prescribing physicians to remember critical information (warnings, toxicity, monitoring) is vast and taxes the memory of busy clinicians. A number of drugs have recently been withdrawn because of operational failure in meeting such expectations (eg troglitazone-hepatic toxicity monitoring). To better understand the extent of such information, and because the Physicians Desk Reference (PDR) is the most widely used drug information source and represents legally-mandated prescribing labeling for drugs marketed in the U.S, we analyzed the content of tab-pharmacy interactions and warnings for frequently prescribed and recently approved drugs. METHODS: We developed a taxonomy of critical lab-pharmacy interaction categories, and reviewed all solid oral medications for the most frequently prescribed drugs (N = 40), plus new molecular entities (NME's) marketed in 1998 ± 99(N = 37). We conservatively counted multiple warnings as a single test whenever possible (eg thrombocytopenia, anemia, leukopenia = CBC). RESULTS:\nCONCLUSION: Officially labeled lab-pharmacy interactions/warnings are frequent±totaling 249 (6.2/drug for commonly prescribed drugs, and 238 (6.4/drug) for NME's)±a number that exceeds the unaided prescribers' abilities to remember and monitor. Automated screening and reminders will need to be programmed into routine prescribing processes to prevent errors related to such interactions. A noteworthy discrepancy exists between number of toxicities and recommended monitoring, and relatively few drugs (especially NME's) appear to have lab interference evaluated, suggesting a need for additional research to ensure safe prescribing and error-free lab test interpretation. To assess racial disparities in influenza vaccination among Medicare beneficiaries and evaluate whether or not the magnitude of racial disparities is smaller for those enrolled in managed care than among those with fee-for-service insurance. METHODS: We analyzed 13,674 responses to the 1996 Medicare Current Beneficiary Survey of African American and white Medicare beneficiaries with managed care and fee-for-service insurance. We compared sociodemographic characteristics and health attitudes among the groups, the proportion receiving influenza vaccination, and differences in racial disparities in influenza vaccination between those with managed care and fee-for-service insurance. Using a propensity model based on sociodemographics, comorbid illnesses, attitudes toward health care, and their interactions with race, we adjusted for differences in the two insurance groups and compared the adjusted racial disparities. RESULTS: Eight percent were African American. Eleven percent were enrolled in managed care. On average, African Americans and fee-for-service enrollees were older, had worse health status, had a higher prevalence of diabetes, and were more likely to avoid doctor visits. Overall, 65.8% of Medicare beneficiaries received influenza vaccination. Whites were much more likely than African Americans to receive influenza vaccination (67.7% vs. 45.1%, absolute disparity = +21.6%; 95% C.I. 18.2% to 25.0%). Managed care enrollees were more likely to receive influenza vaccination than those with fee-for-service insurance (71.2% vs. 65.4%, difference = +5.8%; 95% C.I. 3.3% to 8.3%); however racial disparities in influenza vaccination were nearly identical among beneficiaries both with managed care and with fee-for-service insurance. These disparities were not significantly different after adjustment for propensity to enroll in managed care. CONCLUSION: While managed care is associated with higher rates of influenza vaccination, we found no evidence that managed care reduces racial disparities in influenza vaccination. Our results suggest that health plans are not yet adequately addressing racial disparities in the use of preventive services.\nTEACHING HOSPITAL. J.L. Schnipper 1 , R.S. Stafford 1 ; 1 Massachusetts General Hospital, Boston, MA PURPOSE: To determine the extent to which outpatients with coronary artery disease (CAD) receive adequate secondary prevention measures, and to identify the factors that predict variations in the provision of these services. METHODS: Cross-sectional study of 3920 patients with coronary disease (ICD-9 codes 410.0 ± 414.99) who were seen in one of ten outpatient practices affiliated with Massachusetts General Hospital (MGH) during fiscal year 1997. Demographic and laboratory information were collected on all patients, and a medical record review was performed on a subset of patients to confirm CAD and to determine blood pressures, medical conditions, and medications during fiscal years 1997 and 1998 (N = 302). RESULTS: Our study showed evidence of variable short-comings among different CAD prevention strategies. 17% of patients never received a low density lipoprotein (LDL) cholesterol test during the study period, and only 19% of patients had an LDL level 100 mg/ dL. Among those with an LDL ! 130 mg/dL, 41% received no lipid-lowering medication and only 5% were on a standard maximum dose of an HMG CoA reductase inhibitor (statin). The average of the last two blood pressure readings was less than 130/85 mm Hg in 42% of patients, and 53% of patients met JNC VI guidelines for blood pressure control. Among hypertensive patients, 94% were on some type of anti-hypertensive medication. 75% of all patients were prescribed a beta-blocker during the time period, and 86% were prescribed aspirin or another antithrombotic medication.\nIn multivariable models adjusted for several patient and physician characteristics, having an LDL < 130 mg/dL was less likely for patients in the oldest age quartile (odds ratio (OR) 0.59, 95% confidence interval 0.49 ± 0.72), female patients (OR 0.60 [0.51 ± 0.69]), those without insurance coverage of medications (OR 0.54 [0.37 ± 0.79]), and those whose type of insurance indicated low socioeconomic status (OR 0.85 [0.70 ± 1.01]). Older patients and women were less likely to have a blood pressure < 140/90 mm Hg. Younger age was of borderline significance in predicting beta-blocker use. CONCLUSION: At outpatient practices affiliated with a major academic medical center, there is still much room for improvement in the secondary prevention of CAD. Deficiencies were greatest in the area of lipid management. Lipid management also was the most susceptible to practice variation on the basis of socioeconomic status. Older patients and women also were less likely to receive adequate secondary prevention. Interventions such as physician education, computerized reminder systems, and population management may be effective in correcting some of these shortcomings.\nFOR-PROFIT HOSPITALS AND DRG``UPCODING''. E. Silverman 1 , J. Skinner 2 ;\n1 Dartmouth Hitchcock Medical Center, Lebanon, NH; 2 Dartmouth College, Hanover, NH PURPOSE: One large for-profit hospital chain has admitted to inflating the severity of discharge diagnoses (``upcoding'') for economic gain. It is unknown how widespread this practice is, or whether it is endemic to for-profit hospitals. We examined the association between for-profit hospital ownership and upcoding for Medicare discharges for pneumonia, the most widely recognized condition under which upcoding seems to occur. METHODS: To analyze time trends in coding patterns, we used a 20 percent national sample of Medicare inpatient claims for 1989 through 1997 with the hospital as the unit of analysis. Hospital ownership was defined as for-profit, not-for-profit, or government based on the American Hospital Association's Annual Survey of Hospitals for each year. The relevant diagnosis related groups (DRGs) were for simple pneumonia with and without complications (DRGs 89 and 90), and for complex pneumonia with and without complications (DRGs 79 and 80). The dollar reimbursement for complex pneumonia is approximately 50 percent higher than for simple pneumonia. For each year and ownership group we calculated an upcoding ratio defined as the percentage of disharges coded for complex pneumonia out of the total discharges for simple and complex pneumonia combined. RESULTS: Crude upcoding ratios for pneumonia were highest among for-profit hospitals for each year between 1989 and 1997. In addition, between 1989 and 1996, the upcoding ratio increased from 0.20 to 0.30 in not-for-profit hospitals, 0.21 to 0.30 in government hospitals, and from 0.28 to 0.51 in for-profit hospitals. Between 1996 and 1997 the ratio fell slightly for each group, which corresponds to a period of great publicity and legal charges of fraud. Average thirty-day mortality rates for the DRGs under consideration were similar across ownership categories, and in fact declined over the study period, suggesting that differences in the percentage of pneumonia discharges coded as complex was not related to case-mix. CONCLUSION: For-profit hospital ownership was associated with higher upcoding ratios among Medicare discharges for pneumonia than in not-for-profit hospitals. PURPOSE: Physicians' attitudes are important because of the possible impact they may have on the structure and quality of patient care [1] . In 1982 Mathews et al [2] , surveyed San Diego County Medical Society's physicians about their attitudes toward homosexuality. They found significant differences in prevalence of homophobic attitudes by gender, year of medical school graduation, specialty and practice setting [2] . Our objective was to assess physicians' attitudes toward homosexuality and toward persons with HIV infection and to compare the current prevalence of homophobia to that measured seventeen years previously. METHODS: An anonymous, self-administered, 17-item survey was mailed to all 4,385 members of the SDCMS and 1,271 UCSD physicians. The survey included 8 likert-scaled items measuring general attitudes toward homosexuality (Cronbach's alpha = 0.88). Additional items assessed attitudes toward entry to medical school and consultative referral patterns, conditional on sexual orientation and HIV status of hypothetical referents. PURPOSE: Somatization is a common costly problem with great morbidity, but there has been no effective screening method to identify these patients and target them for treatment. We tested an hypothesis that we could identify high utilizing somatizing patients from a management information system (MIS) by total number of visits and what we termed`s omatization potential,'' the percentage of visits for which ICD-9 primary diagnosis codes represented disorders in the musculoskeletal, nervous, or gastrointestinal systems or ill-defined complaints. METHODS: We identified high utilizing patients from the MIS of a large staff model HMO as those having 6 or more visits during the year studied (65th percentile). A physician rater then reviewed the medical records of these patients and identified somatizing and nonsomatizing patients. In two-thirds of the population (the derivation set), we used logistic regression to refine our hypothesis and identify predictors of somatization available from the MIS: demographic data, all medical encounters, and primary diagnoses made by usual care physicians [ICD-9 codes]. We then tested our prediction model in the remaining one-third of the population (the validation set) to validate its utility. RESULTS: The derivation set contained the following significant correlates of somatization: gender, total number of visits, and percent of visits with somatization potential. The c-statistic was .90. In the validation set, the explanatory power was less with a c-statistic of .78. A predicted probability of .04 identified almost all somatizers, while a predicted probability of .40 identified about half of all somatizers but produced few false positives. CONCLUSION: We have developed and validated a prediction model from the MIS that helps to distinguish chronic somatizing patients from other high utilizing patients. Our method requires corroboration but carries the promise of providing health plan directors with an inexpensive, simple approach for identifying the common somatizing patient and, in turn, targeting them for treatment. The screener does not require clinicians' time. PURPOSE: High utilizing patients with medically unexplained symptoms often are believed to be somatizing patients, defined here as no organic disease explanation for symptoms of at least 6 months duration. High utilizing patients with unexplained symptoms of shorter duration have not been studied and there is no effective treatment for this group, which we call``minor acute illness.'' METHODS: Our aim was to determine how many high utilizing patients with medically unexplained symptoms met our chart-rating criteria for somatization and or minor acute illness and what the stability of these diagnoses was over time. We performed chart review at baseline; one and two years later, we re-rated the charts of patients initially rated as having somatization as well as a 15% sample of those with minor acute illness. Subjects were a random sample of high utilizing staff model HMO patients (6 or more visits/year) from 21 ± 55 years old, identified from the management information system (MIS). Chart review designations as organic disease, somatization, or minor acute illness were the measurements made. RESULTS: Among 883 high utilizing patients, 35% had organic diseases, 14% had somatization, and 51% had minor acute illness as their primary problems. No patients with initial minor acute diagnoses were reclassified as somatization one and two years later and all but 2 patients had minor acute illness in one or both follow-up years. Among somatizers, 54% were re-classified as minor acute illness the following year and, of these, 50% remained as minor acute illness in the second follow-up year. 70% of initial somatizers and 45% of initial minor acute patients continued to be high utilizers in one or both follow-up years. Persistent high utilization in both follow-up years occurred in 17% of minor acute patients and33% of somatizing patients. CONCLUSION: Minor acute illness appears more common among high utilizing patients than somatization and organic diseases combined. It has not previously been studied. Diagnoses of somatization were unstable over two years follow-up while minor acute diagnoses were stable, supporting the latter as a valid entity. Utilization did not remain high in either group. We recommend further study of minor acute illness to determine its relationship to somatization, to determine if it is a valid entity, and to better understand the broad spectrum of patients with medically unexplained symptoms.\nAVOIDANCE OF REGRET AS A MOTIVE FOR ORDERING PROSTATIC SPECIFIC ANTIGEN TESTS. P.C. Sorum 1 , E. Mullet 2 , G. Chasseigne 3 , S. Bonnin-scaon 2 , J. Shim 4 , J. Cogneau 5 ; 1 Albany Medical Center, Schenectady, NY; 2 Ecole Pratique des Hautes Etudes; 3 Universite  Franc Ëois-Rabelais, Tours; 4 University at Albany, Albany, NY; 5 MG France, St Avertin PURPOSE: When making management decisions, physicians try to avoid the regret that would be provoked by unwanted consequences of their actions or inactions. We explored the strength and determinants of such anticipated regret in a study of decisions to order prostatic specific antigen (PSA) tests for hypothetical patients. METHODS: 32 U.S. and 33 French primary care physicians looked at 32 patient scenarios and estimated the probability each patient had prostate cancer and the likelihood the participant would order a PSA. They were then given 12 scenarios and were told to suppose they had performed routine exams on these patients, had not ordered PSAs, and now several years later found advanced prostate cancer in these patients. They were asked to indicate how much regret they would feel that they had not ordered PSAs. The 12 hypothetical patients differed according to age (55, 65, or 75) , the request to be screened for prostate cancer (no or yes), and the shape of the prostate on the earlier rectal exam (no irregularity or some irregularity). The assessments of regret were analyzed by ANOVA (with significance set at p < .02). For each group of physicians, a correlation was calculated between the mean rating for regret and the mean likelihood of ordering a PSA. RESULTS: The regret scores were correlated with the likelihood of ordering PSAs for both the French (r = .64, p = < .005) and the Americans (r = .42, p < .02). The level of regret was higher when the patient had requested a PSA, when the shape had some irregularity, and when the patient was younger. When the patient had requested a PSA, the shape of the prostate had less effect than when the patient had not requested it. The U.S. physicians indicated a higher level of regret than did the French. A patient request had a greater impact on U.S. than on French physicians. Increasing patient age reduced regret more among the French than among the Americans. CONCLUSION: Anticipated regret over a failure to have diagnosed an aggressive prostate cancer is correlated with a policy of ordering of PSAs. The extent and determinants of this regret appear to be culturally sensitive. PURPOSE: Several studies have suggested that a central medical organizational structure can influence processes and outcomes, for example with audit and feedback, economic incentives, and implementation of guidelines. One form of organizational structure is clinical structure, the application of organizational policies which, in aggregate, allow medical organizations to improve the efficiency of care and outcomes delivered to cohorts of patients. We used literature review supplemented by clinical and administrative input to design a model of clinical structure which was tested in a 45 minute telephone survey by physician interviewers of medical directors of 56 medical organizations. METHODS: Responses were obtained from the directors of 53 medical organizations (response rate 95%) providing data from 29 medical groups (MGs) and 24 independent practice associations (IPAs) from 3 west coast states. The survey queried directors about whether their organization had in place corporate-wide policies to standardize clinical operations across the offices and providers that define their corporate entity. The survey asked about each of 30 dimensions of clinical corporate structure distributed across 183 individual survey items We considered an organization to have corporate policy (CP) if the respondent indicated CP for at least one item associated with a dimension of clinical structure. RESULTS: There was substantial variation across organizations in the extent to which the directors reported corporate policies. On average, organizations had CP for 49% of the 183 items across 30 dimensions of corporate structure. The clinical corporate policies most frequently cited by respondents pertained to the use of clinical guidelines (94%); specifications regarding the exact content of the ambulatory medical record (92%), and whether or not prospective review was required prior to specialty consultation (91%). Organizations infrequently cited having clinical corporate policies for assigning new patients to providers according to clinical need (19%), for office staff counseling patients following MD visits regarding next steps (21%), and use of multidisciplinary team meetings (25%). Of the 30 dimensions of clinical structure studied, CP was more prevalent in MGs than IPAs (p < .05) for 17 (57%) dimensions. For example, 66% of MGs and 33% of IPAs reported CP regarding written educational materials being available to patients (p < .05). CONCLUSION: While some medical organizations have developed sophisticated systems to organize the delivery of clinical care to cohorts of patients using corporate administrative policies, most have not. For most dimensions for which the literature suggests care could be improved with organizational support, most organizations, especially IPAs, do not report corporate policies in place.\nnot accept a diagnosis of CHF based upon ejection fraction alone. Thus, it is important for both clinical and research purposes to estimate the agreement between quantitative measurements and the cardiologists' qualitative interpretation reported on echo METHODS: All patients at a university-affiliated VAMC who had an echo between 12/96 and 12/99 were identified by electronic medical records (N = 5,505). One record per patient was identified and for each echo, we abstracted both quantitative (fractional shortening and fractional area change) and qualitative (cardiologists' interpretation) data. We excluded reports with missing quantitative (N = 912) or qualitative (N = 85) data. Abnormal numeric data was defined as fractional shortening less than 0. 1 8 or fractional area change less than 0.35. Abnormal textual report was defined as left ventricular systolic dysfunction or generalized wall motion abnormality. We then calculated agreement from a 2Â2 table. RESULTS: Of 4,508 patients with echos in our study, 1237 had an abnormal echo by quantitative and/or qualitative data (Table) . Of these 1237 patients, an abnormality was identified by both strategies in only 548 (44%) of patients. Discrepancies rates between qualitative and quantitative reports were similar. The cardiologists' interpretation of the echo was normal when the numeric data were abnormal 41% (386/937) and the cardiologist's interpretation was abnormal with normal numeric data 35% (303/851).\nCONCLUSION: There is significant discrepancy between qualitative and quantitative strategies to diagnose CHF, which may explain non-compliance with AHRQ recommendations. Physicians who use either standard alone, risk undertreating or overtreating patients which may lead to morbidity. We conclude that CHF guidelines must develop a clinically acceptable way of identifying patients with CHF in order to insure that appropriate care is delivered and that patient outcomes are maximized. PURPOSE: Studies examining some of the clinical and non-clinical determinants of outcomes of acute myocardial in the VA suggest that age; race, co-morbid illnesses. and availability of cardiac surgical services all play a role. However, few data are available on the regional variation in health care utilization and outcomes during and after acute myocardial infarction. The purpose of our study was to examine regional variation in utilization and outcomes during the acute and chronic care of patients with acute myocardial infarction (AMI). Examining these differences will lead to a better understanding of potentially modifiable variables and processes of care that influence patient outcomes METHODS: Using national VA databases, we identified all veterans who were hospitalized for AMI at any VA Medical center between 10/90 to 10/97. Demographic, inpatient, outpatient, mortality and readmission data were extracted from the VA's administrative databases for the four regions: Northeast, South, Midwest and West. Multivariable Cox proportional hazards models, controlled for comorbidity, were used to assess the effect of independent variables on time to death and readmission. RESULTS: We identified 67,889 patients with AMI who were 98% male at mean age of65 years. There were no significant regional differences in patients' demographic data. Patients in the Northeast had a greater prevalence of comorbid conditions and longer lengths of stay during the index AMI hospitalization (14 days in the NE Vs 10 days in the W). There was no variation in the rates of cardiac procedures (PTCA and/or CABG) among the regions during the index admission. Region of the country was an independent predictor of mortality. Subsequent adjusted all-cause mortality after discharge from the index hospitalization was lower in the Northeast (OR = .910 CI .86 ± .95) and the West (OR = 0.871 CI .87 ± . 95). Patients in the Northeast and West had greater cardiology or primary care follow up at 60 days and 1 year post discharge than patients in the South. Outpatient follow up appeared to account for a substantial amount of the regional variation in all-cause mortality.\nCONCLUSION: There was substantial geographic variation in clinical care and outcomes among veterans with AMI. Outpatient follow-up was highly variable and associated with mortality. Further studies are needed to determine the most effective strategies for improving outcomes after AMI. METHODS: We recently described a new, valid, and reliable instrument called QUEST, which has 4 scales measuring patients' perceptions of quality and their satisfaction with care by physicians (MDs) and nurses (RNs). We administered QUEST and other standardized instruments to 84 medical inpatients (45 terminally ill and 39 comparison) at 2 hospitals. We used correlations, t-tests, and ANOVA to test associations between sociodemographic/clinical factors and QUEST scores. RESULTS: Mean patient age was 60, APACHE-III physiology score 16.6; 71% were white, 60% men, 54% Catholic, 28% Medicaid/uninsured, 38% had cancer or HIV, 41% moderate/ severe pain, 19% depressed, and 58% private (PVT). Age, APACHE, race, gender, religion, insurance, and diagnosis were unassociated with quality or satisfaction. Quality and satisfaction scores for MDs were lower among``service'' (SERV) than PVT patients and lower among terminal than non-terminal patients. Quality and satisfaction scores for RNs were lower among SERV patients and depressed patients. P-values for ANOVA models for QUEST were: MD Quality = .01, MD Satisfaction (adjusted for pain) = .007, RN Quality = .16, RN Satisfaction = .002. CONCLUSION: Terminally ill SERV patients rated MD quality and satisfaction lower than PVT patients and lower than SERV patients not terminally ill. RN satisfaction scores were high only among PVT patients who were not depressed. Understanding these apparent differences in end-of life care will require further investigation. PURPOSE: At the end of life, when cure is no longer possible, perhaps the most important medical intervention that can be offered is time. However, almost nothing is known about the time health professionals spend with dying inpatients. METHODS: We asked day-shift nurses to use a diary to record how much time they spent in various activities for 47 medical inpatients at 2 teaching hospitals (25 terminally ill patients with Do Not Resucitate (DNR) orders and 22 comparison patients). To minimize Hawthorne effects, nurses were told to record multiple categories of activities and were only informed that this was a``time-motion study for seriously ill patients.'' This method has been validated against video-recordings of actual time spent in the room (r = .70, p < .0001). Non-English speaking patients, those whose clinical condition prohibited interview, and those with a reduced set minimental status (MMS) score indicating dementia were excluded. Charts were reviewed and patients were interviewed using a battery of standardized instruments. Because nursing time was not normally distributed, we used log-transformed time as our dependent variable. We then used t-tests, correlations, and ANOVA to test associations between sociodemographic/clinical data and bedside nursing time. RESULTS: The mean patient age was 59, and the mean APACHE-III physiology score 16.3; 64% were white, 19% African-American, 11% Latino, and 6% Asian; 60% were men, 39% Protestant, 41% had cancer or HIV, and 23% were depressed. The mean amount of time nurses spent with patients per day shift was 27 minutes. In bivariate analyes, age (p = .23), APACHE-III physiology score (p = .78), gender (p = .81), religion (p = .27), diagnosis (p = .99), and DNR status (p = .26) were unassociated with nursing bedside time. Nurses spent more time at the bedsides of patients with lower reduced-set MMS scores (indicating worse mental function (r = .26, p = .08)), more time with white patients compared with non-white minorities (31 minutes vs. 22 minutes, p = .08), and more time with depressed compared with non-depressed patients (43 minutes vs. 23 minutes, p = .02). In an ANOVA model, lower reduced-set MMS scores (p = .02), white race (p = .02), depression (p = .15), and a race-depression interaction term (.045) were associated with more nursing bedside time. CONCLUSION: In this population, nurses spent more time with patients who had worse mental function (even after excluding those who were frankly demented), and more time with white patients who were depressed compared with both non-depressed white patients and minority patients regardless of depression score. DNR orders were not associated with less nursing contact. While some of these results appear to suggest unexpected differences in the time nurses give to patients, firm conclusions will require further study. Ryukyu University Hospital, Nakagami, Okinawa PURPOSE: General internal medicine (GIM) as an academic discipline was recently introduced to medical schools in Japan. Residency programs are still quite young and fully trained primary care physicians are in shortage. In May 1999, the Ryukyu University Hospital started a general medicine clinic to provide better care for its patients. However, since there were no staff trained in GIM, specialists from the Department of Medicine and Department of Psychiatry were assigned to the clinic. We conducted two surveys to assess the attitudes toward and perceptions of GIM practice among specialists at different academic disciplines in the hospital. METHODS: First a survey was carried out using a questionnaire that was distributed to all physicians who work at the Ryukyu University Hospital. We also conducted a 2nd survey which focused on the experience at the GIM clinic for specialty physicians who had ever practiced at the clinic. RESULTS: Overall response rates were 66.3% (n = 236) and 75% (n = 48), respectively. The clinic performance over the first year was evaluated and rated from À3 (extremely poor) to +3 (excellent). Respondents whose specialty was not in internal medicine perceived the GIM clinic more positively (average +1.0) compared to internists with and without GIM clinic experience(average À1.9 and À1.6, respectively), which reached statistical significance (p < 0.001). Among residents, fellows and faculties, there was no statistical difference regarding attitudes toward the competence of primary care physicians, encouragement and positive regard for GIM. Eighty percent of internists who had practiced at the GIM clinic agreed that their clinical competencies as a specialist were not sufficient to be a primary care physician. 92% of them disagreed with the hospital's continued operation of the GIM clinic under current administration, however, if GIM trained physicians were stationed at the clinic, the same number of internists agreed that the clinic would play an important role at their hospital. Psychiatrists were more willing to cooperate with GIM clinic compared to other specialties. CONCLUSION: Historically, the culture of academic health centers had not been hospitable toward primary care. However, our survey demonstrated that the chilly climate has been changing. Only very few physicians perceive primary care tasks as not requiring a high levels of expertise. In many schools, increased attention to education for primary care practice is evident in Japan. A more generalist workforce with competence and expertise in primary care is essential. PURPOSE: Previous work has demonstrated that African American elders experience higher functional and cognitive disability than White elders. The Program of All-Inclusive Care for the Elderly (PACE) provides comprehensive community-based long-term care services for nursing home eligible elders who remain in the community. Once enrolled patients have equal access to all needed medical and long-term care services at no additional cost. We examined the impact of enrollment in the PACE system on mortality differences between African American and White patients. METHODS: 2002 White patients and 859 African American patients enrolled at 12 nationwide PACE sites from 1990 ± 1996. All patients were followed from enrollment through death, or 12/31/97. On enrollment, measures of demographic characteristics, functional status, and comorbid medical diagnoses were obtained by social workers, nurses, and physicians. We used the method of Kaplan-Meier to describe mortality rates in African American and White patients. We used the log-rank test and Cox-proportional hazards models for bivariate and multivariate comparisons of mortality rates for African American patients vs. White patients. RESULTS: On enrollment into PACE, African American patients were younger than White patients (77.4 v 79.6, P < .01). However, African American patients had worse functional status (mean score on 10 point ADL scale, 6.5 v 7.2, p < .01) and were more likely to be diagnosed with dementia (56% v. 45% p < .01). 74% of both African American and White patients were women. A survival advantage for African Americans emerged after 9 months of enrollment in PACE.\nRates of mortality at 1, 2, and 3 years after enrollment were 13%, 24%, and 33% for African American patients and 16%, 29%, 39% for White patients (HR for African American patients = 0.76; 95% CI 0.66 ± 0.88). After adjustment for PACE site, age, gender, education, caregiver support, ADL function, presence of dementia and other comorbid conditions, African American patients continued to have a lower rate of mortality than White patients (HR = 0.79; 95% CI 0.66 ± 0.95). CONCLUSION: In a system providing equal access to comprehensive medical and long-term care services to frail elders, African American patients have substantially lower rates of mortality than White patients after adjustment for age, comorbidity, and ADL function on enrollment.\nPOTENTIALLY AVOIDABLE HOSPITALIZATION DAYS: A PROSPECTIVE ANALYSIS. A. Torn 1 , A. Pichon-Riviere 2 , C. Romero 1 ; 1 Sanatorio de la Trinidad, Capital Federal, Buenos Aires; 2 Programa de Efectividad Clinica -Argentina, Capital Federal, Buenos Aires PURPOSE: Analyze the distribution and causes of the potentially avoidable additional hospitalization days (PAAD) in an institution that has a hospitalist program. METHODS: From 06/99 to 01/00 the surgical and medical admissions of adult patients to a private hospital in Buenos Aires, Argentina were followed prospectively to detect PAAD. The additional days were defined using the Milliman and Robertson's Optimal Recovery Guidelines. The case management nurse assigned a cause from a set of 16 possibilities to each additional day and categorized the admissions in 5 severity levels. RESULTS: There were 1812 medical and surgical admissions of adult patients; 626 (34.55%) were medical and 1186 (65.45%) surgical from which 27.4% were emergency and 72.6% programmed procedures. The mean age was 56.3 years with a 25% of the patients older than 74 years; 58.5% were women. The average length of stay (ALOS) was 4.1 days with a median of 2.1; 5.8 for the medical admissions and 3.2 for the surgical ones. The difference between the programmed and emergency operations was significant: 2.6 vs. 4.9. There was also a significant increase of the ALOS with categories of age and severity. The inpatient mortality was 3.5%, 93.4% were discharged and 3% were transferred to a rehabilitation center or to home care. A total of 723 PAAD, representing a 9.64% of the hospitalization days, were generated by 600 admissions. The physicians were responsible of 668 PAAD (92.40%). Inappropriate admission (potentially ambulatory procedures), was the cause of 392 (54.22%) PAAD, 181 (46.17%) were due to gynecological procedures, 73 (18.62%) to general surgical and 62 (15.82%) to traumatological ones. Prolongation of hospital stay was the cause of 331 (45.78%) PAAD divided in 55 days (16.62%) attributed to the health care system and 276 (83.38%) to the physicians. Of the latter 106 (38.40%) were responsibility of traumatology and 82 (29.71%) of gynecology. A logistic regression model adjusted for severity, age, sex, type of admission and specialty was analyzed. The following specialties were positive predictors of PAAD compared to general surgery: gynecology (OR 7.84), traumatology (OR 2.75) and neurosurgery (OR 4.58) . Emergency surgery (OR 0.61) was a negative predictor of PAAD and age (OR 1.01) was a positive one. CONCLUSION: Even though this study was conducted in an institution where a hospitalist program is in place, there are still a significant number of potentially avoidable additional days. They are concentrated in programmed surgical procedures mainly gynecological and orthopedic, and in the lower levels of severity. PURPOSE: Primary care physicians (PCPs) are often required to act as gatekeepers and or patient advocates for mental health services in an extremely complex administrative environment that includes managed care organizations with and without mental health carve-outs. This analysis addresses two aspects of this environment: (1) number of managed care contracts per practice (2) percentage of patients for whom the physician serves as managed care gatekeeper. METHODS: Data from the 1996 ± 97 Physician Community Tracking Study performed by the Center for Studying Health System Change were analyzed. This cross-sectional survey consisted of telephone interviews of 12,385 physicians (5,583 primary care physicians caring for adults) and had a response rate of 65 percent. Access to high quality mental health services was defined as PCPs indicating that they always or almost always could obtain high quality mental health services for their patients. RESULTS: Perceived availability of high quality services was much lower for outpatient mental health than for outpatient medical specialists (30% vs. 81 %, p-value < 0.001). Perceived access to high quality outpatient mental health services declined with increasing numbers of managed care contracts: zero (44%, Odd Ratio 2.3, CI 1.8, 3.1), one (37%, OR=1.8), two to ten (27%, OR=1.1), eleven to twenty-five (29%, OR=1.2) and more than twenty five (25%, OR=1.0). These findings persisted after adjustment for patient demographics (Medicare percentage, Medicaid percentage), physician demographics (age, gender, foreign vs. US medical graduate and overall career satisfaction) and practice characteristics (type of practice and concern about financial penalties for practice decisions) using multiple logistic regression analysis. An adjusted OR of 1.9 (p-value of < 0.001) was observed for zero contracts compared to twenty-five or more. A U-shaped distribution in perceived access was observed for the percent of patients in the practice for whom physician served as gatekeeper: three percent or fewer patients in gatekeeper status (42%, OR 2.6), more than ninety percent of patients (43%, OR 2.6) and fiftytwo to sixty percent of patients were in gate-keeping status (22%, OR 1.0). These findings persisted after adjustment using the same model described above (p-value < 0.001). The same patterns were observed for inpatient psychiatric services. The same relationships between access to high quality services and number of managed care contracts and gatekeeper percentage were not observed for medical services. CONCLUSION: The mental health referral process may be uniquely vulnerable to the administrative burdens imposed by the current managed care environment. Paradoxically, while large numbers of managed care plans with a variety of options are thought to increase choice, they may actually decrease access to high quality mental health services. PURPOSE: For many chronic diseases, effective drug therapy is now available. However, patients often fail to adhere to prescribed regimens with suboptimal clinical results. We surveyed resident and faculty physicians (MD) of a university-based internal medicine clinic serving an indigent population to determine MD attitudes, beliefs and behaviors with respect to patient drug adherence (DA). METHODS: One hundred two MDs completed a 14 question survey asking them to definè good' adherence, estimate the proportion of their patients who achieve 'good' patient DA, and to respond to a series of statements regarding DA. Two mailings produced an 86% response rate. At least 20 responses were obtained from each of PGY1,2,3 and attending MD levels. RESULTS: Seventy nine percent of MDs defined`good' adherence' as patients taking at least 80% of prescribed doses. However, MDs estimated that only 66+/À15% (mean/SD) of their patients acheived`good' adherence. In the prior 6 months, 50% of MDs discussed DA with at least 80% of patients, and 91% of MDs reported discussing DA with at least 50% of patients. Despite these discussions, only 32% of MDs felt that at least half of their patients had a good understanding of the importance of drug therapy to avert adverse outcomes. Almost all (97%) MDs felt that DA assessment and intervention was an important component of the office visit and 100% felt that prescription refill feedback would help them target patients for adherence intervention. There was no association between year of training and DA responses. CONCLUSION: In this indigent care setting, MDs indicated that they frequently discussed DA with patients but felt that patients lacked a good understanding of the role of their medication and that many were not adherent to their prescribed regimens. All MDs felt that specific DA feedback based on prescription refill data would assist them to identify and manage non-adherent patients. Since automated pharmacy claims systems cover the majority of the US population, such feedback is feasible and worthy of future study in both indigent and more affluent populations.\nPURPOSE: Despite mounting evidence that beta-adrenergic antagonists (beta-blockers) are beneficial immediately following acute myocardial infarction (AMI), a median of only 64% of Medicare beneficiaries nationwide receive beta-blocker within 24 hours of AMI. Measuring use of beta-blockers in AMI usually requires expensive chart reviews. Using a comprehensive electronic medical records system instead of chart review, we sought to measure use of betablockers in AMI as well as differences in charges between those who received beta-blocker and those who did not. METHODS: We examined data from all 15,610 Medicare-covered hospitalizations during 1995 ± 99 for 7,251 patients in an urban academic medical center, evaluating for International Classification of Diseases codes for AMI. Restricting study to patients 65 years of age or older, we collated and merged into this all available prescription drug data pertaining to these patients, separating inpatient from outpatient drugs based on date. We then calculated rates of betablocker use in AMI and differences in estimates of total hospital charges and length of stay depending on whether beta-blocker were prescribed. RESULTS: Of 9,191 hospitalizations among 4,490 older adults, 385 (4%) contained a diagnostic code for AMI. Beta-blockers were prescribed in 252 (65%) of the encounters, and 85% of prescriptions occurred on the date of admission or one day later. Overall, beta-blocker prescriptions occurred on one of these days in 55% of hospitalizations for AMI. Metoprolol was the beta-blocker prescribed most often (77% of beta-blocker use in AMI). Of those receiving any beta-blocker, 3% had a diagnostic code for obstructive lung disease, compared to 5% among those not receiving beta-blockers (p = 0.37). Those receiving beta-blockers had mean length of stay 1.5 days longer (p < 0.05) but mean charges not significantly different from those not receiving beta-blockers. CONCLUSION: A comprehensive, electronic medical records system can be used instead of resource-intensive chart review to measure use of beta-blockers after AMI. Uncertainty about the precise relationship between the two methods should prompt, as a next investigative step, a more direct comparison of chart review to the use of electronic records to measure indicators of quality of healthcare. shown that these agents are underutilized in practice. Contributing to this finding is the perception that other agents may work better or have fewer side effects. Under the assumption that a medication class switch represents a physician's belief that the medication was of little benefit, or was not well-tolerated by the patient, this study compares the degree to which each of the major classes of antihypertensive medications is discontinued in favor of another class of antihypertensive. METHODS: We examined pharmacy data from a population of Medicare patients enrolled in a managed care organization (MCO) between December 1996 and October 2000. An overall interval of antihypertensive medication usage was determined for each patient. The subset of this time interval in which the patient received a beta blocker (BB), calcium channel blocker (CCB), ACE inhibitor (ACEI), or diuretic was determined. A therapy class switch was defined as prescribing a new class of antihypertensive medication more than one month after the end of the original antihypertensive class interval ended. In this manner, the addition of a second antihypertensive would not be considered a class switch, unless the first medication was stopped permanently, and the second medicine continued for more than a month. RESULTS: Of the 9798 patients who filled at least 3 antihypertensive prescriptions over at least 90 days, 4260 received a BB, 4225 received a CCB, 4533 received an ACEI and 3480 received a diuretic (sum exceeds total since patients could have been on more than one class). Kaplan Meyer survival analysis indicates that after 1000 days, a higher percentage of patients continue to take BB (68.5%) than ACEI (63.8%), CCB (61.8%) or diuretics (53.4%). The length of time until 25% of patients had switched their medication class was longest for the group taking BB (760 days), then ACEI (589 days), CCB (568 days) and diuretics (387 days). This trend favoring BB is also observed when limiting the analysis to classes of antihypertensives that were initiated in the observation period. CONCLUSION: The results of the study suggest that, contrary to common perceptions, among a Medicare population, beta blockers are not more commonly discontinued than the more modern, and expensive antihypertensive agents. While the clinical factors that influence a physician's decision to start a particular medication class are not known, this potential bias is mitigated by the common tenets of good clinical practice. These tenets dictate that when choosing to prescribe any class of drug, the physician believes it would be safe, effective and well tolerated. The study results suggest that, regardless of the initial indications for choosing a particular therapeutic class of antihypertensive, or the reason for eventually changing the class (non-compliance, patient complaints, ineffectiveness, expense, etc.), BB seem less likely than ACEI and CCBs to be switched, while diuretics are more likely than the other classes to be discontinued. PURPOSE: Attention to issues of autonomy, surrogates and treatment preferences are important for vulnerable elders, who are at risk of decisional incapacity and death. We investigated these elements of end-of-life care among a cohort of vulnerable elders. METHODS: The Assessing Care of Vulnerable Elders project developed and tested quality indicators (QIs) for community-based personsÊ65 years at increased risk of death or functional decline. Based on a systematic literature review, an expert panel rated 14 QIs related to documentation about surrogates and preferences, and translation of preferences into care as valid process measures for end-of-life care. We measured these QIs by reviewing 13 months of inpatient and outpatient charts of patients in 2 managed health plans. RESULTS: Of 3206 seniors randomly selected from the plans, 2,278 (71%) were interviewed and 475 were identified as``vulnerable''. Of 400 (84%) who consented to study participation and had abstractable medical records, 376 were eligible for at least one QI. Fourteen elders (4%) had any documentation (formal or informal) about surrogate decision makers in their outpatient charts. Charts for 103 hospitalizations yielded 8 advance directives, 23 noted a surrogate or an attempt to find one, and 72 (70%) contained no documentation. When hospitalized with altered mental status, 5 of 20 elders had any documentation about a surrogate within the first 3 hospital days and 1 of 6 elders admitted to an ICU had any documentation about preferences within 3 days. Seventy percent of orders to limit care in the hospital considered patient participation in decision-making and 2 of 2 ventilated patients had documentation about preferences. CONCLUSION: Despite much recent attention to end-of-life care, surrogates and preferences are not elicited prospectively from vulnerable elders. Improvement in the quality of end-of-life care is needed, particularly for persons at risk of life-threatening events. PURPOSE: Measurement of quality of care (QOC) for mainstream medical conditions has received much attention, but no method exists to measure QOC for geriatric conditions. We developed a system to comprehensively measure QOC provided to vulnerable elders and compared QOC for geriatric and medical conditions. METHODS: Based on structured literature reviews, content experts developed 239 quality indicators (QIs) for the process of medical care provided to``vulnerable elders'', communitybased persons > 64 years old at increased risk of death or functional decline. These QIs assessed care for 22 conditions. We applied these QIs to a community sample of vulnerable elders by abstracting their outpatient and inpatient charts, comparing QOC provided for medical conditions (depression, diabetes, heart failure, hypertension, coronary disease, osteoarthritis, osteoporosis, pneumonia and stroke) to geriatric conditions (delirium, dementia, falls, geriatric assessment, incontinence, malnutrition and pressure ulcers) RESULTS: Of 3206 seniors randomly selected from two managed senior health plans, 2,278 (71%) were interviewed and 475 were identified as``vulnerable''. Of these, 400 (84%) consented to participate and had abstractable charts. For medical conditions, 52% of 1897 QIs were satisfied (e.g. diabetes 58%, heart failure 72%). This was significantly better (p < 0.01) than the 37% of 2036 QIs satisfied for geriatric conditions. Compliance rates for geriatric conditions included falls 26%, dementia 35% and incontinence 29%. CONCLUSION: QOC for geriatric conditions falls far below that of QOC for medical conditions. Quality assessment and assurance systems should incorporate QIs measuring geriatric syndrome care. Care for geriatric conditions ± common among vulnerable elders and associated with functional decline and mortality ± must be improved. PURPOSE: Continuity and coordination (CC) of care are core elements of general internal medicine. This is particularly important for``vulnerable elders'' who may be at high risk of adverse events due to poor CC. Yet there is no method of measuring CC in medical care. We developed and tested a system to measure CC of care among a cohort of vulnerable elders. METHODS: The Assessing Care of Vulnerable Elders project developed and tested quality indicators (QIs) for community-based persons > 64 years at increased risk of death or functional decline. Based on a systematic literature review, an expert panel rated 13 QIs as valid process measures of care CC. Six of these could be measured by abstracting information from medical records. QIs evaluated medication, test and information continuity, within and between providers, across the inpatient and outpatient settings.\nRESULTS: Of 3206 seniors randomly selected from two managed health plans, 2,278 (71%) were interviewed and 475 were identified as``vulnerable''. For the 400 (84%) patients who consented to study participation and had abstractable medical records, 67% of 324 CC QIs were satisfied. Of 185 seniors started on new medications during the study, 67% had follow-up of this medication at the next physician visit. Of 72 patients who were hospitalized, 89% received postdischarge follow-up within 6 weeks; 6 of 11 had follow-up of a medication started in the hospital and 10 of 14 had follow-up of a test pending at hospital discharge, but only 41% had a copy of the discharge summary in the outpatient chart. CONCLUSION: Vulnerable elders at two managed care plans received a high level of CC of care. Measuring CC care identifies aspects that require intervention. In an effort to control health care costs, many third party payers limited physicians' abilities to order expensive tests for their patients, requiring the use of an appeals process to obtain restricted medical services. Studies suggest that, at times, physicians are willing to misrepresent clinical information to third party payers in order to obtain these services for their patients. Little is known about whether characteristics of the appeals process influence physicians' willingness to sanction deception. METHODS: We surveyed a random sample of 1,617 physicians in the U.S., and presented each with a hypothetical scenario in which an insurance company refused to pay for a medically important service. We varied the``hassle'' of the appeals process by varying the time required to make an appeal and the likelihood of having a successful appeal. In addition, we varied the severity of the patient's health condition from moderate low back pain to severe angina.\nPhysicians were asked if the patient's physician should accept the insurance company's restriction, appeal the restriction or misrepresent the facts to the insurance company in order to obtain coverage for the patient. RESULTS: Overall, most physicians responded that they would appeal (77%) rather than accept (12%) or misrepresent (11%) a restriction on medically necessary care. Physicians were more likely to misrepresent if the appeals process was longer (OR=1.68, CI 1.09 ± 2.59), less likely to succeed (OR=1.57, CI 1.02 ± 2.42), or involved a more severe condition (OR=2.19, CI 1.39 ± 3.45). Among physicians asked about severe angina, as the appeals process became more cumbersome, they were more likely to choose to misrepresent the facts to the insurer rather than appeal. When asked about a patient with moderate low back pain, physicians' decisions about how to respond to an insurance company restriction were not significantly affected by the hassle associated with the appeals process. CONCLUSION: Physicians' willingness to sanction deception of insurance companies varies according to length of the appeals process, the likelihood of a successful appeal, and condition severity. When the stakes are high, and the hassle of appeals is great, physicians become increasingly willing to sanction deception. Changes in the difficulty of appeals processes may ease the tension physicians' face between patient advocacy and honesty. PURPOSE: To assess general internists' (GIM) satisfaction and recruitment of medical students and compare these to family physicians (FP). METHODS: 5704 physicians were surveyed (adjusted response rate 52%) from a national random stratified sample-19% GIM (n=450, 108 women, 75 were``young'' (age < 45); 341 men, 109 were young) and 22% FP (n=502). Mean global job and specialty satisfaction as well as likelihood of recommending their specialty to medical students were compared by specialty. All were assessed on a 5 point Likert scale (3 = neither agree nor disagree). For general internists, further analyses were done by age and gender and logistic regression was used to model predictors of medical student recruitment. RESULTS: GIM had significantly lower job and specialty satisfaction vs FP (job: 3.52 vs 3.77; specialty: 3.18 vs 3.69, both p < .001). Global satisfaction did not differ by gender alone for GIM. However, younger women and older men had higher job satisfaction then their same gender counterparts (women 3.75 vs 3.28, p=.043, men 3.63 vs 3.28, p=.015) with no difference in specialty satisfaction. GIM was less likely than FP to recruit medical students to their specialty (3.26 vs 3.85, p < .001). Women GIM recruited with more enthusiasm than men (3.56 vs 3.16, p=.022) with younger women the most enthusiastic (young 3.67 vs old 3.35, p=.038). Global specialty satisfaction and female gender were positively associated with medical student recruitment by internists. CONCLUSION: General internists have lower job and specialty satisfaction than family physicians. This dissatisfaction leads to less recruitment of medical students into internal medicine. Increasing numbers of young women in GIM may change this trend but recruitment needs to be seen as an important outcome of physician satisfaction to keep the specialty viable. (27); plan cost rating, 64 (29); service restrictions 71 (24). TC rates were: HAART, 68%; PPD,14%; and flu shots, 32%. In multivariable models there were no significant differences between MC and FFS coverage for any of 7 IPC, 4 HPQ, or 4 TC quality indicators. Models controlling for race, education, sexual preference, and duration of physician ± patient relationship, which varied by plan type, were no different. Tests for an interaction between physician and FFS/MC differences were non-significant. CONCLUSION: For patients with HIV in Boston, managed care coverage was not associated with lower quality care. These experienced HIV providers do not appear to practice differently depending on insurance type. Our findings suggest that managed care coverage can be as effective as FFS coverage even for patients with a complex, expensive, chronic illness. We hypothesized that physicians would frequently field questions about CAM from their patients, but, due to a lack of education about these treatments, would feel uncomfortable discussing them. We therefore surveyed a metropolitan area group of physicians to ascertain their patterns of communication with patients regarding CAM and the factors that influence them to discuss CAM with their patients or refer them for CAM treatments. METHODS: Seven hundred fifty one randomly selected physicians in all specialties in the Denver, Colorado metropolitan area were asked about their exposure to, knowledge of, and belief in 16 CAM modalities and patterns of physician Ð patient communication regarding CAM. Analyses were conducted using the SAS system (version 6, fourth edition, Cary, NC, 1989) and included the two-tailed t-test, Pearson correlation coefficients, linear regression, and multivariate analysis. RESULTS: Three hundred and two of 705 deliverable surveys (43%) were returned. Responders averaged 44 years old (+/À 12 years), 63% were male, and 48% were selfdesignated primary care physicians. Seventy-six percent of physicians reported having at least one patient using a CAM therapy, 59% had received queries from patients about specific CAM treatments, 48% had recommended a specific CAM therapy to at least one patient, and 24% had used a CAM modality for themselves. While patients frequently sought out information about CAM modalities from their physicians, only 31% of physicians routinely ask their patients about their use of CAM modalities, and 17% never ask. Physicians most frequently recommended massage, relaxation, acupuncture, and biofeedback to patients, while the modalities physicians used most frequently for themselves were massage, relaxation, herbs, and yoga. Physician recommendation of CAM use to patients was most strongly associated with physician self-use (odds ratio 6.98,p=0.0001). Only 45% of physicians felt at least somewhat comfortable discussing the risks and benefits of CAM with their patients; the overwhelming majority (84%) felt they needed to learn more about CAM modalities to adequately care for patients. CONCLUSION: Education about CAM modalities is a significant unmet need among physicians residing in the Denver metropolitan area, and education may help alleviate the discomfort physicians have when fielding questions about CAM from patients. Physicians who already use CAM treatments for themselves are much more likely to recommend CAM for their patients than those physicians who don't personally use CAM. (1346) responded. The questionnaire included demographic items, the SF-12 to assess physical and psychological function and their satisfaction with, need for and importance of both their VA and non-VA sources of care. Non-VA care was identified using the HCFA enrollment database. RESULTS: Among the entire population, 303 (17%) belonged to a Medicare HMO for the entire 24 months, and 130 (7%) belonged to an HMO for part of the period. Questionnaire responders did not differ from non-responders by marital status, gender, service connected status or HMO enrollment. Among the HMO enrollees, 66% reported having a VA primary care provider and 46% reported receiving all their medical care from the VA. However, 72% reported having a non-VA primary care provider. When comparing VA users who belonged to a Medicare HMO to those who did not enroll in an HMO, the HMO members reported significantly higher levels of educational attainment and financial resources. The psychological and physical function scores did not differ amongst the various Medicare enrolled veterans. CONCLUSION: Users of VA medical care frequently belong to Medicare HMOs, and nearly half of these HMO enrollees report receiving most of their medical care from the VA. VA users who belong to HMOs tend to be better educated and more financially secure. VA care appears to provide a substantial financial benefit to Medicare HMO plans. Little is known about the extent to which characteristics of health care organizations foster or hinder such performance. We evaluated variations in tobacco counseling rates among VA medical centers (VAMC's) nationwide and assessed the medical center and primary care program attributes related to high and low performance. METHODS: We assessed VAMC organizational characteristics (e.g., region, urban location, primary care features) using a previously validated mailout survey fielded to VA primary care directors (100% response). We matched these survey-based measures to two centrally available databases: (1) organizational culture scores derived from the first wave of the National VA Quality Improvement Survey (G. Young, PI) administered to over 100 employees at each VAMC, and (2) tobacco counseling rates for each VAMC from externally-performed chart reviews of randomly sampled users with 1+ primary care and 1+ subspecialty visits (88% match). We used bivariate analyses to examine the relationships between discrete organizational features and counseling rates and multiple linear regression using a forward stepwise algorithm to assess the independent predictors of counseling rates. RESULTS: Counseling rates varied by region (p < .01), with lowest rates in the East (0.73) and highest in the Western states (0.83). More complex VAMC's had lower tobacco counseling rates (p < .0001), consistent with lower rates among urban (p < .0001), academic (p < .01) VA's, with more internal medicine house officers (p < .01) and larger patient caseloads (p < .001). VA's with higher self-reported levels of primary care implementation, higher proportions of veteran users receiving all or most of their care in primary care, and where primary care physicians had responsibility for their patients as inpatients had higher tobacco counseling rates (all p < .05).\nHigher rates also occurred in VA's with higher staff ratings of leadership quality (p < .001) and marginally higher group (p=.054) and risk-taking cultures (p=.055), while those VA's with higher staff orientation to quality improvement (p < .05) and more primary care-based process action teams (p < .05) had higher rates. CONCLUSION: Achieving more guideline-adherent smoking cessation practices in large, academic medical centers may pose significant challenges for primary care managers, but appears more likely among medical centers with more highly organized primary care practice. Improving counseling rates requires systems approaches to address both structural and provider-related constraints. Our objective was to describe duration of visit to chiropractors (CP) and acupuncturists (AC), the factors associated with these visit times, and to compare visit duration among physicians (MD), CP, and AC. METHODS: A random sample of licensed CP (N=130) from Arizona and Massachusetts, and licensed AC (N=133) from Washington and Massachusetts, was surveyed on consecutive office visits in 1998 ± 99 regarding visit time, patient demographics, reason for visit, acuity of problem, payment source, diagnostic and therapeutic techniques performed. (N=2550 CP encounters, mean age 44.9 years, 57% female; N=2561 AC encounters, mean age 46.2 years, 68% female).\nWe used linear regression to analyze factors associated with visit duration and to calculate the minutes attributed to each factor. Models were adjusted for specific therapeutic and diagnostic techniques performed at the visit. MD visit duration was obtained from published 1995 ± 96 National Ambulatory Medical Care Survey results. RESULTS: Mean visit duration for CP was 21.5 minutes (SE=0.8) and for AC 56.6 minutes (SE=0.7), compared to 18.9 minutes (SE=0.5) for a visit to an internist (p=.006 for CP vs. MD, p < .001 for AC vs. MD). AC spent more time with patients at self-pay visits, new patient visits, and visits for flares of chronic problems, adding 4.7, 4.0, and 2.4 minutes respectively. AC visits for anxiety/depression were 2.6 minutes longer, with a mean duration of 50.2 minutes, compared to 32.6 minutes for MD visits for the same problem (p < .001). For CP visits, significantly more time was spent with new patients, when there was communication with other providers, and with patients receiving concurrent care from an MD, adding 6.7, 3.2 and 2.3 minutes to each visit, respectively. CP visits for wellness and chronic problems were shorter than acute visits, by 3.2 and 4.3 minutes, respectively. Patient age, sex, and reason for visit (i.e. back or neck symptom) did not affect CP visit duration. Visits in Arizona were, on average, 3.0 minutes longer than those in Massachusetts. CONCLUSION: Acupuncturists spend significantly more time with patients on an average visit compared to physicians. Chiropractors report spending more time than physicians, although this difference is small and may be due to differences in how the data were collected. CP visit duration suggests regional variation. Payment source, communication with other providers, psychosocial reason for visit, and acuity of visit are important correlates of CP or AC visit duration. These trends towards``vertical integration'' (VI) have been studied primarily in terms of their financial causes and impact. Effect on clinical care is unknown. This study examines whether hospital VI impacts continuity and timing of clinical process of care for acute stroke patients. METHODS: A survey was fielded in mid-2000 of the primary stroke providers at all acute care hospitals with acute stroke discharges in California in 1998. Survey findings were linked to California state hospital facility reports and individual patient discharge records for 1998. Multiple regression models were used to examine the association of highest level of integration (internal rehab unit (IR), formal control of external unit (FER), informal agreement of external unit (IER), and no agreement (NoR)) with service availability, reported interventions, and patient outcome, controlling for other hospital features and patient demographics. RESULTS: Surveys were completed for 256/374 (68%) of hospitals that admitted stroke patients in 1998 and that were still open in 2000. Non-respondent hospitals were smaller, rural, or independent. Among respondent hospitals, level of integration was 20% IR, 7% FER, 15% IER, and 58% NoR. Greater integration was significantly associated with stated greater use of acute care guidelines, hours of rehab care during acute hospitalization, continuity of providers between acute and post-acute care, management by specialists, and use of common medical information systems. When linked to patient records, level of integration did not predict initiation of rehab during hospitalization or in-hospital mortality, but was associated with increased likelihood of discharge to post-acute rehab.\nCONCLUSION: VI appears to have clinical advantages of greater underlying consistency and continuity of care, but does not appear associated with early (acute) rehab intervention. Shortterm mortality does not appear to be associated with VI, but other outcome measures such as functional recovery may be more sensitive and appropriate. As trends towards vertical integration continue, a better understanding of VI's clinical impact is imperative. PURPOSE: One barrier to screening and disclosure of Intimate Partner Violence (IPV) is the patient's lack of trust in their health care provider. The objective of this study was to identify characteristics that facilitate trust in the patient-provider relationship among survivors of IPV. METHODS: Qualitative data were collected using semi-structured, open-ended interviews with a sample of 28 female survivors of IPV. Participants were recruited from community-based IPV organizations in the Boston area. A one hour face-to-face interview explored the woman's beliefs and attitudes about trust in her interactions with health care providers. Interviews were audiotaped and transcribed. Using Grounded Theory Methods, the transcripts were analyzed for significant themes, from which a conceptualization of trust was developed. RESULTS: Among the participants, ages ranged from 18 to 56, 36% were Black, 32% Hispanic and 18% white. All suffered physical violence, but only 60% ever sought medical care as a result. We identified five dominant concepts that described dimensions of provider behavior on which trust was based: 1) OPEN COMMUNICATION Ð listening to patients and keeping them well informed of their medical condition; 2) PROFESSIONAL COMPETENCY Ð appearing knowledgeable, performing thorough medical evaluations and being familiar with the medical and social histories; 3) PERSONAL COMMITMENT Ð being consistently accessible, both physically and emotionally (``always been there for me''), respecting confidentiality, spending ample time with participants and demonstrating personal concern outside the traditional provider role; 4) EMOTIONAL SAFETY Ð sharing personal information and establishing a climate of safety for the participants through nonjudgmental and compassionate gestures (``he had tears in his eyes'') or comforting nonverbal mannerisms; 5) EMPOWERMENT Ð promoting self-efficacy and shared decision-making. Gender and ethnic characteristics of the providers had variable effects on trust. CONCLUSION: These survivors of IPV reported several aspects of provider behavior that facilitate trust in their clinical relationship, including open and explicit communication, professional competence, personal commitment, establishing a safe emotional environment and patient empowerment. Strengthening these provider behaviors may increase trust with patients and thus improve screening and disclosure of IPV. PURPOSE:``Turfing'' is the transfer or triage of a patient to a physician for reasons the receiving physician deems inappropriate.``Turfing'' has been identified as a troubling source of professional and ethical conflict for housestaff and medical students. Prior work has reported that``turfed'' patients have a worse hospital experience than appropriately transferred patients. We wondered whether attendings' perceptions of``turfing'' supported patients' perspectives. METHODS: One trained research assistant conducted in-depth, semi-structured, open-ended individual interviews with a convenience sample of 16 attending physicians from internal medicine, emergency medicine and general surgery. The interviews were meant to elicit attendings' views on the quality of``turfed'' patients' care as well as other dimensions of this triage phenomenon. Audiotapes of interviews were subjected to content and thematic analysis. Results presented here include only attendings' comments about the quality of``turfed'' patients' care. RESULTS: All attendings interviewed in emergency medicine (n=4) and general surgery (n = 4) felt that``turfed'' patients received inferior care. Of the 8 internal medicine attendings interviewed, 5 felt``turfed'' patients received inferior care, 1 was equivocal, and 2 did not comment on this specific issue. Subjects considered quality of care to include length of stay, interpersonal interactions between patients and housestaff, and delays in management plans and bed assignments. Subjects stated:``There is always hostility toward the patient from the housestaff in these situations If you go watch [the housestaff] and see how they interact with the patient, they are abrupt with them, dismissive toward them.''``Patients are put in harm's way.''` [ Housestaff] probably don't take as inquisitive an approach [to history-taking] which is not in the patient's best interest.''``We avoid people who have been in the ED for too long [because no one will accept them on their service].'' Three themes emerged as causes for this inferior care: lack of``ownership'' of``turfed'' patients by physicians, the transient nature of physician relationships with``turfed'' patients, and feelings that``turfs'' are unwanted. CONCLUSION: Attendings' interviews substantiate that the care of``turfed'' patients suffers. Interviewees recommended remedies for these troubling findings, such as stronger moral leadership from department chairs and improved role modeling by clinical faculty. The purpose of this pilot study was to determine whether there are differences in the way Caucasians and African Americans would present facts about prostate cancer screening in a culturally appropriate brochure to men considering screening. Because screening with prostate specific antigen (PSA) is controversial, several professional organizations recommend informing men about the risks and benefits of screening. Compared to Caucasians, African Americans have a higher risk for prostate cancer and are less knowledgeable about screening. In a previous study, experts in prostate cancer and Caucasian and African American couples with screened and unscreened men identified 17 key facts about PSA they believe men ought to know. METHODS: We convened a focus group of 5 African American couples and a focus group of 5 Caucasian couples who each met twice. At the first meeting couples viewed a videotape about prostate cancer and screening and were asked to discuss how to present the 17 key facts in a culturally-sensitive way in terms of content and format. Mock ups of brochures were developed for discussion at the second meeting. We analyzed transcripts of the focus group meetings using content analysis to identify differences between African American and Caucasian groups in how to present this information. RESULTS: We found differences in content emphasis and in graphic design choices between the brochures that African Americans and Caucasians would design for members of their racial/ethnic group. Because of the perceived discomfort and embarrassment associated with the digital rectal exam (DRE), African American men felt strongly that it was important to emphasize the advantages and disadvantages of screening with the DRE, as well as the PSA. Caucasian men did not discuss the DRE at all. African Americans believed it was important to emphasize epidemiologic data specific to African American men, such as their higher risk of prostate cancer. They also preferred images and symbols rooted in African American culture (e.g. Kente cloth). CONCLUSION: African Americans and Caucasians differed in the way they chose to present facts about prostate cancer screening to members of their racial/ethnic group. Cultural differences in format and content need to be considered when designing educational materials such as brochures.\nRACIAL PROFILING, CLINICAL EPIDEMIOLOGY, AND CULTURAL COMPETENCE: WHEN IS RISK STRATIFICATION BY RACE OR ETHNICITY JUSTIFIED? M.H. Chin 1 , C.A. Humikowski 1 ; 1 University of Chicago, Chicago, IL PURPOSE: Population-based, probabilistic approaches to medicine have become more prominent with the rise of the evidence-based medicine movement. Simultaneously, diverse patient populations have spurred development of curricula in cultural competence. Building upon lessons from the racial profiling debate in the legal and political realms, we aimed to develop a conceptual framework for exploring whether race and ethnicity should be used as clinical tools or if dangers of stereotyping are too great. METHODS: We reviewed key literature pertinent to racial profiling, race and medicine, and curricula in cultural competence. We explored issues of race and medicine through a regular seminar series with medical housestaff, and extensive feedback from colleagues. We developed a conceptual framework that balances clinical epidemiological principles with lessons learned from social and political racial profiling. RESULTS: We propose that viewing the patient within a wider population-based cultural setting can help guide the initial clinical approach, but individualized care is mandatory. The use of race and ethnicity as clinical tools is on a slippery slope Ð they may be useful or dangerous. Biological examples are rare in which race or ethnicity are relevant (e.g. Ð Tay ± Sachs, sickle cell). However, ethnicity may have a statistical association with a variety of factors. The potential benefit of using ethnicity as a tool is to aid an individual patient by making medical care better tailored to him or her. The potential detriments are reinforcement and perpetuation of partial myths, encouragement of undesirable behavior by creating negative expectations, and avoidance of addressing the underlying individual factors. Racial profiling has taught us that the costs of using ethnicity as a proxy for behavior are too high. But, ethnicity may appropriately be used as an initial proxy for history, language, culture, and health beliefs, as long as individualization of care is rapid. The predominant approach to cultural competence in medical education today teaches a consideration of individual patients as opposed to a rigid checklist of ethnic traits. For example, providers are advised to inquire about the meaning of illness to the patients, and the social context such as family, literacy, and finances. However, given that physicians have limited time and energy, context including race and ethnicity can be helpful in guiding initial inquiry and raising awareness that an issue (e.g. Ð strong distrust of the health care system) may be important.\nCONCLUSION: In the specific situations outlined, race or ethnicity may be useful clinical tools as long as individualization of care is quick. However, vigilance is required as medicine has not been immune to overt or subconscious racism. Case examples are used to define our conceptual framework in more detail.\nMOOD DISORDERS. W. Resnick 1 , T. Houston 2 , K. Swartz 2 , L.A. Cooper 2 ; DRADA, Baltimore, MD; 1 Johns Hopkins University, Baltimore, MD PURPOSE: To describe how mutual help support groups are used by individuals affected by mood disorders. METHODS: Six focus groups, including 39 participants (19 women, 20 men), were conducted. Participants were recruited through Depression and Related Affective Disorders Association (DRADA) support groups. Discussions addressed what if any influence support groups have had on their lives; and the relationship of the groups to medical treatment and/or therapy. Discussions were audiotaped, transcribed verbatim, and reviewed independently by two investigators to group distinct comments into categories with specific themes. Differences were resolved after a joint review of themes. Comments within categories were then checked for relevance and consistency by two second reviewers Ð a general internist, and a psychiatrist. Adjustments to categories were made until consensus was reached. RESULTS: Comments fell into 10 categories: 1) Patient Activation/Empowerment in their treatment, 2) Using support groups to complement treatment, 3) Obtaining education and information, 4) Learning to interact positively with family and co-workers, 5) Socializing with support group members, 6) Sharing Ð to understand each other, 7) Helping others, 8) Increasing self-esteem, 9) Emotional Support, and 10) General comments. All participants were positive about the influence of support groups in their lives. A sense of belonging, talking about problems, and feeling understood by others, were seen as a source of comfort and well-being. Participants felt their family and work relationships, and communication skills were improved by participation in support groups. They also reported an increase in self-esteem and participation in medical decision-making. All agreed that exchange of information about their illness and exchange of ideas with other people about coping, contributed to their health. Emotional support from peers, destigmatization of their illness, and a sense of hope were all benefits from the groups. Most individuals did not use the group as replacement for treatment. CONCLUSION: Health professionals should consider recommending mutual-help support groups as an adjunct to treatment. The combination of medical treatment and peer support may enhance patient participation in care, treatment adherence and social skills, and provide patients with positive coping strategies. Baltimore Mental Health System, Baltimore, MD PURPOSE: Studies document underutilization of outpatient specialty mental health services by African Americans (AAs). However, AAs with depression are just as likely as whites to receive care in primary care. Despite their use of primary care, AAs are less likely than whites to be recognized as depressed, offered pharmacotherapy, and to initiate or complete guidelineconcordant depression care. AAs express preferences for counseling and negative attitudes toward antidepressant medication, the most common form of depression treatment used by primary care physicians. Few depression education programs and materials target cultural beliefs and values of AAs. The purpose of this study was to determine the acceptability and usefulness of an educational videotape for AAs with depression. METHODS: After showing the videotape, we held four focus group discussions in two community settings and at an historically black university. Subjects included twenty-four AAs, aged 18 ± 76 years, who screened positive for depression. Thirty-eight percent reported a history of previous treatment. Focus group discussion questions addressed the usefulness of the videotape for helping viewers understand depression and its treatment, the most and least effective parts of the videotape, and the cultural appropriateness of the information presented. Participants took pre-and post-tests on attitudes about depression. Discussions were audiotaped, transcribed, and reviewed independently by two investigators to identify and group comments into specific themes. Two other investigators reviewed the themes and comments for consistency. Changes were incorporated to achieve consensus. RESULTS: In addition to comments specifically related to positive and negative aspects of the videotape, the following themes were identified: concerns about antidepressant medication; identification with people in the videotape; racial and cultural issues; stigma and stereotypes; spirituality; and validation and support from the focus group. The videotape was well received and rated by most viewers as effective in improving knowledge and alleviating concerns about depression and its treatment. After watching the videotape, attitudes improved in several areas: depression as a medical illness, effectiveness of treatment, negative perceptions of antidepressant medication and reliance upon spirituality alone to heal depression. CONCLUSION: This culturally tailored videotape about depression is deemed acceptable and effective to AAs with depression participating in focus groups. It also improves knowledge and attitudes about depression. Incorporating culturally-tailored educational materials and messages into community and primary care depression programs may reduce barriers to appropriate depression care for AAs.\nTHE SIGNIFICANCE OF LONG-TERM DOCTOR-PATIENT RELATIONSHIPS AMONG OLDER PATIENTS. C. Crenner 1 , K.A. Greiner 2 ; 1 University of Kansas Medical Center, Kansas City, KS; 2 Kansas University Medical Center, Kansas City, KS PURPOSE: Many older Americans have a personal stake in a relationship to a single physician that extends back decades. Changes in our healthcare system and in chronic care facilities may increasingly disrupt these connections. People who have preserved such long-term relationships have much to tell us about the significance of these relationships and their relevance to care. METHODS: We invited participation at four sites: a rural nursing home; an ambulatory clinic in an urban medical center; a suburban, assisted-living facility; and a private clinic in a lowincome, urban neighborhood. We enrolled subjects who could give an account of past medical care and who had kept the same primary physician for seven years or longer. We sampled to increase variability. We recorded and transcribed 17 semi-structured interviews and conducted a focus group with seven participants including 2 subjects not interviewed individually. We elicited open-ended discussion of each subject's relationship to a long-term doctor, asking about the features that interfered with or helped to sustain the relationship, and the significance that the relationship held. Immersion in the transcribed material and simultaneous reading in social history and anthropology produced a theoretical model based on the distinction between giftexchange qualities and commodity-exchange qualities in these relationships. RESULTS: Subjects were 6 men and 13 women, with a mean age of 75 years, and a mean duration of medical relationship of 19 years. Eighteen subjects were white and one was African American. Subjects attributed much of the durability of these relationships to the positive personal and professional characteristics of their doctors. There was variability in characteristics described as important. Few informants recalled their own active role in selecting their current physicians, and some had been in practices that were``handed-down'' from doctor to doctor. Distance and transportation were very important considerations for continuing care. Subjects were generally able to describe a rational process for selecting their next physician. CONCLUSION: Our theoretical model suggested that gift-exchange qualities in these relationships implied mutual obligations between recipients and donors that extended beyond the simple transaction of services. In contrast, commodity-exchange relationships existed solely for the exchange of specific services. Services in gift-exchange relationships had a value partly determined by the value attached to the relationship, while in commodity-exchange the value was independent. Our subjects favored characterizations of their long-term relationships as forms of gift-exchange, but they demonstrated an ability to shift to a commodity-exchange model. Statements consistent with a commodity-exchange model appeared especially in references to relationships of shorter duration, or to the conclusion of relationships to long-term providers. The day-to-day practice of medicine entails many ethical quandaries and yet little is known about practicing physicians' encounters with ethical dilemmas or how they resolve them. METHODS: A randomized, national telephone survey of internists including generalists was conducted to determine the ethical dilemmas encountered, the strategies used to address them, and the use and value of ethics consultation. Coding schemes were used to categorize verbatim descriptions of ethical dilemmas. RESULTS: Responding generalists (N=95) reflected the demographic characteristics of U.S. general internists (20% female, 23% non-white, 33% foreign born). One quarter of the ethical dilemmas encountered by general internists were related to justice issues concerning uninsured patients, practice in managed care, or allocation of limited resources. During the previous 2 years respondents had requested an average of 2 consultations. Physicians were most hesitant to request consultations because they are time-consuming. While many of the ethical dilemmas encountered were related to justice issues, no justice-related dilemmas were referred for ethics consultation. Physicians handled justice related dilemmas by strategies that included explicitly discussing limits with patients, acquiescing to limits, overstepping limits, searching for alternative sources, or providing free care. Physicians were often dissatisfied with resolution of justice questions and believed institutional or policy changes were required to achieve better resolution. CONCLUSION: General internists encounter a wide array of ethical dilemmas and address the vast majority of them on their own. Medical ethicists need to be aware that justice related dilemmas are often unsatisfactorily resolved and should explore ways to help in addressing them. Recent data suggest that this funding is becoming more concentrated at the medical schools most active in research, which tend to be in urban centers. I hypothesized that over the past 20 years, NIH research funding has increased in the states and regions with the highest per-capita NIH funding and highest population density, relative to the other states and regions. If present, the hypothesized shift in research funding may hinder efforts to reduce regional disparities in medical care. METHODS: Data from the NIH and US census bureau were used to calculate the per-capita NIH funding received by each state during 1980 ± 2000. The per-capita NIH funding amount for each state was obtained by dividing the annual funding amount by the population that year. To enable comparisons between years, per-capita funding for each state was standardized by dividing by the mean for all states in the same year. Regional disparities in per-capita NIH funding were evaluated in 2 ways: standardized funding amounts and their changes over 20 years were calculated for each formal US census region (Northeast, Midwest, South, and West), and standardized values and 20-year changes for each state were plotted on US maps. To determine whether per-capita funding shifted from rural to populous states over the past 20 years, the temporal trends in correlation between per-capita funding and population density were examined. RESULTS: Year 2000 per-capita NIH funding ranged from $3 in Idaho, to $247 in Massachusetts. Over the past 20 years, 7 of the 10 states with the highest per-capita NIH funding in 1980 had relative decreases, and 7 of the 10 states with the lowest per-capita NIH funding had relative increases in per-capita NIH funding. During this time, the Northeastern region of the US received the highest relative per-capita funding (1.6 times the national average), but the proportion (34%) of total research dollars did not significantly change during that time. Analysis according to formal US census region, and by US map inspection, suggested that per-capita funding was relatively stable for large regions, but considerable shifting of percapita funding occurred within states of every region. States with relatively higher population density tended to receive higher funding per capita (r=0.59, p < 0.01), but this relationship did not substantially change during the past 20 years, suggesting that per-capita NIH funding is not moving toward populous states at the expense of more rural states. CONCLUSION: During the past 20 years, disparities in relative per-capita NIH funding have shifted substantially among states, but changed little among major regions of the US. There is no evidence to suggest that NIH funding became more concentrated in the states with the highest per-capita funding or population density during that time. PURPOSE: Repeatedly, race has been shown to be a powerful predictor of health and access to care, but the reasons for racial disparities in health often remain obscure and speculative. Recognizing many concerns about vague and essentialist notions of race in epidemiological and clinical research, the Institute of Medicine, the American Academy of Pediatrics and the National Cancer Institute have recently called for more cautious use of the race variable. The goal of this paper is to provide a much-needed synthesis of key critiques of race, some of which go beyond the concerns expressed by the aforementioned medical bodies. Armed with more complete knowledge about race, investigators should be better able to evaluate, plan, and conduct studies on health differences among human populations. METHODS: In an iterative fashion, and on the basis of clarity and logic, important critiques of race were identified through online and bibliographic searches and reviews of biomedical and social science publications and through discussions with colleagues. Subsequently, recently published studies in highly-regarded medical journals were selected to illustrate major themes. RESULTS: Delineating the causes of health disparities is often limited by misunderstandings of race and inadequate attention to its fundamental confounders, including: BIOLOGY (e.g. race is a social construct, not a biological one); SOCIAL CLASS (e.g. emphasizing biology diverts attention from considerations of SES, and SES is frequently gauged using an inadequate number of measures); CULTURE (e.g. biomedical studies of patients' culturally-based preferences and practices may rely on notions of culture that are overly deterministic, ethnocentric, and unduly constrained by the use of questionnaire data); ETHNICITY (e.g. self-reported ethnicity has become a common substitute for race, but although it offers some advantages, researchers should bear the ultimate responsibility for specifying membership in groups, ethnic or otherwise); RACISM (e.g. the most disturbing explanation for health disparities, racism is also one of the most difficult to study because it is politically charged and open to competing interpretations). CONCLUSION: A great deal of important work has been accomplished in terms of documenting the variety and widespread nature of health disparities, generally, and racial disparities in health, more specifically. The next step is extremely challenging: we must discover more precise information regarding causes and mechanisms. This can be accomplished by remaining aware of the shortcomings of overarching categories of race/ethnicity, focusing on more narrowly defined subgroups, and giving more explicit attention to the confounders of race.\nTRENDS OF ORGANOPHOSPHATE POISONING IN URBAN ZIMBABWE. X. Dong 1 , M.A. Simon 2 ; 1 Yale University, New Haven, Connecticut; 2 Yale Medical Center, New Haven, CT PURPOSE: There have been 3 million reported pesticide poisonings and 200,000 deaths worldwide. In developing countries, poisonings are mostly associated with prescription medications, agrochemicals and household chemicals which have posed major global heath problems. Organophosphates are the most commonly formulated, packaged and used pesticides in Zimbabwe. However, there are no studies elucidating the current trends of organophosphate poisoning. The objectives of this study were to examine the mortality, trends and causes of organophosphate poisoning in an urban hospital in Zimbabwe. METHODS: We conducted a cross sectional descriptive study to examine the occurrence and trends of admissions for organophosphate poisoning in Parirenyatwa, one of the two major urban hospitals in Harare, Zimbabwe. We examined a total 185,828 patients' records and 599 cases on organophosphate poisoning from January 1995 through November 2000. Trends in admissions of the organophosphate poisoning were recorded. Other variables such as sex, age, season and geographic area were examined. Total mortality rates were calculated. The intent of the poisoning was also compared. RESULTS: These data reveal a steady increase in organophosphate poisoning during these years with the exception of 1999. The numbers of admissions for organophosphate poisoning have increased by more than 320% compared to six years ago. There is no difference in the male and female ratio for admission(48% vs. 52%). The majority of the cases admitted were below age thirty-one (82%)and were from urban settings (86%). Suicide is the predominant reason for poisoning (74%). There was a marked peak in the number of cases of children under age eleven (62%) that were due to accidental organophosphate poisoning. Mortality of organophosphate poisoning is 8.3% over the last six years. CONCLUSION: These results reveal startling realities. Organophosphate poisoning has been escalating throughout the world, especially in developing countries, such as Africa, South America and Asia, where agriculture demands large supplies of pesticides. Our data, although representing a very small microcosm of the extent of poisoning and in particular organophosphate poisoning, are consistent with multiple prior studies. Organophosphate poisoning is increasing uncontrollably and is being used at high rates as a suicidal agent in the young urban population, and contribute to the accidental ingestion in the pediatric population. Further research should concentrate on behaviors and situations that lead to poisoning. PURPOSE: Patients with medically unexplained symptoms (MUS) present a major problem for healthcare, yet they have not been adequately described. Physician perspective is especially important in providing the context for these patients' health perceptions, behaviors and outcomes. METHODS: A focus group of 6 internal medicine resident physicians was conducted to provide preliminary data for subsequent focus groups of primary care physicians of a panel of managed care patients with MUS. The discussion was tape-recorded, transcribed and analyzed via content analysis. RESULTS: Participants defined high utilization in terms of time and resource utilization and high physician emotional investment and not number of visits. Most participants described a typical high utilizing patient as one with multiple visits for uncontrolled organic disease or chronic, MUS. These patients were considered extremely difficult, demanding and unpleasant with a sense of entitlement. They believed that patients with somatization were often condescending, unappreciative and litigious. Interaction with these patients left the participants feeling frustrated, helpless, inadequate and powerless. Other emotions expressed include anger, guilt and apprehension about the possibility of missing an organic disease. Participants reported positive reactions to high utilizing patients (defined as more than 8 visits/year) with transient, minor acute illness. These patients were described as nice people with simple, straightforward problems, which got better regardless of what they did. Although they believed that the real motivation behind their visit was something other than their physical complaints, (such as stress, or the need for support and validation), they neither saw these patients' visits as problematic nor perceived a real need to address their possible underlying problems. Some participants considered these patients a major source of practice income and not high consumers of resources. Caring for high utilizing patients with organic disease was most gratifying. Most participants would rather not see patients with somatization; and the possibility of having to care for these patients as primary care physicians is a major factor in choosing to pursue subspecialization. CONCLUSION: This group of internal medicine residents did not consider high utilizing patient with MUS to be problematic if they had multiple transient minor acute illnesses. Conversely they found chronic somatization patients to be extremely problematic and would rather not care for them. Caring for patients with documented organic disease was gratifying regardless of utilization.\nCenter, Denver, CO PURPOSE: Among patients with hypoxemic chronic obstructive pulmonary disease (COPD), supplemental oxygen provides significant physical and cognitive benefits. Despite these benefits to therapy, no trial has demonstrated an improvement in subjective quality of life and patient adherence to therapy is low. The purpose of this study was to describe the experience of using supplemental oxygen therapy to identify barriers to its use and aid in improving adherence to therapy and quality of life among oxygen users. METHODS: 27 participants with hypoxemic COPD underwent semi-structured interviews to explore their experiences with supplemental oxygen therapy. Interviews were taped, transcribed, and analyzed for thematic content in a manner informed by grounded theory. RESULTS: The most commonly described barrier to oxygen use among participants was a fear of dependency. Two metaphors were used to describe this fear: crutches and addiction. As described by participants, a crutch was an external device or a behavior used to compensate for some weakness. Achievements and activities accomplished while using a crutch were considered tainted and illegitimate. Subjects feared that using a crutch made them more dependent on the crutch and less able to function without it. They feared that accepting dependency on a crutch was accepting a lesser state of being and a sign of moral weakness. Respondents described addiction as dependency on some substance used to illegitimately induce a sense of well-being or to reduce physical or psychic discomfort. Oxygen was likened to alcohol, psychotropic prescription medications, and illicit drugs. Participants equated using oxygen to relieve breathlessness with using alcohol or other drugs to alleviate psychic or physical discomfort. They feared that their need for oxygen would increase if they allowed themselves to become dependent on it and they considered using a substance to relieve their symptoms a sign of moral weakness. CONCLUSION: Fear of dependency is a significant barrier to oxygen use among patients who will benefit from supplemental oxygen therapy. In many patients, improving adherence to oxygen therapy may require exploring the metaphors of crutches and addiction with patients to help them see the flaws in these analogies. PURPOSE: The current procedures for procuring tissues and organs for donation have resulted in both organ and tissue shortages and general dissatisfaction with the current system. A new system for allowing individuals to delineate the treatment of their organs and tissues after death could improve donation and assure patient autonomy. METHODS: We reviewed and analyzed current research and opinions on upholding the wishes of patients regarding the treatment of the body after death. This included a review of literature regarding advance directives, organ and tissue donation, and burial preferences. RESULTS: It has been shown that for psychological health, individuals need to believe that they can decide what will happen to their bodies after their deaths. Many proposed systems for the procurement of organs and tissues for donation, including both presumed consent and the requirement of familial consent, directly or indirectly deprive the individual of control over the fate of his or her body after death. In addition, the fear caused by misconceptions regarding organ donation leads otherwise willing people to refuse to donate their own or their relatives' organs. CONCLUSION: In order to improve the current system of organ and tissue procurement, we propose the concept of the``Dying Will,'' a legal document in which an individual may delineate his or her wishes regarding organ and tissue donation, funeral procedures, and the like. In order to alleviate the fears of the individual, physicians could be responsible for helping their patients to write their dying wills. This might increase the number of individuals willing to donate their organs or tissues. In addition, with such a system, the permission of family members would be unnecessary for organ donation. This could further increase the number of available donors and prevent family members from overriding the wishes of the deceased. To expedite the transferal of dying will information to medical personnel, computerized databases such as the one recently initiated by Medic Alert could be used. PURPOSE: Since the beginning of the practice of medicine, family members have played a role in the treatment of a patient. Although this role has traditionally been limited to providing emotional support, loved ones can also become directly involved in doctor-patient communication or in the medical treatment of a patient. But there may be detriment to patient care due to the involvement of friends or family. METHODS: We reviewed and analyzed both literary and scholarly opinion on the role of the family in the practice of medicine. RESULTS: Family members have been described as both helping and hurting the quality of care received by patients. They can provide health care in the home, especially for chronically or terminally ill patients, often facing emotionally or technically trying situations. When a patient is mentally or emotionally compromised, a loved one can speak on the patient's behalf. But this kind of involvement can easily be detrimental to the patient if a friend or relative tries to control the patient or makes decisions based on his or her own values rather than on those of the patient. CONCLUSION: Friends and relatives can help a patient receive the best care possible; but when these individuals have selfish motivations or feel the need to control their loved one, they can do more harm than good. Physicians should take steps to encourage positive family participation while preventing this involvement from interfering in the patient's care. The physician must be able to tell whether a friend or relative is truly helping the patient or merely acting in his or her own interests. In addition, efforts should be made to contact supportive friends or family members for patients. When no loved one is available, a nurse, social worker, or appointed guardian could play the part of a third party in the doctor ± patient relationship. As indicated by the literature, the doctor ± patient relationship can greatly benefit from the involvement of a patient advocate. University of Michigan, Ann Arbor, MI PURPOSE: Physicians may occasionally encounter an ethical dilemma about whether to protect the rights of an individual patient, versus acting in the interests of society. When physicians act on the behest of society, such as in cases of impaired airline pilots, it is usually on the basis of legal imperatives or clearly stated normative guidelines. However, there may be cases where no such guidelines exist, or those in which physicians act to help society despite professional values to the contrary. We examine these situations. METHODS: We have conducted several studies in which physicians were asked about hypothetical dilemmas involving the potential for harm to the society, but in which there are no clear guidelines; or in which guidelines state that physicians should not act in behest of the society due to harm to the individual. These cases involve the report of a past crime by the patient, the issue of patient-initiated health insurance fraud, and the issue of physician involvement in lethal injection for capital punishment. We also conducted a literature review on this subject. RESULTS: A number of physicians in each of these studies would be willing to act on behalf of society and against the individual patient despite no clear normative values or those actually contrary to the physicians' decisions. When analyzed for the factors being most likely to influence the respondents, duty to society and the values of the overall population were most associated with the decision to act on behalf of society rather than in the interest of the individual patient. CONCLUSION: In some settings, a number of physicians are willing to act on behalf of society rather than on behalf of the patient. In such circumstances, physicians rely on their personal and societal values, rather on those promulgated within the medical profession. Some of these decisions may actually be harmful to the patient, and therefore contrary to ethical guidelines established by the medical profession. The impact of societal values on physician decision making needs further exploration.\nPERKS AND PITFALLS: THE EXPERIENCE OF PHYSICIAN ± PATIENTS WITH CANCER. E.K. Fromme 1 , R.S. Hebert 1 , J.A. Carrese 1 ; 1 Johns Hopkins University, Baltimore, MD PURPOSE: Physicians have been described as the`worst' patients. Despite this, several prominent medical journals routinely feature writings by physician patients about their illnesses. This qualitative study examined the experience of physician patients treated for cancer. METHODS: Physicians who had a previous diagnosis of cancer other than non-melanoma skin cancer were recruited by their oncologists or radiation oncologists. 24 physician subjects representing diverse spectra of race, age, specialty, career stage and practice setting were identified. Various disease stages and tumor types were represented, from incidentally identified carcinoma to imminently terminal metastatic carcinoma. Subjects participated in in-depth semistructured interviews lasting an hour and a half. Interview transcripts were independently coded by two readers and compared for agreement. Content analysis identified several major categories of themes. RESULTS: 5 themes representing experiences specific to physician patients are presented. (1) Trusting others to provide care presented a particular challenge to our physician subjects who were well aware of the potential for error and were used to being in control in the medical environment. Physician subjects ranged from total trust in their providers and a desire to`just be a patient' to getting care only in institutions where they had access to their own lab results and computerized records. (2) Physicians have the ability to doctor themselves, and our physician subjects varied in their willingness to do so. In one instance, a physician found himself ordering his CT scans, referring himself to specialists and administering his own chemotherapy. Others, unable to wait for their physicians, broke their own bad news by checking lab results. (3) Physicians with a serious illness are faced with a choice about disclosing their illness to colleagues. Our physician subjects who chose to do so elicited reactions ranging from strong support to total avoidance. (4) Similarly, physicians are faced with a choice about disclosing their illness to patients. Our physician subjects who chose to tell their patients reported that their patients often responded with interest and support, and that they used self-disclosure to facilitate some aspect of the medical encounter. (5) Our physician subjects perceived clear advantages as patients such as easier access to their records, better understanding of their conditions, and particularly prompt and supportive follow up by their care providers. CONCLUSION: Our physician subjects identified advantages and challenges in their experiences as patients with cancer. In some cases, their illnesses affected the way that they interacted with colleagues and patients, whereas their physician status affected the way that they interacted with their physicians and the health care system. The insights of these physician patients may be helpful to physicians who are seriously ill and to those who are currently healthy.\nTHE INNER CURRICULUM: LEARNING AND CHANGE BY PHYSICIAN ± PATIENTS WITH CANCER. E.K. Fromme 1 , J.A. Carrese 1 , R.S. Hebert 1 ; 1 Johns Hopkins University, Baltimore, MD PURPOSE: A common notion, exemplified in the 1991 motion picture The Doctor, is that physicians who experience serious illness themselves can learn to be more caring and therefore better physicians. This qualitative study examined what physicians with cancer learned from their illnesses and whether they felt the way they practiced medicine changed as a result. METHODS: Physicians who had a previous diagnosis of cancer other than non-melanoma skin cancer were recruited by their oncologists or radiation oncologists. 24 physician subjects representing diverse spectra of race, age, specialty, career stage and practice setting were identified. Various disease stages and tumor types were represented, from incidentally identified carcinoma to imminently terminal metastatic carcinoma. Subjects participated in in-depth semistructured interviews lasting an hour and a half. The interview questions were based on a literature review of writings by physicians with cancer and other illnesses in journal citations identified in MEDLINE. Interview transcripts were independently coded by two readers and compared for agreement. Content analysis identified several major learning themes. Relationships between themes were examined and organized conceptually. RESULTS: Physicians' learning led to perceived changes in 4 main areas: (1) Understanding what patients are going through. Physicians better understood the patient's perspective, for example the anxiety created by waiting for test results or delayed surgery. (2) In some cases, this understanding was accompanied by changes in empathy that manifested as a felt connection to patients or a greater sensitivity to patients' emotions. (3) Changes in communication such as repeating important information, being careful not to minimize illness, and striving to deliver bad news skillfully. (4) Changes in values and priorities such as setting limits with employers and difficult patients, appreciating family, and acknowledging the possibility of death. CONCLUSION: Experience with a serious illness is a powerful source of learning for many physicians. In some cases, their experiences caused changes in their attitudes, their communication, and other aspects of their medical practice. Educators interested in teaching about patient centered care might utilize the experience and insights of physician patients. PURPOSE: There is a well-documented interest in genetic susceptibility testing among firstdegree relatives of women with breast cancer. Interest in, and uptake of, testing has been shown to remain high despite education regarding risks and limitations of testing suggesting unknown factors influencing the decision to test. The purpose of this study was to explore factors influencing interest in genetic testing for breast cancer. METHODS: A qualitative study design exploring interest in genetic testing was chosen to allow us to establish the appropriate parameters for future intervention research. We conducted 4 focus groups with 10 ± 12 participants each. Participants were recruited from the general population. Women included in the study were 18 ± 74 years old and had at least one firstdegree relative with breast cancer by self-report. Focus groups were videotaped and transcribed. Content analysis was performed including independent extraction of themes by 2 authors and confirmation of themes by a third. A computerized software program was used to systematize the process. RESULTS: Two critical domains influencing interest in testing were identified: 1) Selfperceived risk, and 2) Perception of testing as loss or gain of control. Accurate self-perceived risk required an understanding of critical genetic concepts such as sporadic vs. hereditary cancer. Women who categorized themselves as low risk for hereditary cancer had less interest in testing while those who categorized themselves as moderate to high risk were more interested in testing. Women who were interested in testing viewed it as gaining control,``knowing what you're facing,'' and``taking measures to prevent it.'' Women who were not interested in testing were fearful of losing control over their lives, of genetic determinism. Women were able to understand the probabilistic nature of genetic concepts discussed. Paradoxically, understanding that genetic testing would provide probabilities rather than``black and white answers'' made testing more acceptable to some women by allowing them to maintain``a percentage of control'' over their lives. Willingness-to-pay for testing was explored as a proxy for interest in testing. The cost of testing was prohibitive for all but those women who considered themselves at high risk. If cost were not an issue many women would choose to get the test. Concern for genetic discrimination was raised. CONCLUSION: Interest in genetic susceptibility testing for breast cancer is influenced by selfperceived risk and knowledge and beliefs about genetic testing. Comprehension of critical genetic concepts is necessary to accurately assess risk and understand the risks and limitations of testing. Cost and concern for genetic discrimination will affect uptake of testing.\nTRUST AS A FRAMEWORK FOR ETHICAL ANALYSIS. S.D. Goold 1 ; 1 University of Michigan, Ann Arbor, Michigan PURPOSE: Examine trust as a sociological and philosophical construct. Argue that trust can serve as a valid and useful conceptual framework to examine ethical issues in healthcare. METHODS: It is widely acknowledged that trust is a vital component of and basis for relationships between clinicians and patients and that these interpersonal trust relationships have moral content. Trust is especially important in health care, and is present in relationships with organizations (e.g., hospitals, insurers) as well as with clinicians. In this paper I will describe the nature and elements of trust-based relationships, both between individuals (i.e., interpersonal trust) and between individuals and organizations (i.e., institutional trust), and the relationship between interpersonal and institutional trust. I will argue that trust is a valid foundation for healthcare ethics because healthcare is relational and serves vulnerable parties. I will discuss conditions and actions that may increase or decrease actual trust. I will then compare and contrast these with actions and conditions Ð both for individuals and organizations Ð that may influence trustworthiness. Texas A&M University, College Station, TX PURPOSE: Anthropologists have proposed the concept of explanatory models of illness to explain discrepancies between patients' and physicians' views of the illness experience. Because large socioeconomic differences often exist between VA physicians and patients, we performed this qualitative study to explore variations and common tendencies among veterans' explanatory models of illness and the impact that these models have on veterans' perceptions of health and healthcare. METHODS: We developed an interview guide to elicit patients' perspectives about the cause of, meaning of, severity of, amount of control over, and perceived treatment efficacy for their illness. Using this guide, we conducted 10 in-depth, semi-structured interviews with patients at the Houston VAMC one hour prior to their regularly scheduled primary care visits. We audiotaped and transcribed the interviews. We analyzed these transcripts through 3 iterations of individual reading and team discussion to identify common themes that occurred in our 5 areas of inquiry. RESULTS: All participants identified themselves as Caucasian, with ages ranging from 52 to 81. Most participants were seeking care for at least one chronic illness. While some veterans' responses to our specific questions could be considered to fit a biomedical paradigm (e.g. Interviewer:``What do you think caused your diabetes?'', Patient:``My pancreas quit working.''), we discovered that broader, non-biomedical stories of experience, or metanarratives, existed across our categories of questions. These meta-narratives described themes pertaining to patient fatalism and self-efficacy and preferences for physician roles of acknowledgement, action, and communication during the healing process. In cases where veterans perceived their physicians to not attend to these meta-narratives, veterans were dissatisfied and perceived a poorer quality of life. CONCLUSION: While veterans' answers to specific questions about the experience of illness may follow a biomedical paradigm, broader, non-biomedical narratives emerge when these answers are viewed in aggregate. Physician understanding of these narratives during the medical interview is important for enhancing the patient's ability to cope with chronic illness. PURPOSE: Repeated calls have been made for educators to foster students' personal awareness of attitudes and perspectives that influence their professional practices. As part of an exercise intended to foster such personal awareness, all students at Baylor College of Medicine (BCM) complete several standardized instruments (SI) that measure their views toward the physician ± patient relationship, psychosocial care, and stress from uncertainty. Students complete these instruments in their first and third years and, after an explanation of the constructs that are measured by the instruments, receive feedback consisting of their own scores, the average scores of their classes, and published normative data from physicians in various specialties of medicine. We conducted this qualitative study to explore the meanings that students attribute to this feedback. METHODS: We conducted focus groups with students in each of the classes of 2000, -01, -02, and -03 at BCM (one group for each class). We developed guiding questions that explored the effects of the SI scores on students' perceptions of self-knowledge, abilities, and attitudes (a conceptual framework for personal awareness training proposed by Novack, et al). Focus groups were co-moderated by a sociologist and a physician. All focus groups were audiotaped and transcribed. We developed categories for transcript coding by noting the patterned occurrence of specific behaviors, attitudes, and values in the transcripts. RESULTS: We conducted 4 focus groups with 22 students; focus groups lasted an average of 90 minutes. Students from different classes framed the information provided by SI scores differently. Preclinical students expressed a lack of experience with patient care, and therefore completed the questionnaires relative to their best guess of the answers of an 'ideal' physician. This led to anxiety when these 'ideal' physician scores differed unexpectedly from the normative scores of the class or of practicing physicians. Clinical students completed the SIs relative to their own patient-care experiences and devalued scores that differed from norms that they saw as different from their own emerging professional identity. CONCLUSION: Using a 'trigger' such as SI scores may be a useful means to stimulate student self-reflection. Such feedback, though, should be framed in the context of student experience. Preclinical students struggle with the meaning of feedback given their perceptions of how an 'ideal' physician thinks and acts. Clinical students possess an emerging professional identity and may devalue feedback that is inconsistent with this identity. In order to foster professional attitudes and behaviors using feedback, educators should take into account differences in preclinical and clinical students' perceptions of the meaning and value of feedback.\nOLDER HEMODIALYSIS PATIENTS PERSPECTIVES ON QUALITY OF LIFE. S.E. Hardy 1 , S.T. Crowley 1 , T.R. Fried 1 ; 1 Yale University, New Haven, CT PURPOSE: As the dialysis population has aged, quality of life (QOL) has become an increasingly important outcome for both clinicians and researchers in end-stage renal disease. Unfortunately, most QOL scales, whether generic or renal-specific, have been developed with limited input from patients, particularly older patients. The aim of this study was to identify those factors most important to older hemodialysis patients in determining their QOL. METHODS: Semi-structured interviews lasting 20 to 60 minutes were conducted with 27 hemodialysis patients, aged 65 years or older, who had been on dialysis for at least 90 days.\nThe interviews used open-ended questions, such as``How has kidney disease requiring dialysis affected your quality of life?'' Two coders independently reviewed the interview transcripts to identify themes, resolving disagreements by consensus. Themes were compared within and across subjects to identify and organize important concepts. Subjects were enrolled until the point of thematic saturation, when further interviews revealed no new themes. RESULTS: The 27 subjects interviewed had a mean(SD) age of 747 years and had been on hemodialysis for an average of 3.53.1 years. They identified five domains important to QOL: symptoms, burdens of treatment, benefits of treatment, information needs, and relationships with providers. The most common symptoms reported were fatigue, balance problems, weakness, cramps, itching, and nausea. Burdens of treatment included time spent at dialysis, difficulty traveling, problems with vascular access, and giving things up. Benefits of treatment included relief of symptoms, social interaction, and enhanced access to health care providers. Subjects noted the importance of adequate information about how to care for themselves and what symptoms or problems to expect, and of competent and caring providers who treat them with respect and acknowledge their individuality. Symptoms and burdens of treatment affected QOL negatively and benefits of treatment affected QOL positively, while the remaining domains could have either positive or negative effects. The magnitude of these effects was influenced by non-medical factors, such as social support, family relationships, formal instrumental support, finances, and spirituality. Subjects universally noted the necessity of dialysis for sustaining life, and preferred even a low QOL on dialysis to death. CONCLUSION: When asked about their perspectives, older hemodialysis patients, while acknowledging the necessity of dialysis, identified a variety of domains important to their QOL as dialysis patients. Several of these domains, particularly information needs and the benefits of treatment, are not found in existing generic or renal-specific QOL scales. In developing such scales, it is important to elicit the factors most important to patients and ensure that these are included.\nTAKING CARE OF OUR OWN: WHEN A RESIDENT BECOMES A PATIENT. R. Harrison 1 , S. Desai 1 , D. Webster 1 , A.J. Hunter 1 , J.L. Bowen 1 ; 1 Oregon Health Sciences University, Portland, OR PURPOSE: To date there are no studies discussing the experiences of caring for residents who are hospitalized at their own training institution. While providing care for a hospitalized resident at our institution a great deal of turmoil developed among the housestaff and faculty over the issue of house officers providing care for their peers. Using the critical incident technique we assessed the experiences of the resident patient and the house staff taking care of their peer in the hospital. METHODS: In order to better understand the origins and nature of these conflicts focus groups were performed that included the resident patient and the house staff who provided the care. We convened 3 focus groups with the house staff and a separate session with the resident patient. All five residents who cared for the hospitalized resident were invited to participate in this study on a voluntary basis. Consent was obtained before audio recording each confidential session. Each session facilitated by the investigators consisted of one to three resident physician volunteers who were asked to describe feelings, both positive and negative surrounding care of their peer. The audiotapes were transcribed, independently analyzed and coded by each investigator. The coded transcripts were then compared and discussed generating a coding scheme. Disagreements were discussed and resolved by consensus. OHSU IRB approved this study. RESULTS: One hundred percent of eligible participants completed the study. Caring for a colleague had an overwhelmingly negative impact on the residents routine medical decision making, work structure, and team dynamics. Barriers to routine care of the hospitalized resident included intense emotional experiences, ambiguity in the care structure, and concern for confidentiality and privacy. This resulted in paralysis in care decisions leading to reactionary care or even care avoidance. Lack of communication amongst team members, faculty and patient was a commonly sited experience. The housestaff patient perspective was equally negative. Problems with communication and care delivery were emphasized particularly surrounding the issues of objectivity and confidentiality. Themes of vulnerability, frustration and powerlessness dominated the narratives of the resident patient. CONCLUSION: The critical incident technique offers a unique perspective into the experiences of residents taking care of peers in the hospital. Many of the themes were identical to both the residents and resident patient including issues of confidentiality, communication and intense emotional conflict. While many residents choose medical care at their own training institutions, this study demonstrates the potential difficulties with this approach and suggests the importance of determining a policy of care for the hospitalized resident.\nWOULD YOU BE SURPRISED IF THIS PATIENT DIED: EXPLORING THE TRANSITION BETWEEN AGGRESSIVE AND PALLIATIVE CARE. D.C. Johnson 1 , J.S. Kutner 1 , J.A. Armstrong 1 ; 1 University of Colorado Health Sciences Center, Denver, CO PURPOSE: Multiple studies have shown that physicians inadequately address the suffering of severely ill patients at the end of life. This deficiency, in part, arises from the difficulty in recognizing the dying patient and an overly restricted focus on aggressive care. The purpose of this study was to examine the thought processes of resident physicians taking care of critically ill patients, with a goal to better understand how physicians transition between aggressive and palliative care, and to determine if the explicit consideration of the possibility of death improves attention to the suffering of patients and their families. METHODS: Internal medicine resident physicians (n=8) caring for severely ill patients were sequentially interviewed over a four week period during Medical Intensive Care Unit Ethics and Discharge Planning rounds. The residents were asked a series of questions based on their response to the core question``Would you be surprised if this patient died?'' The interviews were taped, transcribed and qualitatively analyzed for the purpose of identifying common patterns in the decision-making process of physicians when managing severely ill patients. RESULTS: When asked the core question, there were an equal number of cases in which residents responded that they would (n=4) versus would not (n=4) be surprised if their patient died. Reasons for being surprised that a patient would die included the presence of a reversible disease, the rapid onset of an acute illness, situations where a patient was``doing better'' and prior survival under similar circumstances. Among residents who indicated that they would not be surprised, most (n=3) reported that their management would not change knowing that their patient might die. Cited changes, when present, included clarifying goals, improving communication with families, spending more time with patients and ordering fewer labs. Stated or implied barriers to changes in management despite knowing that a patient might die included a lack of time and experience,``not knowing'' a patient, an improvement in medical condition (``doing better''), a lack of clear goals (``doing everything''), the presence of uncertainty and an exclusive focus on technical intervention. CONCLUSION: Asking the question``Would you be surprised if this patient died?'' encourages proactive reflection and dialogue about the potential for death in severely ill patients. An active acknowledgment of the possibility of death may lead to changes in management that would potentially diminish the suffering of patients and their families. To effectively impart such changes, it may be necessary to develop and implement techniques to overcome identified barriers. PURPOSE: A physician's effectiveness depends on the application of good communication, cognitive, and technical skills, guided by maturity, wisdom, compassion, and integrity. Development of the latter attributes requires growth in the awareness and management of one's feelings, attitudes, beliefs, and life experiences. Yet there is a dearth of empirical research related to physicians' personal growth. We used qualitative research methods to analyze stories of personal growth obtained from a selected group of medical faculty, in order to increase understanding of personal growth in this group and to develop a conceptual framework that might be useful to medical educators and researchers. METHODS: Questionnaire survey of facilitators, facilitators-in-training, and members of a personal growth interest group of the American Academy on Physician and Patient, who were chosen because of their interest, knowledge, and experience in the topic area. Respondents were asked to submit one or more stories of growth in their own self-awareness, and how that growth had affected them professionally and personally. Themes were identified in the submitted stories by 5 members of the research team and sorted into categories by 3 members. Slight further revisions were made following discussion by the entire research team and application of the proposed categories to a subset of stories. The findings were verified by two independent reviewers, who reviewed all of the stories. RESULTS: 32 of 64 subjects returned questionnaires containing 42 stories. There were no significant differences between respondents and non-respondents in age (mean 45.3 vs. 44.6 years), gender ( 75% vs. 69% male), or specialty (69% vs. 72% internists, 28% vs. 19% behavioral scientists / psychiatrists). Usually personal growth stories began with a powerful experience and/or helping relationship, proceeded to introspection, and ended in a personal growth outcome. Personal growth outcomes included: changes in values, goals, or direction; healthier behaviors; improved connectedness with others; improved sense of self; and increased productivity, energy, or creativity. CONCLUSION: Powerful experiences, helping relationships, and introspection are processes that preceded personal growth in a subset of medical faculty. The findings, if confirmed in other populations of physicians, may have implications for medical education and practice. The ABIM expects all physicians board certified in internal medicine to posses humanistic qualities of integrity, respect and compassion. To date, no studies have been done comparing humanistic qualities among physicians at different levels of training. We compared humanistic quality scores of fourth year medical students (SI), internal medicine residents (PGYI and II), and attending physicians on a general medicine ward of a teaching hospital. METHODS: A validated seventeen-item nursing survey to assess humanistic qualities among physicians was distributed to randomly selected nurses working day shifts on the medicine wards. The survey measured physician relationships with other medical staff, and the physician relationship with the patient and family. Each item was scored on a 5 point Likert scale. Composite scores for physician to staff relationships (PSR) and physician to patient/family relationships (PFR), as well as an overall evaluation score (OE), were compared across levels of physician training. Student's t-test was done to determine statistical significance across training levels. RESULTS: 33 nurses completed a total of 295 questionnaires. Surveys were performed for each of the 7 sub-interns, 8 PGYI, 4 PGYII, and 11 attending physicians rotating on the medical wards during a month of the ward service. SI's had higher PSR scores (PSR = 24.2) than PGYI (PSR = 21.3, p value = 0.013), PGYII (PSR = 21.9, p = 0.042) , and attending physicians (PSR = 23, p < 0.01). SI's also had higher PFR scores (PFR = 33) compared to PGYI (PFR = 29.4, p = 0.025), PGYII (PFR = 28.3, p < 0.001) , and attendings (PFR = 31.7, p < 0.01). Finally, SI's had higher OE scores (OE = 4.4) when compared to PGYI (OE = 3.8, p < 0.01), PGYII (OE = 3.8, p < 0.01), and attendings (OE = 4.2, p < 0.001). No statistically significant differences were found comparing PGY I, PGY II, and attending levels among each other. CONCLUSION: Sub-interns appear to have better perceived qualities of humanism compared to resident and attending physicians. Because resident and attending physicians play an important role in medical education, efforts should be made to better understand the mechanisms of these differences, and to improve the humanistic qualities of both resident and attending physicians. PURPOSE: Academic Medical Center (AMC) leaders must respond to numerous internal and external challenges. Self reports from leaders have documented these challenges; however, little is known of the daily workings of AMC leaders and the tactics used to run an AMC in the 21st century. METHODS: Ethnographic methods were used to explore the challenges faced by leaders in the top 3 levels of one institution's organizational chart. The study was conducted at a public Midwestern school, with 1600 faculty and $165 million in NIH funding. The data are: 1)10 hrs. of interviews with leaders and 2)field notes and minutes from over 300 hrs. of participant observations in meetings. Field notes captured process (e.g. deference, control, tolerance etc.) and content (e.g. parking, safety, and budget concerns etc.). The team then identified themes by iterative review of field notes, with validity assessed by comparison with interview responses and subsequent member checking. This report focuses on identified content themes. RESULTS: Six broad content domains are recurrent: 1)faculty morale, 2)business issues e.g. intellectual property rights and establishing frameworks for joint ventures between AMCs, business and/or government. 3)developing affiliations with others to remain competitive 4)creating plans of action to improve diversity 5)creating tools and metrics to demonstratè`a cademic productivity'' & assure society that graduates are competent. 6)maneuvering AMCs to adjust and survive a unpredictable environment. CONCLUSION: AMC's are currently facing instability. This study of leaders day to day activities reveals challenges in 3 broad areas: A)human resources, B)institutional fiduciary roles and C)vision setting. Human resources include faculty, student and staff morale, issues of workforce diversity and assessment of``academic productivity''. The institution has a fiduciary responsibility in ensuring financial stability, education and assuring graduate competency. Finally vision setting includes the ability to adjust and survive in an unpredictable environment. Improved understanding of leadership challenges will allow better preparation of new generations of leaders, and will better prepare AMCs for future challenges. Talking Medicine) developed for third year medical students to teach humanism and professionalism, we asked students to define these concepts and use these definitions to spark small group discussion. METHODS: At the beginning of each internal medicine clerkship we asked students to define humanism and professionalism anonymously on sheets of paper to be handed to the preceptors. We conducted a content analysis of 3 small groups' definitions (n=14). RESULTS: In Project Professionalism, the ABIM defines professionalism as: Altruism, Accountability, Excellence, Honor / Integrity, and Respect. The Students, however, saw a broader definition of professionalism. Themes that they identified included: 1) being a physician, student, teacher, philosopher and healer all at once, 2) doing no harm, 3) understanding that``respect for others'' must include tolerance of differences; as well as a focus on collegiality among practitioners at various levels of the medical hierarchy 4) having appropriate speech, dress and emotions'-which should often be held in``check'', 5) honoring the system, but not blindly and 6) stating when a good job has been performed. CONCLUSION: Third year medical students tend to agree broadly with the ABIM components of Professionalism. However, they focus more on tolerance of difference (e.g. race, socioeconomic status, and varying health beliefs), and the importance of collegiality and collaboration in the new environment of patient care. Their vantage point early in training allows them to look critically at the profession which they are joining and view its shortcomings and strengths. Future work will focus on how these definitions change as students progress through 3rd and 4th year and into residency.\nMINORITIES AND EVIDENCE BASED MEDICINE, DO RANDOMIZED CONTROLLED TRIALS PROVIDE THE ANSWER? S. Malhi 1 , S.M. Sandhu 1 , H.S. Gurm 2 ; 1 Fairview Hospital, Fairview Park, OH; 2 Cleveland Clinic Foundation, Cleveland, OH PURPOSE: Race has a significant impact on prevalence, presentation and outcome of disease. While the randomized controlled trials (RCTs) have become the gold standard for evidence based medicine, there is a paucity of data on representation of minorities in RCTs. The purpose of this study was to review minority enrollment in RCTs published in a major medical journal. METHODS: We conducted a systematic search of all articles published in the original articles section of the New England Journal of Medicine from 1993 ± 2000. All randomized control trials done in North America were included for abstraction. We excluded all studies limited to certain ethnic groups, subset analysis of other trials and meta-analyses. Trials were characterized by primary speciality and source of funding. Data were analyzed using Students t test and ANOVA. RESULTS: 1799 original articles were published in the period between 1993 ± 2000 including 535 Randomized controlled trials. Of the 302 RCTs done in North America, only 106 specified the percentage of minorities enrolled. A total of 180,364 patients were enrolled in these 106 trials of which 37,653 were minorities (20.8%) . The maximum number of trials (80) were in cardiovascular medicine. The minority enrollment was specified in only 24, (30%) and in these the minorities constituted only 14% of the enrollees. (4766/34,132) . In contrast to cardiovascular medicine, 15 out of 22 trials (68%) in pregnancy related disorders and 12 out of 27 RCTs (44%) related to pediatric population specified minority enrollment. Similarly, the trials in these groups enrolled a larger percentage of minorities: 40.7% (13928/34220) in pregnancy related trials and 51.3% (3253/6343) in the pediatric trials. After trials in pregnant women and children are excluded only 6% (20,472/293,297) of enrolled subjects were minorities. CONCLUSION: Although minorities now make up almost one third of the American population, they are significantly underrepresented in randomized controlled trials. Most studies fail to specify the ethnic or racial mix of the population and thereby significantly limit the generalizability of the results. At a time when all research is held up to the gold standard of the RCTs, the RCTs fail to provide data on a significant segment of the population. Our data underscores the importance of an ongoing effort to improve minority enrollment and ethnic specific analysis in clinical trials. PURPOSE: Among the rehabilitation options for persons with substance abuse disorders are the prevalent and increasingly publicized faith-based programs. We sought to describe key structural aspects of substance abuse recovery programs within one major national Christian organization. METHODS: A convenience sample of 7 administrators, clinical directors, and counselors from 4 New England-based residential recovery programs was identified. Each subject was interviewed for 60 minutes using a semi-structured protocol. Using qualitative methods, key words and phrases were derived from all instances where aspects of the program structure were mentioned. The key words were placed into groups that categorized the major structural elements of the program RESULTS: The programs in this study provide long-term free services for men and women of all faiths. All programs are self-supporting and receive no governmental funding. Religious (or spiritual) components mentioned were Bible studies, worship and devotional services, vespers and prayer meetings, spiritual growth classes, retreats, and Alcoholics Anonymous or Narcotics Anonymous meetings. Individual and group therapy sessions also included spiritual references and themes. Secular components were medical and dental services, mental health services, Graduate Equivalency Diploma classes and job skills training, work therapy, sponsorship, access to physical fitness facilities, recreational activities, and aftercare program planning. CONCLUSION: Activities relating to religious study, worship, and spiritual growth are critical components of the faith-based recovery programs studied. However, the religious aspects of these programs appear to complement rather than replace the elements seen in secular programs. Further analyses of how their philosophy of faith is translated into practice are needed. PURPOSE: Tamoxifen was FDA approved for breast cancer risk reduction in 1998 for women who have a 5 year risk > 1.7% (average risk of a 60 year old white woman). Healthy women deciding whether to take it for prevention must weigh its potential benefits and adverse effects. Purpose: To develop an understanding of how ethnically diverse risk-eligible women weigh risks and benefits regarding tamoxifen prophylaxis. METHODS: We undertook a qualitative intervention study using a focus group-format to provide general information about the risks and benefits of tamoxifen for breast cancer risk reduction and to assess the concerns of ethnically diverse (Caucasian, African-American, and Latina) women about the use of tamoxifen prophylaxis. Group facilitators were matched to the ethnicity of participants. Focus group discussions involved: (1) exploration of knowledge of the risks and benefits of tamoxifen as a preventive therapy; (2) a standardized educational intervention about tamoxifen's risks and benefits based on findings from the Breast Cancer Prevention Trial; and 3) discussion about participant decision-making and tamoxifen's overall value in prevention. All participants completed a brief questionnaire to assess the impact of the intervention on their understanding of tamoxifen as preventive therapy. Focus groups were audiotaped and later transcribed for analysis. A multi-disciplinary team of investigators identified prominent themes related to health beliefs that emerged from iterative review of focus group transcripts. RESULTS: 27women (age range 61 ± 78) participated in the focus groups. Most reported that they felt more informed after the group. In general, fear of breast cancer was not prominent, and participants were not inclined to take tamoxifen as preventive therapy. Decisions about tamoxifen were based on participants' assessment of the balance of competing risks and competing benefits. Specifically, participants expressed limited willingness to take medication with serious side effects for breast cancer risk reduction, uncertainty about its interaction with other medications, and concern about the need to discontinue hormone replacement therapy. Caucasian and Latina groups expressed concerns about reliability of scientific studies. African ± American women described faith as important to prevention. CONCLUSION: Women were wary of taking a drug for a disease they might not develop. Women felt they had options other than Tamoxifen to reduce risk of breast cancer, including early detection, diet, faith, and alternative medicine. Information about tamoxifen decreased interest in use for breast cancer prevention. PURPOSE: Although shared decision-making has been advocated for screening mammography for women under age 50, little is known about how women decide whether to get screened, what are their preferred sources of information and what are their preferences for involvement in screening decisions. This study explored these issues in patient decision-making. METHODS: We conducted in-depth, semi-structured telephone interviews with 16 randomly selected English-speaking women ages 38-45 who were enrolled at a large New England HMO, and had no prior history of breast cancer. Interviews were transcribed and coded into major themes using QSR NUD*IST. RESULTS: Thirteen participants were aged 41 ± 45 and 3 were aged 38 ± 40. Nine were white and seven were African ± American. The majority (10/13) of women over age 40 had prior mammograms and all intended to have a screening mammogram before age 50. Factors in women's decisions to undergo screening included knowledge of the``recommendation to begin screening at age 40'' obtained either from their physicians or the media and``having a friend or acquaintance with breast cancer.'' Most women stated that screening has a number of benefits, including``early detection,''``peace of mind'' and``it's better knowing.'' Possible risks of mammography, although not considered important, included radiation, pain, and``not being able to catch everything.'' None of the women who heard of or experienced false positive mammograms (8/16) were or would be deterred from future screenings. While all women stated that the physician should be the primary source of information about screening, 11/16 had no discussions with their own physicians regarding the mammography procedure, its risks and/or benefits. Many (12/16) learned such information from the media. Preferences for involvement in decision making varied; 4/16 preferred to leave screening decisions mostly to the physician, 9/16 preferred to make them mostly themselves, and the rest were uncertain. We found no major thematic differences among the white and the African ± American women. CONCLUSION: Our results suggest that in this setting the decisions of women under 50 to undergo screening mammography are based on their own perceptions of risks/benefits or on the recommendation of their physicians. However, in these recommendations, discussion of specific information relating to screening rarely occurs, and shared decision-making is limited. These findings can guide future research and lead to the development of policy that facilitates patient ± physician communication in this area.\nK.E. Olive 1 , J.K. Neumann 2 ; 1 East Tennessee State University, Johnson City, TN; 2 James H. Quillen VA Medical Center, Mountain Home, TN PURPOSE: A previous study indicated that the absolute vs relative ethical construct is a factor influencing physician decisions. The purpose of this study was to confirm, in a different physician population, the previous results Ð that physicians who believe in ethical values which do not change (absolute values) will respond differently to ethically sensitive clinical scenarios than those who affirm changing ethical values (relative values). METHODS: A national sample of family physicians and psychiatrists received a mailed survey. The survey included a four-item scale to determine relative vs absolute values, demographic and psychological measures, and three clinical scenarios involving contraception, physician-assisted suicide, and abortion. The primary analysis compared approval of the clinical scenarios for physicians with absolute values to those with relative values. Approval ratings were determined using a seven point Likert-type scale (1=do not approve, 7=do approve). Data were analyzed using analysis of variance. RESULTS: 317 of 1000 physicians surveyed (32%) responded. Of those responding, 99 met criteria for absolute values and 59 for relative values. Those endorsing absolute values were significantly less approving of all three scenarios than those endorsing relative values: Contraception (5.5 vs 6.4, F=10.17, p=.0017), Physician Assisted Suicide ( 2.1 vs 4.1, F=36.22, p < .00001), Abortion (3.0 vs 5.0, F=28.45, p < .00001). CONCLUSION: Physicians with absolute ethical values are less approving of contraception for single women, physician-assisted suicide, and abortion than those endorsing relative ethical values. The absolute vs relative values ethical construct may be useful in examining physician attitudes which impact health care delivery. These survey results replicate a previous study indicating the absolute vs ethical construct is a factor influencing physician decisions. This construct is directly relevant to clinical care and physicians need to be aware of their own biases in discussions with patients, families, and other health care providers.\nPURPOSE: As of December 2000, 31 states passed mental health parity statutes, laws requiring a health plan, insurer, or employer to provide mental health benefits``equal'' to physical health benefits. With parity, lawmakers are for the first time legislating their own state definitions of mental illness rather than leaving the definition up to insurance plans or covering all disorders in the American Psychiatric Association's Diagnostic and Statistical Manual (DSM-IV) as legislation has traditionally done. My research examines definitions of``mental illness'' used in state parity laws and implications of these definitions including what populations are getting increased access to care. METHODS: I reviewed the statutory language in 31 state parity bills and conducted a series of interviews with stakeholders participating in the national and state mental health parity process. Interviewees included mental health advocacy groups, provider organizations, legislative bill sponsors, health plans, and employer groups. Interview data was supplemented with extensive literature review. RESULTS: The definition of mental illness varies significantly across states, but falls into one of three categories:``broad-based mental illness,''``serious mental illness,'' and``biologically-based mental illness.'' Broad-based mental illness includes all disorders listed in the DSM-IV and/or World Health Organization's ICD-9.``Serious mental illness'' and``biologically-based mental illness'' include from three to eleven specific DSM-IV mental disorders. Several factors influence a state's definition of mental illness including the cost of parity, the leadership role taken by state advocacy group(s), the strength of insurance interests, the reliance on antidiscrimination arguments, and the political feasibility of parity in the state. CONCLUSION: With parity, state lawmakers are not relying on clinically accepted definitions or previous federal and state definitions of mental illness. It appears that political and economic factors influence the statutory definition, rather than needs-based strategies or clinical judgement. Insurers argue that legislating parity for all mental disorders results in tremendous healthcare costs and necessitates the development of definitions that target only the most seriously mentally ill. However, using a``pick and choose'' approach to the DSM-IV as most states have done has little (if any) clinical basis, and can potentially limit access to care. Increased reliance on clinician judgement and further studies measuring the access implications of current parity laws are needed to identify those who would most benefit from parity. PURPOSE: Ethnic culture ± the basic beliefs an ethnic group uses to interpret experience ± affects patients' views on dying. But views within an ethnic group are not all alike. The genders within an ethnic group may share some views but differ on others and, thus, be distinct subcultures for advance care planning purposes. METHODS: We explored this possibility by asking Mexican Americans (14 men, 12 women), Euroamericans (7 men, 11 women), and African Americans (7 men, 7 women) their views on advance directives (ADs). All subjects were inpatients aged 50 to 79. RESULTS: While believing ADs improve the chances a patient's wishes would be followed, the genders within all three ethnic groups differed over the power and trustworthiness of the health care system, treatment wishes and the willingness to express them, and beliefs about ADs. Among Mexican Americans most men but few women believed the system controls treatment (79% versus 33%), believed patients should be allowed to die when treatment is futile (57% versus 42%), wanted no life support (71% versus 42%), had not told anyone their wishes (64% versus 42%), and believed ADs are needed for when a patient is brain dead or cannot function (57% versus 33% for both themes). But few men and most women trusted the system to honor written ADs and believed ADs help the staff know a patient's wishes (43% versus 67% for both themes). Among Euroamericans most men but few women distrusted the system (57% versus 18%) and disliked ADs (57% versus 36%). But few men and most women expressed wishes not to suffer during terminal illness (0% versus 55%), believed patients should be allowed to die when treatment is futile (0% versus 55%), and believed the system honors written ADs (43% versus 73%). Among African Americans most men but few women distrusted the system (57% versus 29%) and wanted to wait until very sick to express their wishes (57% versus 43%). And few men but most women said that the patient deserves a say in treatment (43% versus 57%), wanted no life support (29% versus 57%), and believed ADs help staff know the patient's wishes (0% versus 57%) and can prevent life support (14% versus 57%). CONCLUSION: While sharing some views, men and women of each ethnic group differed on others. Unlike men, women tended to trust the system and to believe ADs will help realize their wishes. Providers should consider gender-specific values within an ethnic culture when doing advance care planning with patients. PURPOSE: Ethnic culture ± the beliefs an ethnic group uses to interpret experience ± affects patients' views on dying. But views within an ethnic culture are not all alike. Age groups within an ethnic culture may share some views but differ on others and, thus, be distinct subcultures for advance care planning purposes. METHODS: To explore this possibility we interviewed Mexican Americans, Euroamericans, and African Americans. Younger subjects (ages 50 ± 64) numbered 15, 11, and 10 respectively; older subjects (ages 65 ± 79) numbered 11, 7, and 4. RESULTS: While all ethnic groups liked some aspects of advance directives (ADs), age groups within each ethnic group differed over trust in the health care system, treatment wishes, and beliefs about ADs. More younger than older Mexican Americans wanted to be allowed to die when treatment is futile (60% versus 36%), had not told their wishes to anyone (60% versus 45%), believed ADs are needed for when a patient cannot voice decisions (60% versus 27%), and trusted the system to honor written ADs (60% versus 45%). Fewer younger than older Mexican Americans expressed wishes unrelated to life support (for example, where or when they wished to die) (33% versus 64%). More younger than older Euroamericans described unacceptable outcomes (55% versus 43%) and believed that ADs help staff know a patient's wishes (91% versus 29%) and are needed for when a patient is brain dead (64% versus 29%). Fewer younger than older Euroamericans disliked some aspects of ADs (36% versus 57%), had not told anyone their wishes (18% versus 71%), and doubted ADs change treatment (27% versus 57%). More younger than older African Americans believed the system controls treatment (80% versus 25%), distrusted the system (50% versus 25%), said the patient deserves a say in treatment (60% versus 25%), wanted no life support (50% versus 25%), doubted ADs change treatment (50% versus 25%), and said ADs just mean the patient is dying (70% versus 0%). Fewer younger than older African Americans trusted the system to serve patients well (30% versus 50%), had not told their wishes to their physicians (10% versus 50%) but planned to tell someone before becoming too sick (0% versus 75%). CONCLUSION: While sharing some views, intraethnic age groups differed in their trust of the health system, their willingness to express treatment wishes, and their confidence in ADs. Providers must consider age-specific beliefs within an ethnic culture when conducting advance care planning with patients.\nMAY IT PLEASE THE COURTS: THE LEGAL CAMPAIGN AGAINST HOSPITAL DISCRIMINATION. P. Reynolds 1 ; 1 Johns Hopkins University, Baltimore, MD PURPOSE: Hospital discrimination was widespread throughout the United States and in many jurisdictions legally sanctioned as late as the mid-1960s. Discrimination most commonly was experienced as the denial of staff privileges to minority physicians and dentists, refusal to admit minority applicants to nursing and residency training positions, and lack of medical and surgical services to minority patients in many urban private and public institutions. A national campaign to eliminate discrimination in hospital clinical services and education programs involved the collaboration among minority medical and activist organizations, and a direct attack against the hospital policies and practices through litigation culminating in two landmark decisions which both emerged from the 4th Circuit Court of the United States. METHODS: Research was conducted in national archives, in manuscript collections of minority and non-minority physicians, in legal documents with review of relevant court cases, and in primary and secondary law and medical journals. RESULTS: The first hospital discrimination case brought before the courts was filed in North Carolina in 1956 by three physicians, Dr. Hubert Eaton, Dr. Daniel Roane, and Dr. Samuel Gray, and their lawyer, Conrad Pearson. While the courts initially denied the plaintiffs the relief they sought, the NAACP lawyers used information emerging from Eaton vs James Walker Memorial Hospital when deciding the most essential elements in constructing a``test case''. Their attack centered on the federal government's use of public funds to construct``Separate but Equal'' hospitals throughout the south. That same year, the City of Chicago passed the Harvey ± Campbell Ordinance to prevent racial discrimination in Chicago hospitals. In 1962 the second hospital discrimination case in North Carolina was filed by George Simkins and the lawyers for the NAACP against the Moses H. Cone Memorial Hospital and Wesley Long Hospital. This case would determine ultimately the legal foundation of``Separate but Equal'' in hospital policies and practices. Two years later African ± American physicians in Chicago sued 56 hospitals throughout the city for refusal to adhere to the Harvey ± Campbell Ordinance charging they had been denied staff appointments to hospitals in the area because of their race. By the time the Medicare Hospital Certification Program was implemented in the summer of 1966, over 35 suits had been filed by physicians and citizens with the assistance of NAACP lawyers against hospitals and nursing homes located in communities throughout the south. The Medicare hospital racial integration guidelines themselves would be tested in the courts through Cypress versus Newport News Hospital Association. CONCLUSION: The legal foundation for``Separate but Equal'' in hospital policies and practices was eroded through a series of court battles developed and argued by NAACP lawyers culminating in Simkins versus Moses H. Cone Memorial Hospital (1963) and Cypress versus Newport News Hospital Association (1967). The first of these two cases challenged the federal government's use of public funds to expand and maintain segregated hospital care. The second case reaffirmed the federal government's use of public funds to force hospitals to open up patient admission policies, education programs and hospital staff privileges to all citizens and physicians regardless of race, gender, age or nationality. Successful pursuit of a legal strategy against racist hospital policies and practices was a critical cornerstone in a national campaign to eliminate discrimination in health care delivery.`I THINK MY HEART GOT A LITTLE STONE IN IT NOW: CONSEQUENCES OF VIOLENT VICTIMIZATION AMONG YOUNG AFRICAN AMERICAN MEN. J. Rich 1 , C. Grey 1 ; 1 Boston University School of Medicine, Boston, MA PURPOSE: Violence remains a leading cause of death and disability for young African American men. Recurrent violent injury is also well documented in the medical literature. The purpose of this study is to understand how the experience of violent injury changes the lives of young black men and predisposes them to recurrent victimization. METHODS: Participants in the study were 34 African American men, between the ages of 18 and 30, who were admitted to an urban medical center for violent injury. In ± depth, semistructured interviews were conducted within 2 weeks of the initial injury and, in a subset of participants, a follow-up interview was conducted 2 months later. Analysis was performed using grounded theory methods and narrative analysis to identify prominent themes common across interviews. RESULTS: The narratives of young black men who are victims of violence highlight the physical pain and suffering that they experience. More prominent, however, are accounts of negative interactions with the police, both prior to the injury and in the immediate aftermath, who treat them as perpetrators regardless of the circumstances of their injuries. Also prominent are vivid descriptions of disabling symptoms of traumatic stress, and profound feelings of fear and vulnerability. These extreme feelings of vulnerability, coupled with mistrust of the police and other community institutions, lead these young men to develop their own strategies to remain safe and to deal with their distress. Some of these strategies either hinder their ability to heal from their emotional trauma (such as self-treating with drugs and/or alcohol) or put them at risk for recurrent injury (such as acquiring a weapon). Few young men expressed knowledge of resources to address their psychoemotional issues. CONCLUSION: The physical and emotional consequences of violent injury, coupled with a social environment that is hostile and unsafe, places young black male victims of violence at risk for recurrent injury. Efforts to reduce recurrent violent injury will be more effective if they address both the emotional consequences of injury and the often oppressive social environments in which these young men live.\nUNDERSTANDING TEAM-BASED QUALITY IMPROVEMENT FOR DEPRESSION IN PRIMARY CARE. L.V. Rubenstein 1 , L.E. Parker 2 , L.S. Meredith 2 , N.P. Gordon 3 ; 1 VA Greater Los Angeles, RAND and UCLA, Sepulveda, CA; 2 RAND, Santa Monica, CA; 3 Kaiser Permanente Division of Research, Oakland, CA PURPOSE: Team-based quality improvement (QI) methods are an attractive option for designing and implementing improvements in depression care in primary care (PC) practices. QI methods experts disagree, however, about whether teams should be centralized (the CT approach), or local (the LT approach). In CTs a few key experts, with input from local clinicians based in the target practice sites, are responsible for developing the QI intervention design. In LTs the local clinicians develop the QI design through on-site interdisciplinary team meetings. The qualitative analyses presented here compare the success of the two approaches and evaluate the characteristics of teams and organizational environments that affect success. METHODS: We partnered with two non-profit staff model managed care organizations ± VA Greater Los Angeles and Kaiser Permanente of Northern California ± to initiate and evaluate five QI teams tasked with improving care for depression. Teams were structured as either CTs or LTs. The three LTs involved three PC practices and the two CTs involved three PC practices. Trained observers transcribed process notes during all team meetings, and carried out structured interviews with team and clinical practice leaders and members at one and two years. Three social scientists with qualitative expertise independently reviewed all study data and rated teams. Six national depression experts independently rated team QI designs (plans). Analysis of team success used qualitative predictor-outcome matrices. RESULTS: The two CTs ranked higher for depression program planning than two of the three LTs, but one LT received equivalently high ratings. Across all teams, team leadership and primary care and mental health top management support for depression QI had the most effect on plan quality and implementation. CTs, however, had better success than LTs in less supportive environments. For example, PC top management for one of the two PC practices linked to one CT was relatively unsupportive, as was mental health top management for the practice linked to the other CT; the two CTs did better than the two LTs that were based in similarly low support organizational environments. The one LT with favorable team leadership and organizational environment did as well as the best performing CT. CONCLUSION: CT and LT approaches have equivalent success when team leadership and organizational support are strong, while CTs have better success than LTs in less supportive environments. Our results suggest that depression QI approaches that depend on local interdisciplinary team design and implementation should be reserved for favorable organizational environments. Less favorable environments are likely to be better served by a more centralized, expert directed QI approach. PURPOSE: Patients fail to recall and comprehend as many as half of the critical pieces of information conveyed during a clinical encounter. To maximize patients' recall, understanding, and ultimately their treatment adherence, it is recommended that clinicians limit the number of new concepts they present and always elicit patients' comprehension of these points. This educational technique may be particularly important for patients with low functional health literacy (FHL) and chronic conditions such as type 2 diabetes (DM2), since these patients grapple with complex clinical management regimens and often have difficulties processing oral communication. In this study, we measured the extent to which primary care physicians (PCPs) assess these patients' recall and comprehension of their advice. METHODS: The study was conducted in primary care centers at a public hospital. Outpatient encounters between 30 English-speaking, DM2 patients with low FHL (score < 23 on shortform Test of Functional Health Literacy) and 22 PCPs were audiotaped. 2 trained coders measured the duration of each encounter, and identified the following communication events: (1) new concepts raised by physicians (such as an explanation of how blood pressure affects the kidneys, or a change in medication regimen); and (2) whether each new concept was followed up by the physician with an attempt to ensure that patients comprehended the key points, e.g. by a request for patient restatements, or eliciting patient perceptions. The percentage of new concepts for which follow-up assessments occurred was then calculated. RESULTS: The mean number of new concepts conveyed by PCPs was 2.0 (range 0 ± 5). The mean number of follow-up assessments was 0.27 (range 0 ± 2) or 13%; in over half of these assessments the patient did not recall or comprehend the new concept, allowing the PCP to tailor subsequent information. Visits including one or more follow-up assessments did not differ in duration when compared to visits that lacked such follow-up (18 vs. 21 min, p=0.2). CONCLUSION: The vast majority of PCPs caring for DM2 patients with low FHL do not assess patient recall or comprehension of new concepts conveyed during the clinical encounter. Overlooking this critical step in the communication process reflects a missed opportunity to enhance patient care. Future studies should evaluate whether more consistent application of this communication technique improves patient recall, adherence, and health outcomes. PURPOSE: Breakdown can be described as a disruption in smooth unreflective practice that forces an individual to adopt a more explicit, deliberate and abstract perspective. This study examined a series of breakdowns in an ambulatory medical clinic. It examined the response of residents to these breakdowns and its effect on patient care and learning. METHODS: Trained observers collected ethnographic data from waiting areas, workstations and exam rooms. Real-time field notes from sixty-eight separate observations (total 130 hours) were immediately transcribed into the data set, 2919 paragraphs of text. Two analysts coded text units at the paragraph level into recurrent themes using template analysis. Breakdown emerged as the major theme, and was fine coded at the phrase level to create a taxonomy. Using this taxonomy, 156 vignettes were selected to represent the full spectrum of breakdown. Recursive analysis of the vignettes was used to create a structural model of breakdown and learning. Triangulation and member checking were used to validate the model. RESULTS: Breakdown occurred in 1255 (43%) of the text units, and was clearly important for learning. Breakdowns occurred both from individual actions and from clinic structure and process. Preliminary results indicate that the computer-based patient record is an important source of breakdown; it interferes with communication and structures the interview, more so for residents than for faculty. The structural model of breakdown has three levels: psychomotor, conceptual and cultural. Breakdown begins at the psychomotor level of automatic behaviors (e.g., withdrawal in response to patient anger). If the breakdown persists, it moves to the conceptual level, where models are shared to determine correct action. At conceptual level, breakdowns often involve heuristic biases (e.g., favor curable diagnoses over chronic ones). Unresolved breakdowns become obtrusive and progress to the cultural level, where the entire process of norms, concepts, actions and consequences are reflected upon. At the cultural level, breakdowns reflect failure to engage with cultural norms. CONCLUSION: Our observations reinforce previous research that indicates a need to provide time and training for communication, cognitive and reflective skills. In addition, new heuristic biases are described, and problems arising from physician interface with computer-based patient records may have implications for clinic structure, process and training needs. Finally, the data suggest a need to align norms, expectations and resources. PURPOSE: Surrogates are often entrusted to make decisions regarding cardiopulmonary resuscitation (CPR) for patients who lack decision making capacity. Not infrequently, surrogates refuse to authorize Do-Not-Resuscitate (DNR) orders, even when clinicians deem CPR futile in a strict biomedical sense. Few states besides NY have legislation to address these cases. In NY, patients or surrogates must generally sign complex DNR consent forms. However, by statute, NY physicians also have the authority to write DNR orders over the objections of surrogates. Before writing such an order, a physician must conduct a``dispute mediation'' meeting with the patient's surrogates. The purpose of this paper is to provide a description and moral analysis of a series of such cases. METHODS: We present a case series of 6 dispute mediation meetings in which surrogates refused to sign for DNR orders when clinicians thought CPR was futile, convened and moderated by the ethics consult service of a single academic medical center. We use casuistic moral analysis to compare what occurred in these cases with the paradigmatic practice of requiring surrogates to provide substituted judgments and to give formal consent for DNR orders. RESULTS: The average patient age in our series was 60. Two of these patients were from nonwhite minority groups, 1 from a recent white immigrant group, 2 had malignancies, 2 had HIV, and 2 had cardiovascular disease. In all 6 cases, surrogates expressed their refusals to sign DNR orders in similar terms. Some voiced religious concerns; others``could not give up hope.'' In 5/6 cases the surrogates refused to sign the DNR order form even after the dispute mediation meeting. Nonetheless, DNR orders were written in all 6 cases with the implicit assent of the surrogates even in the absence of formal consent. After being told that DNR orders would be written even over their objections, surrogates' comments included:``Well, you gotta do what you gotta do.''``I no make decision. Doctor decision.''``I know he is dying. Just don't make me sign. I feel like I'm signing a death warrant.'' A psychiatrist involved in 3 of these cases noted denial, anxiety, pathological ambivalence, and guilt in the surrogates. No attempts were made by surrogates to transfer the patients to other physicians or facilities. All of the patients expired within one week. To date, no lawsuits have occurred. CONCLUSION: These cases suggest that what appear to be``futility'' disputes may in fact represent the complex emotional states that surrogates experience when they believe that they are responsible for making these decisions. All of the surrogates seemed relieved when they were told that a DNR order would be written regardless of whether they had chosen to`sign' the formal document. These cases raise questions for policy and clinical ethics about the extent to which asking surrogates to consent to DNR when CPR is futile imposes unnecessary burdens and no benefits for either patients or their surrogates. PURPOSE: A variety of factors influence clinical ethical decision-making by physicians. The process by which physicians weigh these many factors against one another in order to make a treatment decision is poorly understood and documented. This study examines the influential factors involved in clinical ethical decision-making by using Q Sort by-person factor analysis. METHODS: A convenience sample of internal medicine attending physicians and housestaff (n = 35) at a university affiliated medical center were presented with a series of four hypothetical case vignettes and asked to select a specific treatment action for each case. Case vignettes involved clinical decision-making near the end-of-life. Based on the selected treatment action, participants arranged twenty-five self-referenced opinion items into a unique quasi-normal factor array according to a continuum of preferences from most to least salient. Q sort factor analysis was performed by means of standard factor extraction whereby individuals with correlated Q sorts are clustered into homogenous attitude groups. A second order factor analysis was used to cluster factors to reveal their underlying commonalities. Subjective interpretation of the salient factors in relation to the specific clinical case scenario was performed in order to explain the different ethical perspectives utilized in clinical ethical decision-making. RESULTS: Data analysis revealed four non-correlated salient factors which guided ethical decision making in the four hypothetical cases. The primary salient factors were defined by the perspectives of: 1) beneficence, 2) patient guided decision-making, 3) patient and family focused decision-making, and 4) legally guided decision-making. Normalized factor scores for statements included in each viewpoint had a Z score > 1.00. Non-influential statements noted across all four cases were the economic impact on the physician, expediency in resolving the situation, and the expense of medical treatment (Z < À1.00). CONCLUSION: Q sort factor analysis is a useful tool with which to study clinical ethical decision-making because it provides an analytical method for discerning salient factors selfidentified by physicians in making ethically-challenging treatment decisions. The four factors identified are grounded in current bioethical values. METHODS: A convenience sample of internal medicine attending physicians and housestaff (n = 35) at a university affiliated medical center were presented a series of four hypothetical case vignettes involving clinical decision making near the end of life, and were asked to choose between aggressive, conservative, and consultative courses of action. An aggressive course of action was one in which medical and/or surgical treatment was pursued in spite of poor prognostic outcome. A conservative course of action was a treatment decision that withdrew medical therapy and instituted palliative treatment. A consultative course of action was defined by a request for information and guidance from an ethics committee or family members. RESULTS: Twenty percent (7/35) of respondents demonstrated a consistent pattern of clinical course of action by choosing either an aggressive, conservative, or consultation course of action three or more times. 8.6% (3/35) of respondents chose an aggressive course of action three or more times, whereas 2.9% (1/35) of respondents chose a conservative course of action three or more times. Three respondents (8.6%) chose a consultative course of action three times or more. A majority (80%, 28/35) of respondents did not demonstrate an identifiable pattern of clinical ethical decision-making by choosing an aggressive, conservative, or consultative course of action for more than two out of the four cases presented. CONCLUSION: When confronted with an ethically challenging clinical case, most physicians adapted their responses to the particulars of the case presented by demonstrating variability in their treatment approaches. Only a minority of physicians demonstrated a consistent pattern of behavior by selecting either an aggressive, conservative, or consultative treatment option. Physician interpretation of the specific ethical situation of each case guides their clinical ethical decision-making.`T HEY TREATED ME LIKE A LEPER'' Ð STIGMATIZATION AND THE EMOTIONAL BURDEN OF HEPATITIS. C S. Zickmund 1 ; University of Iowa, Iowa City, IA PURPOSE: Hepatitis C is a chronic progressive disease that is typically acquired through contaminated blood products or needle sharing. While the widely known association between this illness and intravenous drug use may lead to stigmatization, no study has explored the consequences of such stereotyping. The goal of this investigation was to identify the prevalence and impact of stigmatization on patients with chronic hepatitic C virus (HCV) infection. METHODS: Patients with HCV infection attending the University of Iowa Health Care liver clinic were randomly chosen to participate. All patients completed a semi-structured interview and the Sickness Impact Profile (SIP). Two blinded coders analyzed the interviews, identifying the frequency of stigmatization, characteristics of relationships with friends, family and colleagues in the work environment, anxiety, depression, and helplessness. RESULTS: A total of 193 patients (44.80.7 years; 33% women) with HCV infection were enrolled. The analysis of qualitative data demonstrated an excellent intercoder reliability (kappa=0.86). Eighty-five patients (44%) experienced stigmatization which they attributed to the disease. Gender, education, professional status and mode of HCV infection did not influence the likelihood of stigmatization. Negative stereotyping was associated with a higher score of the psychological SIP subscore (11.11.5 vs. 15.61.9; p =0.05). The responses demonstrated that stigmatization affected all facets of social interactions from family life to the work environment, causing a significant emotional burden for the patients. Stereotyping was associated with increased expressions of depression and anxiety (depression: 58% vs. 37%; p < 0.01; anxiety: 72% vs. 47%; p < 0.01), problems with family (38% vs. 15%; p < 0.01), friends (35% vs. 9%; p < 0.01) or coworkers (34% vs. 7%, p < 0.001). These experiences led to a sense of loss of control (38% vs. 19%; p < 0.01) and problems coping with the disease (38% vs. 19%; p < 0.01). CONCLUSION: Stigmatization is a very common experience of patients with HCV infection and erodes important support structures. The resulting distance from family, friends and colleagues increases feelings of anxiety and depression, common emotional disturbances in this group of patients. These data support the need for broad-based educational efforts to reach patients as well as their relatives and friends. Lowering the emotional impact of negative stereotyping may enhance both the quality of life of patients as well as their compliance with the medical therapy and required life style changes." }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: Microarray is a powerful technique for simultaneously determining the expression of thousands of genes [1] [2] [3] . Such studies can quickly yield a genome-wide description of mRNA expression, called transcriptomes, in a given cell or tissue at a given physiologic or pathologic condition [4] . Even though, one of the main challenges in such genome-wide gene-expression profiling is the difficult inspection of genes with rare transcripts.\nOn the other hand, PCR-based suppressive subtractive hybridization (SSH) techniques are highly sensitive for identifying differences in gene expression [5] [6] [7] [8] [9] . However, the potentials of SSH in assaying dynamic changes of gene expression in minute levels have never been addressed before. In addition, SSH techniques are also restricted in terms of limited specificity and difficulties in identifying enriched genes. Combining the SSH technique with highthroughput screening of the harvested clones through the use of cDNA microarrays could greatly reduce the tedious work for northern blot analysis, as well as the likelihood of false-positive clones enriched via SSH [10] . Such combined approaches by printing the clones obtained from the SSH amplicones on chips have been successfully used for profiling the differentiation of gene expression [10] [11] [12] [13] . Nevertheless, in such approaches, the genes of the subtracted clones remain to be sequenced for identification, and a large portion of redundancy in the enriched amplicons must also be identified. Moreover, since the targets used to hybridize the amplicon clones printed on chips were the un-enriched cDNA pools rather than the subtracted, enriched clones, such approaches would not increase the sensitivity for detection of the low-expression genes.\nHerein, we report our modifications using the subtracted amplicons as the targets to hybridize the pre-prepared microarray chips for SSH/microarray analysis. Since all of the probes on the microarray chips have been well characterized, the hundreds and thousands of genes in the subtracted amplicons can be determined by a single hybridization. Moreover, the relative expression status between the compared tissues for each gene can be augmented and easily determined by combining the use of the targets prepared both from forward and reverse subtractions of SSH. We named this modified approach \"SSH/microarray\" and used human hepatocellular carcinoma as a model to demonstrate the feasibility of this approach.\nThe SSH/cDNA microarray versus regular cDNA microarray approaches To conduct the comparative transcriptomic studies on human hepatoma particularly for the genes at low-expres-sion levels, we set out an approach as shown in Figure 1 . We used the PCR-based suppressive subtractive hybridization to enrich the differentially expressed cDNA clones between human hepatoma and non-hepatoma liver tissues. The SSH was conducted both forwards using RNA prepared from hepatoma and non-hepatoma liver tissues as tester and driver, respectively, to yield the hepatoma (T-N) subtracted amplicons, and reversely using the mRNA prepared from non-hepatoma and hepatoma liver tissues as tester and driver respectively to yield the non-hepatoma (N-T) subtracted amplicons as well. Instead of the previously reported combination with cDNA microarray approaches, in which clones in the subtracted cDNA libraries were cloned and then printed on chips, we directly labeled the subtracted T-N and N-T amplicons and then used as targets to hybridize the pre-made microarray chips printed with the known 14,811 cDNA clones. We named this modified approach as \"SSH/microarray\". The same tissue RNA pairs and cDNA microarray chips were also used for the regular cDNA microarray assays for comparison. Since human hepatoma generally occurs in patients of chronic hepatitis with/without cirrhosis, to identify the genes implicated in the common biological processes leading to hepatocellular carcinogenesis, we selected three hepatoma patients with different underlying liver disease. The first was with chronic hepatitis C and cirrhosis, the second had chronic hepatitis B and cirrhosis, and the third had chronic hepatitis B but no clinical or histological evidence of cirrhosis. Figure 2 demonstrates the representative results obtained from the corresponding regular cDNA microarray and the SSH/microarray assays. Of note, in the SSH/microarray assays the genes in the subtracted T-N and N-T amplicons were readily identified without tremendous sequencing works, which were required for the original SSH and microarray combination assays. In addition, many of the clones, including those of the house-keeping genes, such as β-actin and GAPDH, with unremarkable difference in hybridization intensities by the regular cDNA microarray assays were efficiently excluded from the subtracted amplicons ( Figure 2 , indicated by white circles). This indicated the efficiency of subtraction hybridization. Most importantly, many of the clones with hybridization intensity below the evaluation threshold by the regular cDNA microarray approaches (hybridization intensities lower than or close to the noise) became detectable by the SSH/ microarray method ( Figure 2 , indicated with red circles). These observations suggested a more sensitive detection of the low abundance transcripts using the SSH/microarray assays. Figure 1 Experimental flowchart. Messenger RNA was prepared from hepatoma (T) and the corresponding non-hepatoma liver tissues (N), and then subjected to 1) PCR-based suppressive subtractive hybridization (SSH) followed by using the resulted subtracted cDNA libraries as targets for cDNA microarray analysis (the SSH/microarray), and 2) conventional cDNA microarray analysis. SSH was performed in both the forward (T as tester) and reverse (N as tester) direction to enrich up-regulated (T-N amplicon) as well as down-regulated transcriptomes (N-T amplicon) in human hepatoma, respectively. The two subtracted amplicons were labeled with fluorescent cy-dyes as targets for microarray analysis, in which dye-swapping approaches were used. The results thus obtained were then compared to those obtained from the conventional cDNA microarray assays. The differentially expressed genes were categorized into three groups: I) only detected by conventional cDNA microarray approaches, II) identified by both conventional cDNA microarray and SSH/microarray approaches, and III) only obtained from SSH/microarray assays. The results were further confirmed by qRT-PCR in 6 genes randomly selected from group III differentially expressed genes. \nLabeled and used to hybridize chips\nTo further address whether the SSH/microarray approach was able to assay the expression of those genes with rare transcripts, we selectively inspected those genes with low expression. Of the total of 14,811 clones on the chips, 9376 clones that were filtered out for further analyses in the regular cDNA microarray studies due to the low hybridization intensity (intensities <500 unit after the noise subtracted) ( Figure 3A ) were selected for examination of their hybridization intensity in the SSH/microarray assays ( Figure 3B ). Of these, 5015 clones were enriched via the SSH/microarray assays and could be analyzed for differential expression (intensities >500 unit after the noise intensity subtracted) ( Figure 3B &3C ). That is, the SSH/microarray approach allowed about 100% more of the genes, most of which were low abundant, for the subsequent analyses.\nSince SSH specifically amplified the difference of gene expression, the SSH/microarray approach should also be able to identify more differentially expressed genes between hepatoma and non-hepatoma liver tissues, which were originally undetectable by the regular cDNA microarray methods. As shown in Fig 3C, of the 5015 lowexpression genes identified only by the SSH/microarray assays, we identified additional 512 and 1923 genes with at least a 2-fold decrease and increase in hepatoma tissues, respectively.\nWe further examined the sensitivity for detection of differential expression by both approaches. We compared the ratio of Cy5/Cy3 intensity of each clone obtained from the regular cDNA microarray assays to that obtained from the SSH/microarray assays. As shown in Figure 4 , a total of 3028 genes with a ratio lower than 2 folds (-1 < log 2 < 1) using the cDNA microarray assays had a ratio greater than 2 folds (log 2 > 1 or < -1) using the SSH/microarray assays The representative results of the regular cDNA microarray vs Figure 2 The representative results of the regular cDNA microarray vs. SSH/microarray assays. RNA were prepared from the hepatoma and para-hepatoma liver tissues of a patient with HCC. Left panels are the representative results obtained via the regular cDNA microarray assays using the Cy 5 and Cy3 labeled aRNA generated from hepatoma and non-hepatoma liver tissues, respectively. Right panels are the representative results obtained via the SSH/microarray assays using Cy3 and Cy5 to label the forward and reverse subtracted amplicons, respectively. Lower panels are the close views of the upper panels. White circles indicate the clones, which had equal Cy3 and Cy5 hybridization intensities in the conventional cDNA microarray assays but been subtracted away from the subtracted amplicons. Red circles mark the clones, which had low Cy5 and Cy3 hybridization intensities in the conventional cDNA microarray but presented with differential expression in the subtracted amplicons.\nSSH/microarray ( Figure 4 , 1091 and 1937 genes indicated by the red box, respectively). That is, using the modified SSH/microarray approaches resulted in much more differentially expressed genes, particularly for those with rare transcripts that using the regular cDNA microarray approaches.\nA total of 69 and 701 genes differentially expressed in human hepatoma were finally identified (based on the data derived from the three tissue pairs in duplicate with log 2 > 1 or < -1, and P < 0.01) by the regular cDNA microarray assays and by the SSH/microarray assays, respec-tively ( Figure 5 & see additional file 1). That is, the SSH/ microarray assays detected 10-fold more differentially expressed genes, particularly for those low-expression genes, than did the conventional cDNA microarray. To confirm the differential expression of genes with low expression identified by the SSH/microarray assays (including 446 and 255 up-and down-regulated genes, respectively), we quantified the relative expression level of nine randomly selected genes, whose differential expression was detected by the SSH/microarray assays, in 18 pairs of HCC and the matched non-HCC liver tissues. We found the results were consistent with those obtained from SSH/microarray assays ( Figure 6 ).\nDetection of differential low-expression transcriptomes by the SSH/microarray Figure 3 Detection of differential low-expression transcriptomes by the SSH/microarray. A) Of the 14811 distinct genes on the chips, 9376 genes had hybridization intensities lower than 500 units (the defined low intensity threshold for exclusion from further microarray analysis) in a representative set of results of the conventional cDNA microarray assay. B)These low-expression genes were then subjected to re-calibration for their Cy3/Cy5 intensities in the SSH/microarray assay and 5015 genes became detectable in SSH assays (blue spots). C) The distribution of these 5015 low-expression genes identified only in the SSH/microarray assays, including 512 and 1923 genes down-and up-regulated in hepatoma tissues, respectively. The data presented in this figure were based on the results derived from patient 1, a case of chronic hepatitis C with cirrhosis and hepatoma. \nWe compared the HCC up-and down-regulated transcriptomes identified by the SSH/microarray assays in accordance with their potential molecular functions, implicated biological processes and sub-cellular localization. As presented in Table 1 \nMicroarray techniques are limited in the detection of genes with low expression [14] , while subtractive hybridization methods are restricted by their specificity and the tremendous work needed for validating the results, as well as for sequencing to identify genes. There have been many reports using microarray techniques for rapid and high throughput validation of the subtraction specificity of SSH by spotting the enriched clones on the chips or membranes [10] [11] [12] [13] [15] [16] [17] [18] [19] [20] [21] [22] [23] . However, such approaches would not only require tremendously sequencing works to identify genes including a great number of redundant clones in the subtracted amplicons, but also lose the sensitivity of SSH techniques to detect the low abundance transcripts, since the sensitivity of microarray analysis is determined by the targets used to hybridize chips but not by the probes printed on chips.\nHerein we report our modified approaches. Instead of using the subtracted clones as probes spotted on chips, we directly labeled the enriched amplicons and used them as targets to hybridize the pre-made microarray chips for microarray analysis (named as \"SSH/microarray\" in this report). This approach allowed us not only to readily identify genes without tremendous sequencing works in the subtracted amplicons regardless of many redundant clones, but also to easily evaluate the subtraction efficiency and specificity. Moreover, the SSH/microarray approach made it possible to conduct a transcriptomewide identification of differentially expressed genes particularly for those with low expression. It has been reported that the absolute expression level is not a crucial determinant for identifying genes, while the relative difference in expression levels does impact on whether or not a gene is recovered by subtractive hybridization [5, 24] . In this report using the SSH/microarray approach, we identified about ten times more of the differentially expressed genes, particularly for those with low expression, in human hepatoma. Our findings successfully demonstrated the transcriptome-wide assays of differentially expressed genes at low abundance. This simple but very powerful approach would greatly facilitate future researches on functional genomics [15, 19, 25] .\nOne of the main concerns about SSH techniques is their specificity. However, as combined with microarray tech- Figure 4 Comparison of the relatively expression by regular cDNA microarray vs. the SSH/microarray. The distribution of the ratios of Cy5/Cy3 intensities obtained by the conventional cDNA microarray assays vs. the SSH/microarray assays is shown. Only the clones with significant Cy3 and Cy5 intensities in both assays were included in the analysis. The clones inside the red-boxes were those with their log 2 ratios < 1 and > -1 via the conventional cDNA microarray assays, while their log 2 ratios > 1 or < -1 by the SSH/microarray assays. The data presented were based on those derived from patient 1, a case of chronic hepatitis C with cirrhosis and hepatoma.\nLog 2 ( Cy5/Cy3) of SSH Log 2 ( Cy5/Cy3) of cDNA niques, the subtraction efficiency can be readily evaluated. As shown in Figure 2 , the subtraction efficiency was determined by the rare presence of clones with equal hybridization intensities between the forward and reverse amplicons, and by the consistent exclusion of the housekeeping genes in the subtracted amplicons. The specificity was further confirmed by quantification of the differential gene expression between hepatoma and para-hepatoma The differentially expressed genes Figure 5 The differentially expressed genes. The genes differentially expression in human hepatoma were identified by either only the conventional cDNA microarray assays (26 genes) or SSH/microarray assays (658 genes), or both (43 genes). The results were derived from three pairs of hepatoma liver tissues with at least two-fold difference in gene expression and P < 0.01. APEG1, TTN, HTN1, MEST, EVL3, VLDLR, MYOG, TNNI2, HOXC11, IGF2, AHSG, TTID, BIRC1, PAFAH1B1, SYK, LMNA, TNFRSF11B, FALZ, BMP1, ETS2, NRD1, CXCL1, JAG1, SEMA4G, HEY1, ZNF22, CRMP1, TEAD4, IGFBP1, IGFBP3, IGFBP7, PTGS1 , NEUGRIN, CUGBP1) related to the regulation of cell differentiation and embryonic development, and 59 genes (ASGR2, CAP2, RGS5, ITGB4BP, NCK1, EVL3, PLCG2, EPHA7, IGF2, IFNGR2, AHSG, CAP1, PTPRF, CXCL2, GNAI1, TGFBR1, NCSTN, DOK1, FRBB3, SYK, MC1R, CD79B, CSNKIE, CD69, AVPR1A, GNB2L1, ACVR1, BIRC2, FLT4, CXCL1, FAG1, PNOC, SLC9A3R1, LANCL1, GNB3, ADORA2B, FGR, NCK1, RAB11A, PLCG2, LOC91614, PRKAR1A, FLJ22595, ARF3, TYK2, MAP3K7IP1, MC1R, TNFSF10, ARF4, VAV2, AVPR1A, GNB2L1, MAPK10, STMN1, SNX17, ADORA2B, ECT2, RGS5, IGFBP3 ) associated with signal transduction in response to extra-cellular proliferation and growth stimuli were found. Our findings that differentially expressed genes were related to multi-biological processes suggest the complexity of the molecular mechanisms for hepatocellular carcinogenesis.\nIn this study, we modified the previously method of the combination of the suppressive subtractive hybridization and microarray techniques to differentially profile the low-expression transcriptomes of human hepatocellular carcinoma by directly labeling both of the reciprocal subtracted amplicons as target for cDNA microarray assays. Compared to SSH or previous SSH in conjunction with microarray approaches, this modified approach provided us with three additional advantages: 1) easy inspection of the subtraction efficiency, 2) avoidance of tremendous sequencing work for gene identification, 3) high sensitiv-ity for identifying the low-expression, differentially expressed genes. This approach allowed for the detection of about ten times more of the differentially expressed genes than did the regular cDNA microarray approach in human hepatoma, particularly for those with low expression. Using this approach, we identified many genes potentially implicated in human hepatocarcinogenesis, which were not identified before. For its high efficiency and high sensitivity, this SSH/microarray approach is powerful for the rapid differentially profiling the lowexpression transcriptomes, and most adequate for applying to functional genomic studies.\nFor SSH and cDNA microarray analysis, hepatoma and the corresponding non-cancerous liver tissues were obtained from 3 patients who had liver surgery at the Chang Gung Memorial Hospital. For reverse-transcription real-time PCR, hepatoma and the matched non-hepatoma liver tissues were obtained from additional 18 patients of hepatoma. Diagnoses of HCC and non-hepatoma liver tissues were based on histo-pathologic findings. The Internal Review Board for Medical Ethics of Chang Gung Memorial Hospital approved the specimen collection procedures and informed consent was obtained from each subject or subject's family.\nTotal RNA from hepatoma and the para-hepatoma liver tissues and the poly(A) RNA was prepared as described before [26, 27] . SSH was performed with the Clontech PCR-Select cDNA Subtraction Kit (Clontech Laboratories Inc., Palo Alto, CA) as described by the manufacturer but with the following modifications. Starting material consisted of 2 μg hepatoma mRNA as tester and 2 μg nonhepatoma liver tissue mRNA as driver, and vice versa. Primary and secondary PCR conditions were altered to increase specificity of amplification according to either plan A or B. Both A and B reduced the extension time and the number of cycles of the primary PCR to 2 min and 26 cycles, respectively. The primary PCR products were diluted 1/50 prior to use in the secondary PCR. All other aspects of plan A were as per the instructions of the manufacturer. Plan B diverged from plan A in two ways. First, the initial cycle of primary PCR was performed using annealing and extension times that had been reduced to 15 s and 1.5 min, respectively. Second, for subsequent cycles, the denaturing time was increased to 10 s while the annealing and extension times were reduced to 15 s and 1.5 min, respectively.\nIn this study, we used the GMRCL Human 15 K set, Version 2 chips as previously described [28] , which contained 14,811 sequence-verified, human cDNA clones mapped to 12,530 distinct genes. All of the samples for the regular cDNA microarray or SSH/microarray assays were performed with the dye-swapping microarray design for minimizing labeling bias and statistical variances of data. For the regular cDNA microarray experiment, we used 2 μg of the total RNA for labeling and hybridization using a 3DNA Array 350RP Detection kit (Genisphere, PA, USA).\nFor the SSH experiment, 1 μl subtractive PCR products were labeled with Cy3 and Cy 5-dCTP (NEN, Boston, MA) using random primers. Unincorporated fluorescent nucle-otides were removed using a Qiaquick PCR purification kit (Qiagen). The fluorescent-labeled DNAs were mixed with 30 μg of human cot-1 DNA (Invitrogen) and 100 μg yeast tRNA, precipitated and then resuspended in 30 μl of Microarray Hybridization Buffer Version 2 (Amersham Pharmacia). The hybridization solution was heated to 80°C for 10 min to denature the DNA and was then incubated for 30 min at 37°C, allowing cot-1DNA and yeast tRNA to block the repetitive sequences in genome probes. The probes were hybridized to a human cDNA microarray (GMRCL Human 15 K). We scanned the slides with a confocal scanner ChipReader (Virtek, Canada) and acquired\nQuantification of relatively gene expression in human hepatoma Figure 6 Quantification of relatively gene expression in human hepatoma. A total of nine differentially expressed genes including six (GBA, PTPRF, GNL3, ERBB3, OGDHL, FMO3 belonging to Class III) identified only by the SSH/microarray assays and three (C9, MT1F, MT1X belonging to class II) identified by both of the SSH/microarray and regular cDNA microarray assays were assayed for their relative expression between HCC and the corresponding non-HCC liver tissues of 18 patients of hepatoma using real-time semi-quantitative RT-PCR. The results are presented with the log 2 values of the gene expression ratio's between HCC vs. the matched non-HCC liver tissues. Left panel is the relatively expression level of these genes initially determined by the SSH/microarray and regular cDNA microarray assays, respectively. Of note, the difference of the expression for the six genes belonging to Class III was originally only identified by the SSH/microarray assays, but not by the regular cDNA microarray assays. the spot and background intensities with the GenePix Pro 4.1 software (Axon Instruments, Inc., CA, USA). The within-slide normalization was done using programs written with MATLAB 6.5 software (The MathWorks, Inc., MA, USA).\nTo validate the results obtained from the SSH/microarray assays, we randomly selected six differentially expressed genes identified only by the SSH/microarray assays and three differentially expressed genes identified by both the regular cDNA microarray and SSH/microarray assays for the comparison of gene expression between hepatoma and the corresponding non-hepatoma liver tissues in eighteen patients of HCC using reverse transcription realtime PCR (qRT-PCR). Total RNA was extracted from tissues with Trizol reagents and reverse transcribed using the SuperScript III first strand synthesis system (Invitrogen, Carlsbad, CA). qRT-PCR was conducted using the ABI PRISM 7000 sequence detection system (Applied Biosystems). Pre-designed Assays on Demand TaqMan probes and primer pairs for these 9 genes were obtained from Applied Biosystems Incoporated (ABI) (Foster City, CA). For each gene, two to four sets of Taq-Man probes and primers were tested. The probes contained a 6-carboxyfluorescein phosphoramidite (FAM dye) label at the 5' end of the gene and a minor groove binder and non-fluorescent quencher at the 3' end. These were designed to hybridize across exon junctions. As a result, no fluorescent signal was generated by these assays when genomic DNA was used as a substrate, which confirmed that the assays measured only mRNA. Equal amounts of RNA were used for all qRT-PCR reactions, which were performed in triplicate, and 18S ribosomal RNAs were used as internal controls.\nAll data (derived from three pairs of HCC and non-HCC liver tissues in a dye-swapping approach) were filtered so that 446 up-regulated and 255 down-regulated genes in HCC with at least two-fold difference and p value less than 0.01 were included in the further studies. After removal of the un-annotated genes, a total of 360 and 202 HCC up-and down-regulated genes were subjected to the subsequent gene ontology analyses. The two lists of the differentially expressed genes were analyzed using the online software FatiGo for comparative gene ontology categories including molecular function, biological process and cellular component [29] . They were also imported into the on-line software KEGG2 for pathway mapping [30, 31] . The statistical significance was defined as P < 0.01 between the HCC up-and down-regulated gene groups using Fisher's exact test." }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: During the past decade, the highly successful field of functional genomics experienced huge growth as a result of the development of DNA microarray technology [1] [2] [3] [4] , which made it possible for the first time to measure the RNA expression of thousands of genes in parallel, in a single assay. Immune responses are complex phenomena that supervene on genomics, that is, immune responses ultimately depend on the expression of genes inside a variety of cells, but explaining the function of the immune system only in terms of gene expression in those cells would constitute a reductionist approach. While studying the immune system in terms of genomics is an important goal [5, 6] , the function of the immune system, from antigen processing to epitopespecific immune responses, may be better understood through an integrated approach that takes into account properties of the immune system as a whole.\nWe quote from [7] , ''The immunome is the detailed map of immune reactions of a given host interacting with a foreign antigen, and immunomics is the study of immunomes.'' Whereas functional genomics strives to identify the role of genes in cellular processes via the paradigm of hybridization of mRNA to complementary DNA, functional immunomics aims to identify the roles of chemical/biological targets involved in immunological processes via the paradigm of specific cellular and humoral immune responses elicited by antigens presented to the immune system [8] [9] [10] [11] . This is an effort that promises great rewards, both in terms of our basic understanding of the immune system and in terms of disease diagnosis/prognosis [12] and the design of vaccines [13] [14] [15] to combat a variety of human infirmities ranging from pathogenic infections to allergies and cancer.\nEnabling technologies. Functional genomics was made possible by the significant advances that had previously been made in sequential genomics, including not only the massive efforts required to identify genome-wide DNA sequences [16] , but also the computational methods used to parse and align those sequences [17] . Sequential genomic data are deposited in large public-access databanks such as GenBank [18] , and researchers or companies who make DNA microarrays use the sequences in these databases as probes. In a similar fashion, the field of functional immunomics has now come of age as a result of advances in sequential immunomics, which consists of methods to catalogue the chemical/biological targets capable of eliciting an immune response, also known as epitopes. Computational and statistical methods are now available for automated large-scale epitope prediction (please see the next subsection), in addition to classical immunoassays such as ELISPOT [19] and tetramer staining by flow cytometry [20] that together enable high throughput identification of epitopes. Recently, a coordinated effort has been initiated by the National Institute of Allergy and Infectious Disease at the US National Institutes of Health, under the auspices of the Large-Scale Antibody and T Cell Epitope Discovery Program (in which the authors of this paper participate), to create an integrated immunome database and resources such as a toolbox of epitope prediction methods. This initiative is designed to identify immune epitopes from selected infectious agents; the information will be made freely available to scientists worldwide through the Immune Epitope Database and Analysis Resource (IEDB) [7, 21, 22] (http://www. immuneepitope.org). The sequencing information produced by this and other epitope mapping efforts being carried out will be essential for the construction of immunomic microarrays (discussed in detail below), leading to an experimental paradigm similar to that employed in functional genomics.\nComputational epitope prediction methods. The immune system recognizes antigen via binding of antibody (humoral response) or T cell receptors (cellular response) to self or foreign proteins. B cell epitopes correspond in general to the 3-D features on the surface of antigen where recognition by the immune system occurs; a continuous or linear epitope is a sequential fragment from the protein sequence, while a discontinuous or conformational epitope is composed of several fragments scattered along the protein sequence and brought together in spatial proximity when the protein is folded [23] . Humoral response is targeted mainly at conformational epitopes, which may represent up to 90% of the total B cell responses. This makes prediction of B cell epitopes a hard problem [24] , even more so because B cell responses are virtually only restricted by immunoglobulin access to the epitope, B cell receptor activation, and self versus no-self discrimination rules of the immune system. Ideally B cell prediction systems would use 3-D surface models of the protein antigens and measure surface energy interactions of variable regions of the immunoglobulins that correlate with B cell activation. However, so far B cell prediction systems make estimations of the probability of a primary peptide sequence being present at the surface of a protein based on hydrophilicity and secondary structures [25] . Cellular responses, on the other hand, are restricted through the binding of T cell receptors to short linear peptides, which are bound by a specific groove in two main classes of major histocompatibility complex (MHC) molecules, and presented on the surface of cells to the T cell receptors of CD4 þ and CD8 þ cells [26] . Binding affinity between the peptide and the MHC molecule is therefore a necessary requirement for effective cellular immune response. A complicating factor is the highly polymorphic nature of the MHC molecule, which displays large variability in human populations. Using experimental affinity data deposited in public databases as training data, researchers have developed statistical methods to predict the MHC affinity of a given unknown peptide [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40] [41] [42] . Typically, such computational epitope prediction methods scan the full length of pathogen or self-immunogenic protein sequences by taking consecutive overlapping peptides. In addition, such methods can predict ''promiscuous'' epitopes, that is, the ones that bind to a large class of different MHC alleles, know as supertypes [43] . Computational MHC-binding prediction methods have become essential for the systematic search for epitopes, in situations where techniques such as ELISPOT and flow cytometry are effectively impractical due to the large number of peptides to be assayed.\nEarly MHC-binding studies identified characteristic amphipathic chemical patterns on the binding peptides [44] , and enhanced versions of these systems continue in use in association with other methods [41, 44, 45] . Today, the most basic MHC-binding prediction methods are based on the identification of specific amino acids commonly found at particular positions, called binding motifs, within peptides that bind to a specific MHC molecule. However, all the amino acids of a peptide bound to an MHC groove (normally 8-10 amino acids for MHC I and 8-14 amino acids for MHC II) can potentially play a positive or negative role in binding, and more complex methods assign positive or negative values for each amino acid at each position of a peptide and combine these values to define scores that predict binding; these ''quantitative-matrix'' approaches have been very successful. One of the limitations of the ''quantitative-matrix'' approach is that it does not take into consideration the influences of interactions between amino acids at different peptide positions of the epitope. The value of these interactions are difficult to measure and have been explored only in a limited fashion by combining pair-wise interactions between two peptide positions [46] . The combination of independent binding calculated by quantitative matrices with coefficients derived from the pair-wise interaction provided better predictions. Moreover, quantitative-matrix scores have been generated for several HLA alleles, and studies using HLA sequence homology have allowed the development of virtual quantitative matrices to be applicable to many more HLA alleles [28, 39] . General methods for MHC-binding prediction systems, such as artificial neural networks, and statistical models such as Hidden Markov, can incorporate nonlinear complex interactions between the MHC molecule and the peptide epitope, and can evolve as more data is included in the training set. These general methods have shown to be potentially superior to the previous ones [27, 37] . The greatest challenge, however, of the general methods is that, to be reliable, they require a larger amount of peptide-binding data. Strategies using query-by-committee approaches to compare predictions from different training sets have been used to identify the most informative peptide-binding data to be determined in the biochemical binding assays in order to more efficiently build a representative training dataset [40] . Recently a collection of more than 48,000 peptide binding affinities of class I molecules were made public [47] . The development of prediction models will be greatly accelerated by this community resource benchmark. We remark that epitope recognition by the immune system involves more than a receptor-ligand problem between the MHC molecule and a peptide, as other biological processes are involved in preparation of the peptide for loading into the MHC molecule. Epitope prediction systems continue to evolve, and many steps in antigen processing and presentation required for development of cognate immune responses are being modeled and combined into rational prediction systems. The greatest current challenge is the development of models incorporating the rules of B cell and T cell receptor engagement and their possible outcomes.\nThe growth ''boom'' of immunomics. While the growth boom in genomics took place in the 1990s and this field has now begun to enter a mature stage of development, a similar growth boom in immunomics is likely to take place over the remaining years of the current decade. We recently searched the PubMed database (with Sente 2.3) using the following query: ''immunomics OR immunomic OR immunome OR (antigen AND microarray AND functional) OR (epitope AND microarray).'' After removing nine irrelevant articles from the output list and adding five articles for the new journal Immunome Research, we obtained a list of 71 articles covering the years from 1999 to the present (please see Figure 1 ). It is clear that interest in this field has accelerated, supporting the expectation of a continuing boom in growth. It is expected that the number of publications will increase at an exponential pace as immunomic microarrays became commercially available for research use. As immunomic array technology evolves, we expect that immunomic arrays with a small number of features will eventually be designed for specific clinical diagnostic purposes and used regularly in medical practice. However, these clinical applications might still be in the distant future.\nThe basic functioning principle behind all microarray technology is the binding, and subsequent measurement, of target biological specimens of interest to complementary probes arrayed in a spatially addressable fashion. Typically, a planar surface, such as a glass slide, is used to support an array of spots containing the probes. As a consequence of using spatially addressable probes, a large number of different targets can be measured in a single experiment. For example, in the case of DNA microarray technology, which provides the basic enabling technology for functional genomics, the targets are fluorescent mRNA molecules (indicators of genomic expression) that are hybridized to gene-specific DNA probes immobilized on a planar surface. In a similar fashion, the enabling technology for functional immunomics is the immunomic microarray. The basic technologies for immunomic microarrays that we consider in detail in this paper are antibody, peptide, and peptide-MHC microarrays (see Table 1 for a summary of these technologies). Other functional immunomic approaches include dissociable antibody microarrays [48] , cell microarrays [49, 50] , serum microarrays [51] , peptide libraries [52, 53] , and serological analysis of cDNA expression libraries (SEREX) [54] [55] [56] [57] [58] . There are significant technological challenges inherent in fabricating immunomic microarrays, including the identification of a workable surface coating for the glass, appropriate probe concentration and target incubation times, and suitable spot size and interdistance [59] [60] [61] [62] [63] [64] .\nAntibody microarrays consist of antibody probes and antigen targets; thus, they can be used to measure concentrations of antigens for which the antibody probes are specific [65, 66] . As such, antibody microarrays are quite useful in proteomic applications, such as in the proteomic profiling of cancer antigens [67] [68] [69] . Antibody microarrays have also been proposed for post-translational functional genomics [70] . The rationale for directly measuring protein concentration, rather than using a traditional DNA microarray format, is the existence of evidence of poor correlation between concentrations of mRNA and its corresponding protein, which reflects post-translational modification of the protein [71] . Given the possibility of measuring antigens or proteins associated with ''foreign agents,'' antibody microarrays can be employed in functional immunomics applications [72, 73] (The application in [73] actually used cells, which display the target protein markers on their surfaces.) As a general rule, however, using antibody microarrays in data-driven functional immunomic applications may be problematic. One of the main reasons is that this approach requires the production of specific antibody sets for use in defining each of the antigen targets, and the development of large numbers of interrogative features (antibody sets) is a tremendous challenge, since humoral responses are much broader than MHC-restricted T cell responses, are highly conformationally dependent, and can be developed against a great variety of chemical/biological elements present in biological fluids, including small molecules. Peptide microarrays use the opposite technical approach; that is, they use antigen peptides as fixed probes and serum antibodies as targets [74] . This format is promising for functional immunomic applications. Published studies using peptide microarrays include applications to autoimmune disease [10, [75] [76] [77] [78] [79] , allergy [80] [81] [82] [83] [84] [85] , B cell epitope mapping [13, [86] [87] [88] , vaccine studies [89, 90] , detection assays [91, 92] , serum diagnostics [12] , characterization of weak protein interactions [93] , and analysis of antibody specificity [94] . Peptide microarrays essentially correspond to highthroughput parallelized ELISA assays [12, [95] [96] [97] [98] and thus can reveal the repertoire status of antigen-specific B cell antibody responses. However, B cell responses are highly dependent on CD4 þ T cell immune responses, and thus peptide microarrays should ideally be used in parallel with extensive analysis of T cell responses (e.g., by using peptide-MHC microarrays; see below). One of the pioneer studies that best depict the usefulness of peptide immunomic technology was performed using an array of 87 protein antigens to search for specific antibody reactivity patterns in the serum of 20 normal health volunteers; these were compared to the patterns of 20 type-1 diabetes mellitus patients, and simple classifiers were designed to discriminate between healthy and diabetic patients, with an overall sensitivity of 95% and specificity of 90% [78] . In a subsequent study, the 87-feature array was able to identify prognostic signatures that could predict the susceptibility of healthy animals to develop diabetes [10] . Another important report describes the use of a panel of 225 selected peptides of several protein antigens known to be recognized by autoimmune disease patients [79] . The autoimmune peptide array was used to study the profile of the autoantibody reactivity pattern of rheumatoid arthritis (RA) patients. The RA study used serum from 18 RA patients, 38 healthy controls, and 58 recently diagnosed RA patients, and found early clinical prognostic markers able to predict which patients are more likely to develop severe RA, and also markers to identify the group of patients with the milder form of the disease [77] . Moreover, an array developed from a panel of 213 peptides derived from allergenic peanut proteins established that patients responding to a greater diversity of peptide peanut epitopes had the worst allergic reactions [80] . The importance of the breadth of antibody response against the simian-human immunodeficiency virus (SHIV), a experimental nonhuman primate model for human immunodeficiency virus (HIV), was demonstrated through studies performed with an array of 430 peptides derived from simian immunodeficiency virus (SIV) and HIV amino acid sequences [90] . This study indicated that the reduction of the repertoire of the antibody response was associated with development of acquired immunodeficiency syndrome (AIDS). These examples underscore the great potential of peptide microarrays to identify several valuable clinical markers.\nThe most recent technology to be proposed is the peptide-MHC microarray or artificial antigen-presenting chip [9, 11, 99] ; in this case, recombinant peptide-MHC complexes and costimulatory molecules are immobilized on a surface, and populations of T cells are incubated with the microarrays, whose spots effectively act as artificial antigen-presenting cells [100] containing a defined MHC-restricted peptide. Different methods have been proposed for detecting T cells expressing receptors with affinity for specific peptide-MHC complexes on the microarray; these can include simple inspection of T cell clusters bound to a spot [9] or identification of activated cells secreting specific cytokines with cytokine-specific capture antibodies [11, 99] . Peptide-MHC microarrays correspond to high-throughput parallelized ELISPOT assays [19] , particularly when low enough densities of cells are used, in which case a direct counting of activated cells is possible [11] . Quantitation can involve cell counts alone, detected cytokine intensities alone, or a combination of both, as in [99] , which used a cell-count score adjusted by an intensity score. The benefit of using peptide-MHC microarrays is that it can map MHC-restricted T cell epitopes, which are involved in several helper and regulatory functions of the immune system, and can be used in conjunction with peptide-based B cell epitope microarrays to study the adaptive system as whole. Figure 2 illustrates the functioning of peptide-MHC microarrays. In Figure 2a , a peptide-MHC microarray is depicted, with an inset showing the probe molecules that are deposited on a microarray spot. Figure 2b depicts T cells that bind to, and are activated by, specific peptide-MHC complexes, with the help of co-stimulatory antibodies; these T cells secrete cytokines that are captured by specific detection antibodies. Finally, as depicted in Figure 2c , the T cells and excess cytokine are washed away, and the bound cytokine is revealed by fluorescent antibody (other methods can be employed to reveal the cytokine [11] ). Therefore, a peptide-MHC spot is designed taking into account two elements: a peptide-MHC complex, and the detection antibody specific to the particular cytokine one wants to measure. A third element can be the kind of T cell population (e.g., T helper or CTL) that is used as a target (i.e., incubated with the microarray). The choice among these three different elements will lead to a vast number of immunomic responses that can be measured.\nAn exciting feature that distinguishes immunomic from DNA microarray data is the possibility of measuring two or more signals simultaneously, determined by a single feature, the epitope. In the case of DNA microarrays, one response value is obtained for each gene per sample, namely the concentration of mRNA produced by the gene (note that twodye experiments use mRNA from two different samples). In the case of peptide-MHC chips, a single epitope can generate different response values corresponding to different cytokines or different target T cell populations, or even different antibody isotypes, in the case of peptide microarrays. In other words, in the case of genomic microarrays, one parameter is measured, namely the level of transcription of each individual gene, whereas in the case of immunomic microarrays, it is possible to measure several parameters regarding immune responses against a single epitope. For instance, a single B cell epitope can be recognized by different isotypes of immunoglobulins, such as IgE or IgG1. Therefore, in this case it is not only the intensity of the antibody response that can be measured, but also the quality of the antibody response. This aspect can be very relevant since a high IgE titer in relation to IgG1 may be associated with allergy, whereas the opposite, a high IgG1 titer in relation to IgE to the same epitope, is not. This situation is even more significant in the case of the peptide-MHC array, where the same peptide-MHC epitope can induce several different cytokine responses. These ''multicolor'' peptide-MHC microarrays have a counterpart in the multicolor ELISPOT assays currently in use [101] . It is known that the combined effect of multiple cytokines is essential to the control of immune responses; this is described by the suggestive term ''cytokine chord'' in [102] . Thus, given a family of epitopes, one may want to simultaneously measure both inflammatory (effector) and anti-inflammatory (regulatory) T cell responses, which are known to be associated with the concentrations of IFN-c and IL-10, respectively [103] . In this case one would have more than one spot on the microarray containing the same epitope (peptide-MHC complex) but use distinct cytokine antibodies for detection (see Figure 3 ). The result of this analysis is not a real-valued profile, as is obtained from functional genomics microarrays, but rather a vector-valued profile. Such profiles are sometimes called ''multispectral'' profiles (see the section on data analysis below). We will, however, adopt the term ''multicolor'' when referring to immunomic data, due to the fact that this term is already used in the similar setting of ELISPOT assays.\nThe technological challenges mentioned previously in connection with antibody and peptide microarrays are much more complex in the case of peptide-MHC microarrays, which in fact involve elements of the two previous technologies, namely presentation of peptide and antibody detection of secreted cytokine. The technology of peptide-MHC microarrays, though still in its infancy, is viewed as a simple and economical method for screening the T cell repertoire of a host [99] , and thus holds great potential. The first clinical research application of peptide-MHC microarray technology was the study of the correlates of protection regarding the effects of an experimental therapeutic cancer vaccine [104] . Ten patients with melanoma were immunized with a peptide vaccine, and their immune responses were examined with a peptide-MHC microarray, which contained seven types of peptide-MHC epitopes and probed for 26 secreted factors. This peptide-MHC array was shown to have the sensitivity to detect one peptide-MHC specific T cell in 10,000, and 10 6 CD8 þ cells were incubated with the array (so, in theory, this peptide-MHC array could detect up to 100 distinct reactive features that have reached minimum frequency of 1:10,000). Analysis of the peptide-MHC microarray response patterns demonstrated that patients who presented both IFN-c and TNF-a secretory responses against a specific epitope remained free of melanoma.\nThe complexity of the statistical analysis with regard to immunomic microarray data is on a whole different level than that of genomic microarray data. The total number of genes in humans is estimated to be ;30,000; in comparison, the total number of different T cell receptors in humans, generated by somatic recombination, which recognize peptides within the context of a major histocompatibility molecule, is estimated to be on the order of 10 7 to 10 15 , and the number of B cell clonotypes, generated by somatic recombination of V(D)J genes, is estimated to be on the order of 10 12 . DNA is based on a four-letter ''alphabet,'' consisting of the four nucleotide bases A,G,C,T, whereas peptide epitopes are based on a 20-letter alphabet, consisting of the amino acids known to be involved in life processes. Clearly, the combinatorial complexity in the case of functional immunomics is several orders of magnitude higher than that of functional genomics. In addition, for functional genomics the number of interrogative features that need to be built on microarrays is on the order of 10 4 to 10 5 . In functional immunomics, the total number of interrogative features included in microarray analysis can be much larger and may be estimated as follows, in the case of peptide-MHC microarrays: analysis of MHC peptide-binding motifs [26] suggests that a core of nine amino acids within a peptide is sufficient for characterization of a T cell epitope. The total number of interrogative features would thus correspond to the number of possible nine-letter words based on a 20-letter alphabet, which is 20 9 ' 10 11 ; fortunately, only ,1% of these are able to bind to MHC molecules, which makes the number of interrogative features more manageable. In addition, B cells and T cells go through a process of clonal selection, where leukocytes that either do not react or react too strongly are eliminated, and dangerous clones that react with self antigens are deleted or anergized as part of the process of immune tolerance. The number of features is still very large, however, and methods to further reduce this number are essential; such methods include the prediction methods and immunomic databases mentioned previously in connection with epitope mapping, as well as the selection of peptides from specific genomes of pathogens, allergens, and self-antigens involved in human infirmities, such as tumor antigens, diabetes, and autoimmune diseases.\nOne additional procedure that we and other groups have used after screening the genomes of pathogens for putative binding peptides is to compare those candidate epitopes with known host protein sequences, and in some cases we have found peptides that are identical to host peptides. This is very important because several critical diseases are caused by pathogen molecular mimicry, that is, some diseases, such as diabetes, dengue hemorrhagic fever, and Guillian Barret syndrome, are hypothesized to be the result of infections that induce self-reactive pathogenic immune responses.\nIt is important to state that the goal of the immunomic array is not to test all possible naïve T cell or B cell clones that can be generated by the somatic, VJ, or V(D)J recombinations against all possible combination of peptides-there simply would not be enough patient blood to do that, even if it were considered to be a relevant pursuit. The goal of the immunomic array is to identify primed cells that have reached a reasonable level of precursor frequency and are thus expected to have biological relevance. If the frequency of a circulating T cell in the peripheral blood is less than 1:100,000 we can expect that the biological relevance of it is small in contrast to a T cell that has a frequency of 1:1,000. In addition, a T cell has a binding affinity for the peptide-MHC that the T cell is specific for, and it should not bind to the ones with which it has no affinity. The current reported limit of detection of the peptide-MHC immunomic array is 1:10,000 cells when using 10 6 CD8 þ cells. So, in theory, a peptide-MHC array incubated with 10 6 purified CD8 þ cells (;10 ml blood) could detect up to 100 distinct reactive features that have reached minimum frequency of 1:10,000. However, if we expect to be able to detect rare clones, with very low frequencies, more cells would be needed. This requirement may be overcome with larger amounts of blood, and it is not unreasonable to collect 100 ml blood, or by adding a T cell expansion step to grow the cell population ex vivo before it is incubated with the array. This technique of T cell clonal amplification is commonly used to detect rare populations of cells by flow cytometry or by ELISPOT and may as well be applied in immunomic studies.\nNormally, 250,000 human PBMCs are used in an ELISPOT assay with one peptide, and the limit of detection is estimated to be 4-fold to 5-fold more sensitive than flow cytometry. However, ELISPOT is quite distinct experimentally from immunomic microarrays. In ELISPOT, T cells and APCs are present in the same mixture, and for a T cell to be activated, it has to be in close contact with its APC. In the peptide-MHC array the spot surface is equivalent to a very large defined APC, completely loaded with one specific peptide epitope, for which a reactive T cell has a binding affinity, so that it adheres to its specific peptide-MHC spot and not to the other spots. In [104] , the authors used 10 6 cells and compared the limit of detection of the array with flow cytometry. It turned out that both had similar limits of sensitivity, approximately 1:10,000 cells or 0.01%. It is possible that in the future the peptide-MHC spot surface can be improved and the sensitivity of the peptide-MHC array may become even greater than the current ELISPOT assay.\nAnother great challenge is the polymorphism of HLA genes, in particular HLA class II, and the several combinations of different alpha and beta chains. Some approaches may be useful to limit the number of features, such as the selection of specific alleles most frequently found in a population to be used in broad screening arrays. A second such approach could be the use of supertype prototype HLA molecules compatible with a set of several HLA alleles. A third approach would be through customization of the arrays, by having many different arrays of single alleles and combining them according to the HLA types of the individuals being tested.\nAn issue that sets immunomic microarray data apart is the availability of vector-valued response profiles (in the case of peptide-MHC microarrays). The statistical challenge here is reminiscent of the data analysis problem in the engineering field of remote sensing [105] , where different materials have characteristic vector responses, called spectral signatures. In the case of immunomic data, the analogous notion to the spectral signature is the cytokine profile associated with a given epitope and T cell population; see Figure 4 for an illustration. One simple technique to address the data analysis problem for multicolor immunomic data is to combine the responses into one long feature vector, by juxtaposing the individual cytokine response profiles for each epitope, with the caveat that there may be systematic correlation among the features in the resulting feature vector.\nThe large number of features that can be measured simultaneously with microarray technology also presents a challenge. On one hand, it is likely that a large number of irrelevant features will be present; on the other hand, the scientist would like to work with a small number of strong, relevant features that can be used for diagnostic/prognostic panels, or as the basis for further biochemical validation studies of the mechanisms involved. This problem of feature selection also arises due to a fundamental limitation in statistics, sometimes called the ''curse of dimensionality'', according to which the existence of a large number of features necessitates an even larger (exponentially larger) number of samples to achieve consistent and accurate results. As the number of patients in microarray-based studies is severely restricted by factors such as the cost of the technology and difficulties in patient enrollment, it is almost always the case that only a small number of samples are available. Thus, only a small number of features at a time can be considered. The recommended approach to feature selection is to consider combinations of m features at a time, do classifier design based on the feature set under consideration, and use an estimate of its probability of error as the performance score. Application of this type of analysis to immunomic microarray data would allow the identification of sets of epitope-specific immune responses, associated for example with distinct disease states. However, feature selection presents an explosive combinatorial problem. For example, in an exhaustive selection of sets of three features among 1,000 initial features, the total number of feature sets to be assessed is equal to 166,167,000. If an initial set of 10,000 features is used instead, the number of feature sets of size three to be searched is larger than 10 11 . The complexity of feature selection is especially crucial in functional immunomics applications, since in this case the number of initial features to be considered is huge. The use of high-performance computing architectures, such as large computer clusters, is almost mandatory.\nGiven that a set of features has been selected, one should be able to design a classifier that takes as input microarray data for an unknown sample and generates as output a predicted class label (e.g., clinical outcome, kind of infection, or other conditions). Figure 5 illustrates this approach in a hypothetical functional immunomics application. In this case, there are two class labels, corresponding to control and protected patients, in a situation in which protection is achieved by immunization with an attenuated-virus vaccine for a given infectious disease. The objective is to identify the epitopes that show a discriminatory response between the two groups and are therefore prime targets for rational epitope-based vaccine design. A set of two features, corresponding to epitopes X and Y, have been identified via feature selection among the thousands of microarray probes. For instance, we can imagine a situation in which the protected individuals presented a higher TNF-a response to epitope X, as well as a higher IFN-c response to epitope Y. Based on the response values observed for each patient (note that a patient corresponds to a point in the plane), a linear classifier is designed. This classifier corresponds simply to two decision regions separated by a line. If a future unknown patient has response values that fall in the upper decision region, he/she is likely to be a protected patient, provided that the classifier has a small probability of error. In this case, the responses to epitopes X and Y (in terms of the TNF-a and IFN-c cytokines) characterize immunological memory induced by the attenuated-virus vaccine: large response values to both epitopes X and Y indicate protected patients (note that the response of neither epitope X nor epitope Y by itself is a good discriminator in this example, indicating the need to consider the multivariate, combined effect of both responses). Note, in Figure 5 , that the apparent error rate (i.e., the number of misclassified sample divided by the total number of samples) is 2 4 20 ¼ 10%; the actual probability of classification error on future data typically exceeds the apparent error rate [106] .\nSystems biology makes use of mathematical modeling in order to provide a theoretical core for biology, analogous to the way that mathematical theories provided that core for physics in the 20th century. The success of engineering and computational methodology in the physical realm is due to the predictive capability of mathematical modeling. We quote from [107] : ''Predictive mathematical models are necessary to move biology in the direction of a predictive science. They are also necessary to the application of engineering methods to translate biological knowledge into therapies with a mathematical and computational basis.'' The large complexity of biological systems, in comparison with most physical systems, makes even more urgent the application of mathematical and computational modeling techniques to them. Dynamical systems provide ''the natural language needed to describe the 'integrated behavior' of systems coordinating the actions of many elements'' [108] , and are also capable of displaying emerging self-order from massively disorganized complexity, which is believed to be a fundamental feature of life. In what follows, we will describe the notion of immunomic regulatory networks, a dynamical system model for immune regulation.\nAn important recent development in immunology has been the discovery of regulatory T cells [103, [109] [110] [111] . These T cells suppress immune responses, helping to stem runaway inflammatory processes and to avoid autoimmune disease. It is thought that this beneficial suppression activity can turn deleterious when it is taken advantage of by pathogens, leading to chronic and abnormal infectious processes. It has been observed that some regulatory T cells are not antigenspecific; these are called natural regulatory T cells [103] . In addition, there exist regulatory T cells, both CD4 þ and CD8 þ , that are antigen-specific and thus epitope-driven [103] . Given the suppressive action of epitope-driven regulatory T cells in conjunction with the promoting activity of epitope-driven helper T cells, it follows that the immunological response to a given epitope may be suppressed or promoted by the immunological response to other epitopes. Thus the notion of regulatory networks arises as a fundamental concept in understanding the functioning of the immune system. In fact, most human diseases are the result of an unbalance in immune system homeostasis.\nIn functional genomics, DNA microarray data is used to infer genomic regulatory networks [112] . For biological and efficiency reasons, gene expression is often quantized to two levels: on and off [113] . The multivariate methods of classification and feature selection discussed in the previous section have proved to be essential in the inference of such Boolean (binary) regulatory networks. By the same token, immunomic microarray data can be used to infer immunomic regulatory networks. As is true for gene expression, one may quantize each epitope response measured with an immunomic microarray at one of two levels-on (immunogenic/responder) and off (nonimmunogenic/ nonresponder)-leading to the inference of Boolean immunomic regulatory networks.\nIn the general case, each node of an immunomic regulatory network represents a combination of the epitope, the cytokine response measured, and the T cell population used as the target; in most cases, each node is in a one-to-one relationship with a single physical spot on an immunomic microarray experiment with a given T cell population. The edges between nodes represent putative regulatory relationships between the cells that respond to the respective epitopes. Figure 6 depicts a simple example with quantized Boolean responses, where peptide-MHC microarrays are used in conjunction with three kinds of T cell targets: CD4 þ helper T cells, CD4 þ regulatory T cells, and CD8 þ cytotoxic T cells. Epitope A is specific to the CD4 þ helper T cells that promote the response to epitope C, which is specific to the CD8 þ effector T cells that produce the actual protective mechanism. In addition, there is an epitope B that activates the CD4 þ regulatory T cells that suppress the effector response to epitope C, thereby producing an antiinflammatory response. In practice, such a model would be derived from microarray data by automatic epitope (feature) selection and determination of predictive relationships (classifier design). In this example, the effector response is activated only in the presence of both help from epitope A and an absence of regulatory response to epitope B. The suppressing response to epitope B is itself promoted by the presence of a response to epitope C, providing a negative feedback mechanism. These relationships can be represented by a wiring diagram and transition rules, depicted in Figure 6a .\nSince the responses have been quantized to two values, on and off, and there are three epitopes, the total number of possible states of the system is 2 3 ¼ 8. Figure 6b depicts the state transition table obtained from the transition rules in Figure 6a . Using this table, one can determine the attractors of the system, which are the states or sets of states in which the system stays in the long run in the absence of external disruptions. For each attractor, there is associated a basin of attraction, containing attractors and transient states, which are the sets of states that lead to the attractor [108] . In the context of biological systems, attractors are a mathematical model for homeostasis. In our minimalist example, we see that there are two different behaviors, corresponding to the two distinct basins of attraction in Figure 6c ; the respective attractors are indicated by dashed rectangles. As can be seen, which of the two behaviors the system is in depends only upon the state of the response to epitope A. If it is off (there is no help), then the system may pass through some irrelevant transient states but it will always tend toward the resting single-state 000 attractor and thus an absence of activity; see the diagram on the left in Figure 6c . If the response to epitope A is on (there is help), then the activity of the system corresponds to that of a cyclic attractor, with the effector DOI: 10.1371/journal.pcbi.0020081.g006 Figure 6 . Example of a Simple Immunomic Network, Consisting of Three Epitopes Epitope A is a promoter (A is specific to CD4 þ helper T cells), epitope B is a suppressor (B is specific to CD4 þ regulatory T cells), while epitope C produces the effector response (C is specific to CD8 þ cytotoxic T cells), while also promoting the suppressing response of epitope B (negative feedback). response being turned on and off cyclically; see the diagram on the right in Figure 6c . This situation corresponds to modulation of the effector response and regulation of the inflammatory response by means of a negative feedback mechanism. Note that when there ceases to be a response to epitope A, the system jumps to the other basin of attraction, and tends to the resting 000 state. The immune response to epitope A in this example determines the behavior of the system, and thus it functions as the master of the overall immunological response, with the individual immune responses to epitopes B and C being slave to it. The concept of master-slave regulatory units is quite important for the understanding of complex regulatory systems and has in fact been considered as a mechanism for genomic regulation (Michael Bittner, unpublished data).\nInference of immunomic regulatory networks from immunomic microarray data constitutes, after proper validation, computational knowledge discovery. There are subtle epistemological issues involved in using data-driven, computer-based methodology to obtain scientific knowledge. As Karl Popper explained in his classic book The Logic of Scientific Discovery [114] , every scientific theory consists of an initial irrational act of creativity (induction) followed by rigorous logical consequences (deduction) and testing of the initial hypothesis. Where is the initial act of creativity, in other words the scientific hypothesis, in computational knowledge discovery? Computers are clearly not capable of irrational creativity. Can this be considered science? We maintain that the answer is yes [115] . In fact, the initial irrational act of creativity is there; it is involved in all the steps of experiment design, selection of patients/samples, and choice of statistical methodology. Once these are settled, the actual data analysis is purely logic deduction via the machinery of mathematical operations, as prescribed by Popper. In this deductive stage the computer plays a critical role, as it facilitates the application of very complex computational methods. Therefore, scientist (and statistician) bias here is in fact unavoidable, as it is in all scientific disciplines. In particular, the term data-driven is really a misnomer in describing computational knowledge discovery.\nFunctional immunomics promises great rewards, both in terms of our basic understanding of the immune system and in disease diagnosis/prognosis and rational epitope-driven vaccine design. Research into the basic biology and statistical methods associated with functional immunomic experiments will lead to the advancement of medical science and public health. Functional immunomics is however still at an early stage of development. In this review we have attempted to provide a coherent vision of this nascent field, and have speculated on future research directions for this technology.\nIn our discussion we have often compared and contrasted immunomics with genomics. Immunomics supervenes on genomics, in the epistemological sense, since immunology ultimately depends on the functioning of genes inside cells, but immunomics has its own independent character and properties. In the same manner that each cell has its own pattern of gene expression that defines its unique cellular properties, each reaction of the cognate immune system to an antigen has its own pattern of epitope-specific responses that define its final outcome.\nThere exists a large collection of mathematical models to describe the immune system [116] . Here, we have proposed Boolean immunomic regulatory networks as a new mathematical model for immune system regulation. This is a dynamical system model, with parameters that can be estimated from immunomic microarray data. A somewhat similar concept was suggested in [102, 117] , where a network model for cytokine action was proposed, albeit without explicit reference to large-scale immunomic technology or regulatory T cell response. In addition, immune system regulatory networks have been previously discussed in the context of genomics [118] . The immunomic regulatory network model may be useful for computational knowledge discovery and simulation of regulatory mechanisms of the immune system in health and disease, which may lead to advances in practical applications (e.g., vaccine design) as well as in the basic scientific understanding of the immune system. \"" }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: The anthrax attacks that followed the events of September 11th, 2001 focused attention on the threat of terrorism with biological agents including variola major, the causative agent of smallpox [1] . There is concern that an attack with smallpox could result in many deaths because of the susceptibility of the U.S. population [2] [3] [4] [5] [6] [7] [8] . A number of issues, such as the amount of virus released and the number of people exposed, need to be considered in order to estimate the impact of a bioterrorist attack with smallpox. However, one key factor in determining the size of an outbreak resulting from such an attack is the transmissibility of smallpox, which can be difficult to estimate. One measure of transmissibility is the effective reproductive rate (R), which is equal to the expected number of infections produced by an infectious host in a real world setting [5, [9] [10] [11] .\nR can be determined in a straightforward manner for individual epidemics and is related to the circumstances, such as population vaccination status, under which the individual epidemics occur. When considering intentional attacks, the greatest interest lies in initial spread, much of which often occurs before an outbreak is recognized. For this reason, we focused on the transmission from index cases to the first generation of secondary cases, which we termed the initial R.\nThe data for this study was obtained from a systematic literature review for reports of smallpox outbreaks. We limited our review to reports of smallpox outbreaks occurring in Europe and North America after 1945 because these societies were much more similar to the modern United States in terms of their demographic, social, and physical structure, than were the pre-World War II western and the developing societies from which most smallpox outbreak reports have emanated. Moreover, population immunity was also relatively more similar to a contemporary US population than might be expected a priori. While early post-war European and North American populations were generally vaccinated in childhood, re-vaccination rates were generally low and few adults had more than remote vaccination [12, 13] . Immunity naturally acquired through infection was also similarly low in both populations because outbreaks were rare. Although terrorist use of smallpox in the 21 st century may have substantially different epidemiological characteristics (see discussion below), the outbreaks analyzed in this paper are the closest actual population experience that may help partially guide modern smallpox control efforts.\nThe following databases were searched for literature on smallpox through 2001: National Library of Medicine 1800 to 2001 (keywords: smallpox or variola), Embase 1974 to 2001, Biosis 1969-2001 (keywords: smallpox or variola virus and human and not in-vitro), Cochrane Controlled Clinical Trials Library (keywords: smallpox or variola) and Defense Technical Information Center 1947 to 2000 (keywords: smallpox). The search was later updated by a PubMed search from 2001 to 2004 (keywords: smallpox or variola). Duplicate titles were removed. Secondary searches included more specific searches for outbreaks in specific countries and sites, and a review of reference lists from publications identified in other searches (Figure 1 ). There was no language restriction. Two authors (SB and VB) independently scanned titles for relevance, and publications chosen by either reviewer were reviewed further. One of the authors (VB) scanned the selected articles to identify post-1945 smallpox outbreaks from Europe and North America. Two abstractors (RB or research associate and VB) independently extracted data from the identified outbreaks. Discrepancies between reviewers were resolved by consensus. Non-English publications were abstracted by one author (RB) or by a research assistant under an author's (VB) direction.\nTo be included in the analysis, the following had to be available: the date of occurrence, and the number of index cases, total cases, cases in each generation, and generations. The following outbreak details were also extracted if available: the number of cases acquired within a hospital, a household, or at a distance (cases not acquired by household or hospital contact); the number of missed cases (cases not diagnosed as smallpox at the time of illness) and deaths; the generation at which the outbreak was identified, and the clinical presentation of the index case. Index cases were classified as having had typical smallpox, atypical smallpox (a milder illness as seen in people with prior smallpox vaccination and/or a rash not characteristic of smallpox), or hemorrhagic smallpox (a usually fatal illness characterized by internal hemorrhage and/or petechiae) [14] . The following data regarding the efficacy of control measures were also extracted if available: the use of ring vaccination (the vaccination of people who were in close contact with a patient with smallpox), case isolation (the separation of suspected cases of smallpox from the general public), quarantine (the restriction of movement in or out of a region that has been exposed to smallpox), mass vaccination (the vaccination of an entire region), and/or prophylactic vaccinia immune globulin administration [15] [16] [17] .\nThe number of outbreaks over the period studied was small (total of 45 outbreaks) and the circumstances varied widely. Therefore, a descriptive and qualitative analysis was emphasized. For this analysis, the initial R was meas-ured by dividing the total number of first generation cases by the number of index (zero th generation) cases. In addition, several outbreaks had sufficient details to be classified by setting. Because hospitals were often the centre of outbreaks, setting was defined according to the proportion of secondary cases (non-index cases) acquired within a hospital [12, 13, [18] [19] [20] . After examination of the distribution of case locations across outbreaks, hospital outbreaks were defined those in which 70% or more of secondary cases were hospital acquired, mixed outbreaks as those in which 30% to 70% of secondary cases were hospital-acquired, and community outbreaks as those in which 30% or less of secondary cases were hospitalacquired.\nBased on the results of the primary searches, 5,976 titles were scanned; 1,389 of these were selected. Despite initial de-duplication, 10 of these were duplicates (Figure 1 ). Secondary searches identified 14 additional publications. Of the resulting 1,393 publications, 1219 (87%) were obtained; 92 journal citations (7% of the selected publica-tions) and 41 government reports (3% of the selected publications) were unobtainable.\nForty-nine post-1945 European and North American outbreaks were identified. Thirty-six of these were included in a single World Health Organization (WHO) publication [21] and prior work by T.M. Mack [12] . Several outbreaks were documented in multiple publications (Tables 1 and 2 ). Four outbreaks were excluded. The source of the first outbreak could not be confirmed as the outbreak may have resulted from the importation of smallpox on a rug (Union of Soviet Socialist Republics or U.S.S.R, 1959). The second and third outbreaks occurred under unique circumstances; one occurred aboard a ship (Poland, 1962) and the other may have been the result of testing aerosolized smallpox (U.S.S.R, 1972) [21] [22] [23] . A fourth outbreak was excluded because the number of cases in each generation was not documented; therefore, the initial reproductive rate (R) could not be derived from the data (United States or U.S., 1946) [24] . The remaining 45 original outbreaks occurred over a thirty-year period in different countries and under different circumstances. The majority of reported outbreaks occurred in three coun- (Tables 1 and 2 ). Most resulted from involved index cases imported from endemic countries, but two outbreaks were the result of laboratory exposure [25, 26] .\nAlthough published or listed as single outbreaks, four outbreaks included multiple epidemiologically distinct components, such as occurred when a case travelled to a different region resulting in a secondary outbreak in the new area (see reference column and footnotes for Table 2 ). The components of these four outbreaks were treated as 10 separate outbreaks so that an effective total of 51 outbreaks were analyzed [21, [27] [28] [29] . For all 51 outbreaks, available information included the total number of generations and cases, the number of cases in each generation, the duration of the outbreak, and the number of deaths (Tables 1 and 2 ). Thirty of the outbreaks could also be categorized by setting and had detailed information on the generation that the outbreak was identified, control measures, clinical presentation of the index case, and the number of missed cases ( Table 2) .\nThe median initial reproduction rate (R) across all 51 outbreaks was 2 with a range of 0 to 38 (Table 3, Figure 2a ). About half had an initial R of 1 or less, and over two-thirds had an initial R of 3 or less. The median duration, as measured by number of generations, was 1 with a range of 0 to 9 (Table 3, Figure 2b ). About a third did not extend beyond the index generation, and nearly three quarters lasted for 3 or fewer generations. The median outbreak size, as measured by the total number of cases, was 4 with a range of 1 to 134 cases (Table 3, Figure 2c ). About half involved 3 or fewer cases, and two-thirds involved 15 or fewer cases. The median number of deaths was 1 with a range of 0 to 26 (Table 3, Figure 2d ). About two-fifths involved no deaths, and three quarters involved 3 or fewer deaths.\nOutbreaks with greater initial R-values tended to be larger and longer. For example, in outbreaks with an initial R of 5 or less versus more than 5, median values for the total number of cases and generations were 2 versus 23 and 1 versus 2.5 respectively.\nThere was no temporal trend in the amount of detail reported, but the thirty outbreaks reported in detail typically had a larger R (median 2 vs.0), lasted more generations (median 2 vs. 0), and had more total cases (median 13 vs. 1) and deaths (median 2 vs. 0) compared to lessdetailed outbreaks (Table 3 , Figures 2a-d) . In the detailed outbreaks, most of the index cases had atypical (mild) or hemorrhagic disease. Because these clinical presentations of smallpox are less common, many of these index cases were missed or not diagnosed with smallpox at the time of illness. The majority of index cases were infected \nThe median initial R across the 30 detailed outbreaks was 2 with a range of 0 to 38 (Table 3 , Figure 2a ). About a third had an initial R of 1 or less, and about two thirds had an initial R of 3 or less. Outbreaks that remained in the community had a lower initial R (median 1) than those that were hospital based from the first generation (median 12) ( Table 3 , Figure 3a ). Mixed outbreaks, which generally started in the community and later spread through hospital contacts, had an intermediate initial R (median 2) ( Table 3 , Figure 3a ). The initial R was smaller when the index case(s) had a typical versus atypical or hemorrhagic presentation (medians 1 vs. 4 or 5, respectively), and when the index case was identified as smallpox versus unrecognized (median 1 vs. 5) ( Table 3 , Figures 3b and 3c) . The effect was most pronounced in hospital outbreaks -median initial R's for identified versus unidentified index cases in hospital outbreaks were 3 versus 15, but the same values for community and mixed outbreaks were 1 versus 4, and 2 versus 2, respectively.\nThe median duration for 30 outbreaks was 2 generations with a range is 0 to 9 generations (Table 3, Figure 2b ). About a third did not extend beyond the first generation, Table 2 ) analyzed by components:\n(1) Outbreak from Marseille, France (1952) broken into two components: Entries 3 and 15 [27] .\n(2)Outbreak from Cardiff, U.K.(1963)broken into three components: Entries7, 24 and 26 [21, 28] .\n(3) Outbreak from Poland (1963) broken into three components: Entries 8, 17 and 27 [41] .\n(4)Outbreak from Kosovo, Yugoslavia (1972) broken into two components: Entries 10 and 19 [21] . Footnotes for Table 2 : a Nature of the index cases was not clear from the text (limited information) [27] . b Total number of cases listed as 74, consistent with text [38] and second reference [27] . c Total number of cases listed as 5 [28, 34] ; 6 is listed in World Health Organization Table 23 .4 [21] . d Proportion of hospital acquired cases listed as 0.44 because three nurses infected as a result of household exposure and were setting excluded as hospital contacts [21, 36] . e Total number of cases listed as 1 [37] ; 2 is listed in World Health Organization Table 23 .4 [21] . and another third lasted for only through the second generation. Outbreaks that remained in the community lasted a median of 1 generation, while those that were hospital-based lasted a median of 2 generations and those that were mixed lasted a median of 3 generations (Table 3) . Outbreaks tended to be shorter when the index case was typical compared to atypical or hemorrhagic (median number of generations 1 vs. 3 vs. 2, respectively) and when the index case was identified (median number of generations 1 vs. 3) ( Table 3 ). The duration of hospital outbreaks was not affected by the identification of the index cases, but the duration of mixed and community outbreaks lasted up to 6 and 9 generations, respectively, when index cases were unidentified.\nThe median outbreak size, as measured by the total number of cases, was 13 with a range of 1 to 134 cases (Table 3, Figure 2c ). There were approximately equal proportions of small (total number of cases less than 5) and large (total over 20) outbreaks ( Table 2 ). Outbreaks that remained in the community involved a median of 2 cases, while those that were hospital based involved a median of 20 cases and those that were mixed had a median of 22 cases (Table 3) . Outbreaks tended to be smaller when the index case was typical versus atypical or hemorrhagic (median number of cases 2 vs. 27 vs. 16.5) and when the index case was identified (median number of cases 3 vs. 27.5) ( Table 3 ). The outbreak size was similarly small across all 3 settings when the index case was identified and similarly large across the settings when it was not.\nThe median number of deaths was 2 with a range of 0 to 26 (Table 3 , Figure 2d ). About one-fifth involved no deaths, and four fifths involved 4 or fewer deaths (Table 2) . Community, hospital based, and mixed outbreaks had medians of 1, 4, and 3 deaths, respectively (Table 3) ; however, case fatality rates were 0.24, 0.20 and 0.17. The number of deaths was smaller if the index case was identified (median equals 1 vs. 6, Table 3 ). The case fatality rate was highest if the index case presented with hemorrhagic-type smallpox.\nThere was a strong association between typical presentation and early identification of index cases: index cases were identified as smallpox in 9 of 9 outbreaks when the presentation was typical, 3 of 13 when it was atypical, and 2 of 6 when it was hemorrhagic. This relationship aids in the understanding of outbreak severity. For example, 93% of outbreaks had greater than 10 cases when the index case was not identified. Moreover, in all of these larger outbreaks, the index cases were atypical or hemorrhagic. When the index case was identified, most outbreaks had less than 10 cases regardless of whether the index case was typical, atypical, or hemorrhagic.\nDescriptions of smallpox transmission are important to biosecurity planning, especially since some planning efforts have posited outbreaks resulting in numbers of cases -hundreds, thousands, or more -that are very large relative to the clinical experience shortly before eradication of the natural disease [32] . In a systematic review designed to provide an understanding of smallpox transmission patterns, we identified and analyzed what were effectively 51 smallpox outbreaks from post-1945 Europe and North America. In these outbreaks, we found a median of 4 total cases and 1 death.\nThe characteristics of the outbreaks varied greatly: one outbreak with an initial R of 11 involved 134 people and caused 26 deaths over only 3 generations of spread; another outbreak with initial R of only 2 involved 28 cases but lasted 9 generations. However, most outbreaks were small and contained within a few generations: 31 percent involved only one case, 41 percent caused no deaths, and 31 percent had an initial R of 0 (i.e., no first generation cases).\nExamination of outbreaks within categories of setting, identification of the index case, and clinical characteristics provided additional insights. By reviewing original publications, we were able to categorize and analyze many outbreaks by setting. For example, were able to review the original publications for eight outbreaks documented by the WHO [25, 26, 28, [33] [34] [35] [36] [37] as well as, identify thirteen additional outbreaks [20, 27, [29] [30] [31] [38] [39] [40] [41] . Several of these outbreaks also had epidemiologically distinct components, resulting in 51 effective outbreaks for analysis.\nMany (six out of eleven) of the hospital outbreaks were only found in the above-mentioned additional outbreaks [20, 27, 29, 30, 38, 39] . Hospital outbreaks had higher initial reproductive rates than either community or mixed outbreaks, and had more cases and deaths than did community types. Moreover, the interaction of identification and setting in the studied outbreaks reveals a pattern similar to that seen in frequently unrecognized infectious diseases, such as tuberculosis and, more recently, SARS [42] . When index cases were identified, control is established early and both the median initial R and the number of generations were similarly low in all 3 settings. When the index cases were unidentified, a large increase in the median initial R was seen only in the hospital setting and a large increase in the number of generations was seen only in community and mixed settings. This is consistent with the observation that, within the close quarters of an institution, the transmission of smallpox is relatively effective before identification and containment measures are quite effective after identification. This impression is reinforced by examination of the two shipboard out-breaks that were excluded from the analysis. In these outbreaks, the initial R's were 6 and 25, respectively, but recognition was followed by control within two generations [22] .\nIn community outbreaks, transmission is intrinsically less effective. The consequence of missed initial identification in the community was not an elevated initial R, but an increase in the length of the outbreaks. The longer outbreaks may be due to a decrease in the effectiveness of containment measures after initial spread in the community, possibly because of the greater difficulty of tracking and managing cases in that setting. Nonetheless, all of the 30 outbreaks reported in detail were brought under control, generally by case isolation and ring vaccination.\nOther measures were rarely used (mass vaccination was used in the New York City, 1947 outbreak and mass vaccination and quarantine were used in the Kosovo, 1972 outbreak [21, 30] ) and there is limited information on the efficacy of these additional measures. It should also be noted that newer treatment modalities, such as Cidofovir, have been shown to be protective in animal studies against vaccinia and other orthopox viruses (monkeypox and cowpox) after exposure [43, 44] .\nOutbreaks beginning with an atypical or hemorrhagic versus a typical presentation of the index case had more cases, a higher initial transmission rate, and were somewhat longer. This is related to the ease of initial diagnosis: only non-typical cases were mis-identified, and outbreaks in which the index case was mis-identified were generally larger, deadlier, longer, and had a median initial R of 5 compared to 1 if the case was identified. We speculate that the large difference in initial R is because, when the index case is identified, this parameter is effectively the initial reproductive rate with early institution of control measures. However, it is clear that the types of outbreaks seen in developed countries in the second half of the 20th century were well and quickly controlled.\nDespite searching several databases, using broad searches, scanning nearly 6,000 titles, and reviewing papers in several languages, our requirement that included epidemics involve post-World War II western populations limited us to identifying only 45 separate outbreaks, some of which were divided for analysis. The majority of the outbreaks were from only three countries, suggesting that outbreaks from certain countries were more likely to have been documented. The 30 outbreaks reported in detail were larger than the 21 that were briefly reported; it is probable that smaller outbreaks were not routinely published in detail because outbreaks were still relatively common and smallpox was still endemic in many parts of the world. This apparent publication bias, however, suggests that there may have been many unpublished small outbreaks and that this analysis may overestimate the transmission potential of smallpox because it is based only on published reports.\nThe recent literature contains several estimates for the reproductive rate of smallpox. It is difficult to directly compare our findings and estimates for initial R, the initial effective reproduction rate for smallpox, to these other studies. Two of the studies do not use contemporary western outbreaks. One estimates the basic reproductive rate, (R 0, a theoretical parameter defined as the expected number of new infected hosts that an infectious host will produce in a large randomly mixed population of susceptible individuals [9] ), rather than effective reproduction rate. Specifically, Gani and Leach used epidemic modelling of data from 19th Century European smallpox outbreaks to estimate a R 0 of 3.5 to 6.0 [19] . Eichner and Dietz obtained an R of 6.9 from data on a 1967 outbreak in Nigeria [5] . Meltzer et al examined post-1961 outbreaks from countries around the world in estimating an R of 3 [8] .\nOur estimate of the overall initial R is quite comparable to Meltzer et al., but use of any single point estimate for R 0 or initial R in policy analysis ignores a main lesson of this review. This work clearly shows that the pattern, speed, and duration of an outbreak's spread vary widely according to the specific circumstances surrounding an outbreak. Different smallpox release scenarios should be expected to yield different results in part because the parameters and modified natural history of the outbreak itself will vary with the scenarios. For example, our previous work modelled the outcome of terrorist attacks under different scenarios [4] . These scenarios included community based outbreaks resulting from infected cases riding a busy transit system and the intentional release of aerosolized smallpox in airport terminals, and a mixed outbreak resulting from the release of aerosolized smallpox in a large office building. Although adjustments had to be made for a poorly vaccinated population and healthcare workforce, the understanding of smallpox transmission patterns in hospital, mixed and community settings, provided important insights into how smallpox may spread under different attack scenarios.\nIn a contemporary US population, one might expect transmission from introduced smallpox to be higher than we found to be typical. Population density is higher; mobility is greater, and, though more similar that one might think, immunity in the contemporary U.S. population is likely somewhat lower than in the populations studied. The healthcare workforce is also poorly vaccinated and inexperienced with smallpox. This will decrease the human resources available for care and containment, and increase the likelihood that initial cases will be missed. Counterbalancing this is the fact that few index cases will be vaccinated recently so the likelihood of more easily recognized typical cases is much higher. As the outbreak scenarios of greatest concern involve intentional release, outbreaks could be larger and more obvious than those reviewed, or could involve strains selected or engineered to be more virulent or contagious.\nFinally, the outbreak from Kosovo deserves notice because this region is not as developed or as wealthy as most of the other countries studied. This outbreak was the largest identified for this review (N = 176 for both combined hospital and mixed components). Moreover, the hospital and mixed component of this outbreak had the second highest (R = 38) and highest (R = 11) initial reproductive rate, and the hospital component lasted 9 generations (median number of generations for a hospital outbreak = 2). This suggests that a smallpox outbreak in a less developed country with limited resources for healthcare, disease surveillance, and case isolation could be potentially more devastating than a bioterrorist attack in a Western/industrialized country [44] .\nIt is clear that most post-World War II outbreaks in Western countries were small and had low transmission rates. This was almost uniformly the case when the index case presented with typical smallpox and was recognized early. However, exceptions were common and heterogeneous, particularly when (usually atypical or hemorrhagic) index cases were not initially recognized. Initially unrecognized community outbreaks lasted several generations despite low initial R values. This suggests that control measures were less effective in this setting, perhaps because of the difficulty tracing and vaccinating all cases and contacts. In contrast, unrecognized hospital outbreaks were controlled quickly despite high initial R values, suggesting that control measures worked very well in a closed, contained setting. Early detection, particularly of patients who do not present with typical smallpox, coupled with early implementation of control measures decreases both the duration and size of outbreaks in all settings.\nPublish with Bio Med Central and every scientist can read your work free of charge " }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: between countries. One of the first case definitions used for national disease reporting was the case definition for AIDS published by the Centers for Disease Control and Prevention (CDC) in 1982 [2] . In 1985 Sacks published a survey among all 50 US states, Puerto Rico, and Washington, DC, that revealed important variations in the case definitions between the different states, and concluded the necessity to unify case definitions if surveillance data between states are to be compared [3] . In 1990 the CDC in collaboration with the Council of State and Territorial Epidemiologists published an edition of case definitions for public health surveillance [4, 5] .\nSince then case definitions have become an important tool of other national surveillance systems and international surveillance networks. Koo and colleagues have analyzed surveillance data for Cholera in Latin America and have described the importance of uniform case definitions to make data comparable between countries [6] . In 2003 the European Union (EU) case definitions for the European networks have reached obligatory status for the member states reporting to the EU [7] . During the SARS epidemic the case definition had a major impact on whether and how countries were considered affected or not, resulting in severe political and economic consequences for a number of countries [8] .\nCoggon and colleagues have demonstrated the difficulties of determining optimal case definitions if a satisfactory diagnostic gold standard is lacking [9] . In sharp contrast to the importance of case definitions hardly any research has been published on the performance of surveillance case definitions. Studies are rare on how local health departments and other health professionals are able to understand case definitions and to what extent case definitions are unambiguous enough to really assure reliability. To our knowledge, the only publication investigating this issue was focused on case definitions for nosocomial infections: Gastmeier and colleagues had investigated how uniform the case definitions of the nosocomial infections surveillance system in Germany had been applied by different investigators using a set of 60 case studies [10] . Due to the general importance of case definition for public health surveillance and the current need for harmonization in international surveillance systems we conducted a systematic evaluation of the national case definitions with the objective to identify general as well as specific criteria and recommendations for improvement of case definitions.\nGermany is a federal republic with 16 states subdivided into 440 counties. As in many countries the local (county) health departments (total number: 425) are the primary recipients of infectious disease notifications made by physicians and laboratories. Local health departments verify the incoming notifications and assess the need for public health action. Local health departments use one of five software products on the market to classify the case reports according to the national edition of case definitions and to report these cases electronically to the state health department. From there the report is being forwarded to the Robert Koch Institute (RKI), the federal institution in charge of national infectious disease surveillance in Germany [11] .\nThe edition of national case definitions for all notifiable infectious diseases was introduced in Germany in 2001, following the implementation of a new law to control infectious diseases (Infektionsschutzgesetz, IfSG) [12] [13] [14] . The IfSG determines the set of diseases and pathogens to be notified by physicians and laboratories throughout the Federal Republic of Germany. The five eastern states, which formerly belonged to the Democratic Republic of Germany (East Germany) and the State of Berlin have enacted complementary rules that make certain diseases additionally notifiable within the state jurisdiction, that are not notifiable in all of Germany.\nThe case definitions were developed by the RKI, using the delphi method including the expertise of state epidemiologists, national reference laboratories and medical and scientific associations for the specific diseases. The case definitions for infectious conditions under public health surveillance published by the CDC were also taken into account [5, 15] . After having published the IfSG case definitions in the fall of 2000 to be implemented with the beginning of 2001 the RKI also published additional case definitions in January 2002 for some of the diseases exclusively notifiable in the eastern states jurisdictions [11, 16] . From June 2002 to September 2003 we had conducted a systematic evaluation of the case definitions with the purpose to revise them by the end of 2003.\nThe German case definitions are divided into three types of evidence: Clinical picture, laboratory detection, and epidemiological confirmation. The types of evidence are specifically defined for each disease (see table 1 ). Based on whether or not requirements for these three types of evidence are fulfilled a case is classified into five categories. In the revised 2004 edition of case definitions these categories are named: A) clinically diagnosed illness (neither epidemiologically nor laboratory-confirmed), B) clinically and epidemiologically confirmed illness (not laboratory-confirmed), C) clinically and laboratory-confirmed illness, D) laboratory-detected infection not fulfilling clinical criteria, E) laboratory-detected infection with unknown clinical picture. (In the 2001 edition of case definitions these five categories were named slightly differently) For most notifiable diseases only categories B, C, D and E are reportable from the local health department to the next level, requiring at least laboratory detection of the pathogen or epidemiological confirmation. For some exceptions (e.g. tuberculosis, polio, measles, Creutzfeldt-Jakob disease), cases are also reported from the local health department to the next level if category A -the clinical picture alone -is fulfilled.\nIn June 2002 we conducted a Round-Robin test in analogy to the established quality control procedure of laboratories and other testing units [17] . Round-Robin tests are mainly used in proficiency tests in order to determine laboratory performance by means of comparing tests on identical items by two or more laboratories in accordance with predetermined conditions [18] .\nWe asked each local and state health department to classify a selection of 68 written case examples on the basis of the case definitions that were implemented in 2001 (2002 respectively for disease only notifiable in East German States). While proficiency tests generally intent to assess the ability of laboratories in finding identical results, we applied this method to assess to which extend the case definitions were unambiguous enough to assure identical classification by the health departments.\nWe applied four different outcome variables in our analysis: 1) Disease identification: A disease was defined as being correctly identified if the participant of the Round-Robin test was able to identify the correct disease of the case example.\n2) Case categorization: A case example was considered correctly categorized if the participant classified the case example with the correct disease and the correct case definition category as defined in the gold standard.\n3) Reporting: The decision on reportability was considered correct if a case that should have been reported to the Clinical picture: Clinical picture compatible with salmonellosis, characterized by diarrhea, abdominal pain, malaise, vomiting, fever. Salmonella can also cause infections outside the intestinal tract (for example: arthritis, endocarditis, pyelonephritis, septicaemia). Laboratory diagnosis: Isolation (culture) of pathogen from stool or other clinical material (e.g. blood, urine). The identification of serogroup has to be attempted.\nClinical picture • Clinical picture of acute salmonellosis, defined as at least one of the following four symptoms: diarrhea* • cramp-like abdominal pain • vomiting • fever* additional information: Samonella can also cause generalized (septicemia) and localized infections outside the intestinal tract (for example: arthritis, endocarditis, pyelonephritis). These should in case of an acute infection also be reported. The reactive arthritis, which may also be caused by Salmonella infection, is not to be reported. Laboratory diagnosed Positive finding using the following method: • Direct detection of pathogen: isolation of pathogen (culture) Additional information: Results of identified serogroup and lysotype should also be reported. Epidemiological confirmation Epidemiological confirmation, defined as at least one of the following three constellations while taking into account the incubation period (about 6 to 72 hours):\n• Epidemiological link to another laboratory-diagnosed human infection through ❍ Person-to-person transmission OR ❍ Same source of exposure (e.g. animal contact*, food*) • Consumption of food (including drinking water), for which Salmonella spp. was laboratory-detected in non-consumed food.\n• Contact to animal (e.g. poultry) with a laboratory-detected infection, or contact to its secretions or consumption of its products (e.g. eggs). * terms marked with an asterix are defined in more detail in a glossary of the case definitions next level would have been forwarded according to the case definition category, given that the correct disease was identified. Inversely decision on reporting was also seen to be correct if a case that should not have been reported to the next level was in fact classified in a way that the case would have been held back. However, cases forwarded with wrong disease identification (see above) were a priori considered incorrect. Thus reporting was based on the question whether the case needed to be forwarded to the state level or not, which is a direct result of the disease identification and the case definition category. Sensitivity of reporting was defined as the number of cases that would have been correctly forwarded divided by the number of cases that should have been forwarded according to the gold standard. The positive predictive value of reporting was defined as the number of cases that should have been forwarded among those that would have been forwarded. Precision of reporting is defined as the number of cases that would have been either correctly forwarded to the state level, or would have been correctly held back at the local health department level, divided by the total number of case examples. Unless stated otherwise, reporting precision was the outcome parameter used in the following analysis.\nTo specifically assess the effect of different styles in formulating case definitions, a fourth outcome variable was used. The clinical classification was considered correct if the part regarding the clinical picture was classified according to the gold standard, regardless whether other parts of the case definition were correctly classified or not. This analysis was done to compare case definitions with narrative description of the clinical pic-ture (as in all former IfSG case definitions) to case definitions with a more explicit check-list format of clinical criteria, that was implemented for diseases additionally notifiable in specific states and for the new IfSG case definitions.\nThe case examples consisted in facsimile excerpts of one or more of the following sources: laboratory report form, physician form, and protocol of the patient interview [see additional file 1]. The case examples were created based on real cases that have come to the attention of the RKI in the quality control process and in the information service hotline that the RKI is offering to the health departments.\nThe case examples were pre-tested among epidemiologists within the RKI and among epidemiologists and public health nurses in the state and local health departments. \nAfter the data of the respondents had been analyzed, the classification originally intended while creating the case definition, was challenged with the results of the respondents. Three epidemiologists then reassessed each individual case example and re-examined whether the classification originally intended was still justified. Based on this process the gold standard was defined for each case example.\nWe compared the responses to the established gold standard and stratified by the following variables: health department being in an East German versus a West German state, disease of the case example, whether or not physicians participate in routine quality control of case reports (versus this being done exclusively by public health nurses), institutional level (local health depart-ment, state health department, RKI), acceptance and style of case definitions (check-list vs. text) and software used at local health department. Because of the selection and distribution of case examples described above, we conducted the individual analyses for each group. After univariate analysis we conducted a multivariate analysis using SPSS 13.0 for Windows (Version 13.0.1).\nThe distribution of the classifications was compared to the gold standard, in order to identify common discrepancies. Based on these discrepancies we identified which part of the case definition was affected and identified specific aspects of the case definitions that had repeatedly been interpreted differently by the participants, indicating failure of the case definition to be unambiguous and reliable. These aspects were then summarized in order to deduct commonalities which could then lead to specific recommendations on how to improve this particular case definition and also on how to improve formulation of case definitions in general.\nIn May 2002 -simultaneously with the Round-Robin test -we conducted a written survey addressed to all 425 local health departments in Germany. Among various questions on the structure and equipment of the local health departments, and their experiences with the new IfSG, we also asked about the profession of the person who had actually filled the questionnaire and about his or her attitudes and experiences towards the case definitions. \nThe multivariate analysis was limited to data from the local health departments and without additional case examples for the East German states (n = 5995). Only statistically significant associations are mentioned in the following.\nThe disease of the case examples was for all groups significantly associated with reporting (p < 0.001 in group 1, 2 and 4, p = 0.022 in group 3).\nLocal health departments using the RKI-software showed a higher chance to identify the disease (disease identification) of the case example according to the gold standard compared to health departments using any of the commercially available software programs (group 2: OR = \nThe administrative level at which the respondents worked, was significantly associated with the outcome reporting.\nFor the analysis we used all cases of set A and set E (without the additionally diseases for the East German States, n = 2213). Adjusted for the diseases the chance of correct reporting to the next level was 1.5 times higher in cases done by state level staff compared to those done by local health department staff (OR = 1.52, CI: 1.14 -2.02).\nThe following observations have been made in the qualitative analysis of the responses:\n• The concept of epidemiological confirmation was not well understood. For example travel in endemic countries was equivocally seen as an epidemiological confirmation (e.g. haemorrhagic fever and travel to Egypt). Re-evaluation of the case definitions showed that in fact there was only a vague definition of the epidemiological confirmation.\n• Participants appeared to have difficulties in deciding whether all clinical signs and symptoms mentioned in the case definition had to be existent in a case, or whether they were only listed as descriptive examples.\n• Case examples of diarrheal disease without any evidence of a specific pathogen, were frequently classified as salmonellosis.\n• Laboratory findings with only one elevated antibody value in serum were repeatedly classified as laboratory detection although the case definition required a rise in antibody level.\n• In some case definitions detection of the pathogen is only accepted if the detection was done in specific materials (normally sterile material such as blood for detecting N. meningitidis). This limitation was frequently neglected.\n• Some of the information in the case definition intended to serve as additional background information was mistakenly used as selection criteria (e.g. statement that clinician described rash as \"very typical\" for measles, but fever was missing).\nWhen asked about the availability of the case definitions, 395 (99%) of 398 local health departments responded that the case definition were accessible at the work place.\nThe case definitions were seen as useful by 377 (95%) of 397 health departments who answered this question and not useful by 20 (5%). The clarity of the individual sections of the case definitions was rated differently: The section on the clinical picture of the case definitions was seen as unambiguous in all case definitions by 72 respondents (18%), in the majority of case definitions by 305 (76%), in the minority by 20 (5%), and in none of the case definitions by one (0.3%) of the respondents (n = 398 respondents). The section on the laboratory confirmation of the case definitions was seen as unambiguous in all case definitions by 137 respondents (34%), in the majority of case definitions by 248 (62%), and in the minority by 11 (3%) (n = 396).\nThree-hundred and three (87%) of 347 health departments stated that case classifications were done exclusively or primarily by public health nurses. With respect to the case examples presented to the participants, 220 (55%) of 396 respondents (from the local health departments) stated that the case examples were realistic.\nThe results of our evaluation have shown that although case definitions may appear to be clearly defined, they may be interpreted quite differently by their users, which may result in severe misclassifications and reduced sensitivity and positive predictive value. This study is believed to be the first to systematically assess these effects Also the complexity of the case definition itself is likely to affect reporting precision. Unfortunately much of the complexity of the case definition is a result of methodological limitations of available laboratory tests and cannot be influenced. The case definition system with its three different types of evidence leading to five different categories may appear very complex and less intuitive that the classical categories of \"suspect\", \"probable\" and \"confirmed\". The detailed differentiation of the German case definitions however enables us to apply computer algorithms in order to translate these to the EU case definitions and thus make the data compatible to the standards of various European surveillance networks and to WHO reports.\nReassessment of the gold standard after receipt of the responses resulted in modifications of 5 of the 68 case examples. This procedure took place in an initial review process of gold standard before the actual analysis was done. We believe it was legitimate and necessary in order to correct for biases caused by unforeseen ambiguity of the case examples.\nThe software used at the local health department was significantly associated with the quality of the data in only some subgroups and outcomes. Apparently the software is not a very strong determinant in the given study design, although our experience in implementing the electronic surveillance system in Germany showed that commercially available software products often do not fully implement the standards published by the RKI for data transmission software or they do so with a delay of several years [21] .\nThe other interesting finding is that the administrative level of the participants was significantly associated with the outcome: Participants from state health departments had a significantly higher rate of agreement with the reporting gold standard than the participants from local health departments. This might be explained by the fact that staff at the state level is generally higher trained in epidemiology and infectious diseases than local health department staff and they are routinely involved in quality control of incoming case reports and also training and supervision of local health departments' staff. \nAll the observed quantitative effects and their propagated explanations merge into the one main conclusion: Case definitions must be very carefully formulated in order to assure their unambiguous interpretation by local health department personnel. The detailed evaluation of our study has resulted in a substantially revised edition of the German case definitions [23,24]:\n• We rephrased the case definitions in a check-list format indicating clearly how many of the symptoms and signs had to be fulfilled in which combination.\n• Some diseases previously jointly described in one case definition were defined separately (Dengue was separated from other haemorrhagic fever; hemolytic uraemic syn-drome was created new, separately from EHEC and Shigella.)\n• We rephrased the definitions in a way that for serologic confirmation the necessity for two samples is clearly apparent at the beginning of the phrase.\n• The material in which the pathogen has to be detected is now highlighted and is only listed if it is relevant for the case definition.\n• A glossary now defines the expressions that are being used repeatedly in the case definitions\n• The case definitions are now limited to criteria relevant for the decision process. All additional explanatory information is clearly indicated as such in a separate section of the case definition • The evidence type \"epidemiological confirmation\" was completely redesigned and replaces the previously used term \"epidemiological link\". The accepted types of epidemiological links are now specified individually for each case definition.\nOne practical implication, that is supported by this analysis is, that software used at the local health department must be designed with strict accordance to the case definitions using identical terminology and structuring which would have been more easily archived if all local health departments had been equipped with one identical software system developed within or under supervision of one institution. Possibly other countries in the process of developing or implementing new electronic surveillance systems might want to learn form this experience [21,25].\nThe case example book, which resulted from this study, constitutes a detailed feed back for the participants of the study and is now being used as training material for public health nurses.\nWe have demonstrated that rigorous reduction of case definitions to testable yes/no-criteria in a check-list format is likely to improve their reliability. Reducing the differential diagnostic complexity of a disease to a limited number of yes/no-criteria, is a major challenge, but it also carries the benefit of facilitating computerized testing algorithms for quality control and for case classifications.\nAs the reliability of epidemiologic surveillance largely depends on the reliability of its case definitions, it is essential to create and revise case definitions based on systematic evaluations [9] . Most of the basic principles for the revision of the German case definition edition deducted from this analysis may also be applicable for case definitions in other countries (such as the United States, Ireland, Sweden, Mexico) or international networks (EU, WHO) as they share the same structural and editorial characteristics that we identified to be problematic in the first edition of the German case definitions [4, 7, 8, 26, 27] . We therefore believe that our findings are highly relevant for many national and international surveillance systems." }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: Nosocomial pneumonia (NP) is defined as parenchymal lung infection, occurring after the first 48 hours of hospital admission [1] . It accounts for 13-18% of all hospitalacquired infections, but leading cause of death from nosocomial infections [2] . It is a major threat to patients admitted intensive care units (ICU) and receiving mechanical ventilation (MV). In the recent studies, it was shown that ventilator associated pneumonia (VAP) was the most common infectious complication among patients admitted ICU [3, 4] . It results in high mortality and morbidity, prolonged lengths of hospitalisation, and also increased cost of hospitalisation. The mortality rates for VAP range from 20% to 75% according to the study population [5] [6] [7] [8] [9] [10] [11] [12] [13] . The average excess cost of pneumonia was estimated to be U.S.$1255 per patient in 1982 (14) , U.S.$2863 per patient in 1985 [15] , and in a recent study in 1999, it was >U.S.$40 000 per patient [16] .\nDespite the clinical experience and major advances in diagnostic techniques and management, VAP remains a significant problem for intensivists. In this review, epidemiology, diagnosis and, mainly, infection control of VAP were discussed.\nIn different studies, the incidence of VAP was reported different, depending on the definition, the type of hospital or ICU, the population studied, and the type of rate calculated and varies from 7% to 70% [17] [18] [19] [20] [21] [22] [23] . In a large database, 1-day point prevalence study, conducted in 1417 European ICUs, pneumonia accounted for 47% of nosocomial infections [4] . In the National Nosocomial Infections Surveillance System (NNIS), NP accounted for 31% of all nosocomial infections in ICU [24] and in another NNIS data in medical ICUs, it was accounted for 27% [25] . The recent studies reported the device-associated incidence rate 13.2-51 per 1000 ventilator days [20, 23, 26, 27] . Generally, the rates of VAP in surgical ICU were higher than in medical ICUs, depending on the differences in the patient population, surgical disorders, the proportion of patients that needed MV and the duration of ventilation. Kollef et al. [28] reported incidences of NP of 21.6% in patients admitted to a cardiothoracic ICU, 14% in other surgical ICU, and 9.3% in a medical ICU.\nNP may occur by four routes; haematogenous spread from a distant focus of infection, contiguous spread, inhalation of infectious aerosols and aspiration. Aspiration of the pathogenic gram-positive and gram-negative bacteria, colonized on the oropharynx and gastrointestinal tract, is the main route. The role of other routes is very rare [1] .\nOnce microorganisms reach the distal lung, they multiply and cause invasive disease. The host defence, including filtration and humidification of air in the upper airways, epiglottic and cough reflexes, ciliary transport by respiratory epithelium, phagocytes and opsonins in distal lung, and systemic cell mediated and humoral immunity, prevent bacterial invasion [29] . In ICU, the host defence of patients are usually altered because of their underlying diseases, and devices that are used. They can not cough efficiently due to sedation or underlying disease. And also, when they are intubated, the endotracheal tube holds the vocal cords open and facilitates aspiration.\nThe most important risk factor for NP is tracheal intubation; associated with a 3 to 21 fold risk [30] [31] [32] [33] . It increases the risk by; 1) causing sinusitis and trauma to nasopharynx (nasotracheal tube), 2) impairing swallowing of secretions, 3) acting as a reservoir for bacterial proliferation, 4) increasing bacterial adherence and colonization of airways, 5) requiring the presence of a foreign body that traumatizes the oropharyngeal epithelium, 6) causing ischemia secondary to cuff pressure, 7) impairing ciliary clearance and cough, 8) causing leakage of secretions around the cuff, and 9) requiring suctioning to remove secretions [34] . Microorganisms can adhere to the surface of the endotracheal tube and some species exude an exopolysaccharide that acts as a slime-like adhesive. That microbial biofilm on the tube surface provides a reservoir of microorganisms, and they are greatly resistant to the action of antimicrobials and host defence [35] . Also, the patient requiring MV exposes to other devices, such as nebulizers, humidifiers, which can be the source of microorganisms.\nThe duration of MV increases the risk of infection. Cook et al. [22] reported a cumulative increased risk of VAP with time, with 3% per day in the first week of MV, 2% per day in the second week, and 1% per day in the third week. In other studies, similarly, it was shown that the risk of pneumonia increased by the duration of MV and the highest risk was during the first 8-10 days [36] [37] [38] . The need for reintubation, urgent intubation and documented massive aspiration are also associated with high incidence of VAP [1, 21, 29, 39] .\nThe effect of prior antibiotic therapy is still controversial. Sirvent et al. [40] reported that a short course of cephalosporin prophylaxis was associated with a lower rate of VAP in patients with structural coma. Also other investigators showed that antibiotics administered during the first days, reduced the risk of early-onset ventilator associated pneumonia [22, 37] . However, prior antibiotic exposure predisposes patients to subsequent colonization and infection with resistant pathogens [28, 41] .\nNasogastric tubes impair the function of the gastroesophageal sphincter and increase the risk of maxillary sinusitis, oropharyngeal colonization and reflux, all of which may lead to migration of bacteria and pneumonia [29, 39] . Enteral nutrition given by nasogastric tube is also associated with increased risk of VAP. Moreover, it may predispose to VAP by elevating gastric pH, leading to gastric colonization and increasing the risk of reflux and aspiration by causing gastric distension [22, 42, 43] . Also patient transportation was found risk factor for VAP by facilitating the aspiration of contaminated secretions from the upper airway and the ventilator circuits in the supine position [44] .\nThe other independent risk factors for VAP are shown in Table 1 [6, 7, 11, 22, 28, 33, 34, 45] . Identifying these risk factors will guide to prevention measures of VAP.\nThe causative organisms vary according to the patients' demographics in the ICU, methods of diagnosis, the durations of hospital and ICU stays, and the antibiotic policy. Gram negative bacteria are the most common pathogens cause VAP in several studies [7, 17, 44] . In NNIS data, although the most frequent reported isolate was Staphylococcus aureus (17%), 59% of all reported isolates were gram-negative. The most common gram-negative species were Pseudomonas aeruginosa (15.6%), Enterobacter species (10.9%), and Klebsiella pneumoniae (7.0%) (24) . In recent years, gram-positive bacteria have become more common in ICU and in the EPIC study, S. aureus accounted for 31% of the 836 cases with identified microorganisms (46) . NNIS data from medical ICU, also, reported high percentage (20%) of S. aureus [25] . Polymicrobial infection rate is usually high in VAP [12, 17, 47, 48] .\nThe duration of MV and the prior exposure to antimicrobials significantly influence the distribution patterns of etiologic agents. In early onset VAP (<5 days), methicillinsensitive S. aureus, Streptococcus pneumoniae, and Haemophilus influenzae are the most common pathogens, whereas methicillin-resistant S. aureus (MRSA), P. aeruginosa, Acinetobacter baumannii and Stenotrophomonas maltophilia are more frequent in late onset VAP (≥ 5 days) [49, 50] . Also, MRSA, P. aeruginosa, A. baumannii and the other multi-resistant gram negative pathogens are the most common pathogens in the patients expose to prior antibiotic.\nThe other special factors that predispose patients to infection with specific microorganisms are summarized in Table 2 [29, [51] [52] [53] [54] [55] [56] . Determining risk factors for microorganisms will help to select appropriate antimicrobial treatment, that improves the outcome.\nThe diagnosis of pneumonia in mechanically ventilated patients is difficult, and still there is no \"gold-standard\" diagnostic method. It is usually based on the combination of clinical, radiological, and microbiological criteria defined by Centers for Disease and Control (CDC) ( Table 3) . But these criteria have low sensitivity and specificity. The systemic signs (fever, leukocytosis, etc.) of infection can be seen by any condition in ICU (pulmonary edema, pulmonary infarction, after surgery, trauma, devascularized tissue, open wounds, etc.). Investigators reported that the clinical diagnosis of VAP is associated 30-35% falsenegative and 20-25% false-positive results [57] [58] [59] . And also, ICU patients do not always have systemic signs of infection due to their underlying disease (chronic renal failure, immunosuppresion, etc.). Radiological infiltration has limited value, mimicking by cardiogenic pulmonary edema, noncardiogenic pulmonary edema, adult respiratory distress syndrome (ARDS), atelectasis, pulmonary contusion, which are not uncommon in ICU [29] . In an autopsy-proven VAP study, Wunderink et al. [1] reported that no radiographic sign had a diagnostic accuracy greater than 68%. The presence of air bronchograms was the only sign that correlated well with pneumonia, correctly predicting 64% of pneumonias in the entire group. The upper respiratory tract of patients is colonized with potential pulmonary pathogens a few hours after intubation [48, 61] . Consequently, isolation of pathogens from tracheal secretions do not always indicate pulmonary infection. But a positive Gram's stain may guide the initial antibiotic therapy. However prior antibiotic and corticosteroid therapy can reduce the sensitivity of this technique [62, 63] .\nPugin et al. [64] proposed to combine the seven variables (temperature, leukocytes, tracheal aspirate volume and purulence of tracheal secretions, chest X-ray, oxygenation-PaO 2 /FiO 2 -and semiquantitative culture of tracheal aspirate) for the diagnosis of VAP, defined as clinical pulmonary infection score (CPIS) ( Table 4 ). The score varied from 0 to 12 points and was reported that a CPIS of more than six was associated with a sensitivity of 93% and a specificity of 100% for the diagnosis of pneumonia. In a post mortem study, Papazian and colleagues [65] reported a high diagnostic accuracy of CPIS at a threshold of 6 (72% sensitivity and 85% specificity). However, the original scoring system has some limitations; that it requires 24-48 hours for the results of tracheal aspirate cultures, and also identifying pulmonary infiltrates progression depends on intensivist experience. Singh et al. [66] used a modified CPIS (calculated at baseline from the first five clinical variables, and CPIS at 72 hours was based on all variables of the score) that antibiotics were stopped in patients with a persistent low score (<6) after 3 days of therapy, avoiding unnecessary use of antibiotics, and all patients who discontinued the therapy improved. In a recent study, Fartoukh et al. [67] reported that the modified CPIS does not perform better when the clinical suspicion of pneumonia is high, so they proposed incorporating the results of specimens gram stain (by adding two more points when gram stains were positive) to modified CPIS to increase the sensitivity of the score and the physicians' diagnostic accuracy.\nQualitative cultures of tracheal aspirate (TA) is not a specific diagnostic method because of the lower respiratory tract colonization and a high percentage of false-positive results [48, 68] . However, investigators reported that quantitative cultures of TA have equal diagnostic accuracy to the other invasive techniques [69] [70] [71] [72] [73] . In a recent study, quantitative cultures of TA were compared with plugged telescoping catheter (PTC). The specificity of TA was similar to PTC when a cut-off point of 10 6 cfu/mL or higher was used, although the sensitivity of TA at ≥ 10 6 cfu/mL was lower than PTC. But when a cut-off point of 10 5 cfu/ mL was used, the sensitivity of TA was not statistically different from that of PTC [74] . Although, quantitative cultures of TA is non-invasive, inexpensive and a simple method, it has some risks, that if the cut-off value ≥ 10 6 cfu/mL is used, sensitivity will be low and some patients with VAP may not be identified or when the cut-off value ≥ 10 5 cfu/mL is used, unnecessary antibiotic treatment will be given because of low specificity [75] .\nIn the recent years, many investigators favour invasive techniques for diagnosis of pneumonia (protected-speci-men brush -PSB-or bronchoalveolar lavage-BAL-) that may have more diagnostic accuracy [76] [77] [78] [79] [80] . In PSB, 0.001 mL of secretions are collected and the presence of >10 3 cfu/mL bacteria has 80-90% sensitivity and 95% specificity for the diagnosis of VAP. In BAL, larger proportion of lung can be sampled and the diagnostic threshold is >10 4 cfu/mL. The sensitivity and specificity of BAL are 86-100% and 95-100%, respectively [76, [81] [82] [83] . Heyland and colleagues [84] proposed that PSB or BAL may increase physician confidence in the diagnosis and management of VAP and allows for greater ability to limit or discontinue antibiotic therapy. Also in this study, patients who underwent bronchoscopy with PSB and BAL had a lower mortality rate compared with patients who did not undergo bronchoscopy. However, a recent meta-analysis concluded that regular use of bronchoscopy for the diagnosis of VAP does not alter mortality, because it does not directly affect the initial antibiotic prescription [85] . The disadvantages of these invasive techniques are [71, 86, 87] ; a) prior antibiotic use may decrease the sensitivity and accuracy of these methods. However, in a recent study, Souweine et al. [88] reported that if a current antibiotic treatment prescribed for a prior infectious disease other than VAP, the diagnostic accuracy of protected specimen brush or bronchoalveolar lavage is not changed, b) these techniques are based on quantitative culture and results of these cultures require 24-48 hours, and, therefore miss early cases, and also give no information about appropriate initial antibiotic therapy, c) these invasive tests may worsen the patient's status (cardiac arrhythmias, hypoxemia, bleeding, etc.), d) increase the costs of caring, e) it has not been proven that the use of these invasive techniques lead to a decrease in patients' mortality.\nThe spread of microorganism to blood or pleural space is <10%, so blood and pleural effusion cultures have low sensitivity and specificity. Luna and colleagues [89] demonstrated that the positive predictive value of blood cultures to detect the etiologic microorganism was 73% and the sensitivity of blood cultures was only 26%. They con- Three or more of the following criteria:\nRectal temperature >38°C or <35.5°C Blood leucocytosis (>10.10 3 /mm 3 ) and/or left shift or blood leukopenia (<3.10 3 /mm 3 ) More than ten leukocytes in Gram stain of tracheal aspirate (in high power field) Positive culture from endotracheal aspirate AND New, persistent, or progressive radiographical infiltrate cluded that blood cultures in patients with VAP are useful if there is suspicion of another probable infectious condition, but the isolation of a microorganism in the blood does not confirm that microorganism as the pathogen causing VAP. Therefore, two sets of blood samples for culture and tapping pleural effusions >10 mm should be performed in patients suspected VAP [50] .\nIn conclusion, microbiological testing should be always performed to decide the appropriate initial empirical antibiotic therapy. Clinicians can choose optimal diagnostic test for specific patients in their clinical setting.\nBecause of longer duration of mechanical ventilation, longer stay in the ICU, increased use of antibiotics, higher costs for healthcare, and most importantly, increased mortality, the prevention of VAP is the major priority. But, despite the advances in the pathogenesis of VAP, intensivists still struggle with the prevention strategy.\nBasic hygiene principles of infection control (hand washing/disinfection just before and after each patient contact, the use of glove and sterile equipment) remain important for the prevention of VAP. Healthcare workers (HCW) can spread microorganisms from patient to patient by their hands easily. Although HCWs realize the importance of handwashing/disinfection, their compliance is still low (25-40%) [90] [91] [92] . Especially their compliance rate is lowest in activities that carried higher risk for transmission and in ICU. High workload decreases their compliance [93] . Wrist watches, bangles, and other jewellery act as reservoirs for organisms, and inhibit effective hand cleaning [94] . Therefore, staff have to take off wrist watch and jewellery to achieve effective hand cleaning. They have to use gowns and gloves when appropriate and must change and wash/disinfect their hands between patients [95] . Bedside hand antiseptics (alcohol-based handrub solution), easier access to sinks and availability of washing equipment, decrease in workload, communication and education tools (posters) and feedback improve compliance and decrease the cross-transmission of nosocomial infection [90, 96] .\nThe internal machinery of mechanical ventilators is not an important risk factor for VAP. Therefore, using a filter between the inspiratory phase circuit and the patient is not necessary. Furthermore, the importance of filters on the expiratory limb of the mechanical-ventilator circuit in preventing cross comtamination has not known and needs further investigation [34] .\nThe devices used on the respiratory tract come into contact with mucous membranes, therefore cleaning and highlevel disinfection (at 75°C for 30 minutes) of reusable equipments are required [97] . Resuscitation bags, spirometers, and oxygen analyzers must be cleaned and disinfected between patients to avoid cross-transmission [95] .\nIn several studies, routine changing of the ventilator circuits is not recommended [26, [98] [99] [100] [101] [102] [103] [104] . Replacement is required when there is gross soilage and mechanical malfunction [105] . The condensate fluid in the ventilator circuit, which contains high concentration of pathogenic bacteria and a risk factor for VAP, has to be removed regularly [106] . And accidental drainage of condensate into the patient's airway and contamination of caregivers during ventilator disconnection or during disposal of condensate should be avoided. In-line devices with one-way valves, put in place into disposable circuits and emptied regularly, are recommended for collecting condensate [75] . Humidification of the inspired air is an important care in the ventilator management. Humidification may be achieved by active humidifiers (bubble-through or wick) or passive humidifiers (hygroscopic condenser-artificial nose-or heat-moisture exchanger-HME-). In humidification, formation of condensate in tubing and colonization of this condensate with microorganisms is an important risk factor for VAP. HME recycle heat and moisture that reduces the condensate formation and also bacterial colonization in the circuit. Moreover they have bacterial filtration characteristics [34, 98] . They do not need to be changed daily and can be used for at least 48 hours, sometimes for up to 1 week [107, 108] . Also with other advantages (reduced nurses workload, reduced financial cost, and better safety), HMEs are favourable devices in many ICUs. Indeed several investigators [109] [110] [111] [112] reported lower rates of VAP in HME groups than conventional heated-water humidification systems, the effect of HME on the prevention of VAP is still controversial and a recent study showed no significant difference in VAP rates [113] . Furthermore, HMEs increase dead space and resistance to breathing, cause airway occlusion, and more tenacious secretions [34, 98, 104, 111] . Additional studies are needed to identify the benefits of HMEs on infection control of VAP.\nNebulizers are used for medication or humidification of air, and inserted into the inspiratory phase of the mechanical ventilator circuit. They can be contaminated by condensate in the tube or by using contaminated solutions, and inoculate infectious aerosol particles directly into the lung parenchyma and can cause outbreaks in ICUs. Recommendations for the infection control of nebulizers are; a) filling immediately before use, b) using sterile water and drugs, c) never refilling the liquid to be nebulised, d) cleaning and disinfecting the receptacle daily, d) using sterile water for rinsing and allowing to dry, e) using patient-specific large-volume nebulizers, f) using patient specific mask, mouthpiece, connecting pieces and medicine cups [34, 114] .\nSuctioning the secretions in the trachea is another approach to VAP prevention. Two types of tracheal suction catheters are used on ventilated patients; the open, single-use catheters and the closed, multiple-use catheters.\nIn single-use system, HCWs have to use sterile solutions during rinsing these catheters and have to care aseptic technique when suctioning endotracheal secretions. In closed suctioning systems, secretions can be suctioned without removal of mechanical ventilation support. This may cause less hypoxia, hypotension and arrhythmias, and also less environmental contamination [115, 116] . However similar VAP rates with closed and open system were suggested in the earlier trials [115, 117] , Combes and colleagues [116] reported a 3.5 times greater risk of VAP in open suctioning system than closed suctioning system in a recent study. Indeed, closed suction catheter is an extension of the ventilator circuit, daily change of this catheter is not necessary for infection control, and in one study no significant difference in VAP rate was reported when daily changes were compared with no routine changes, that may decrease the costs [118] . The use of closed suction system is recommended as part of a VAP prevention program [104] .\nThe relationship between the use of invasive devices and nosocomial pneumonia directed the investigators to use noninvasive ventilation to reduce the VAP rates. In several studies, lower risk of VAP, with less antibiotic use, with a shorter length of ICU stay, and with lower mortality were reported in the use of non-invasive ventilation [119] [120] [121] [122] [123] . Therefore, care can be taken to use non-invasive mechanical ventilation more often, and to reduce the frequency of tracheal intubation.\nEndotracheal tube alters host defences, impairs mechanical clearance from the respiratory tract, causes local trauma and inflammation, and allows pooling of secretions around the cuff. The pressure of the endotracheal tube-cuff should be sufficient to prevent the leakage of colonized subglottic secretions into the lower airway [37] . Also, continuous or intermittent suctioning of oropharyngeal and upper respiratory tract secretions above endotracheal cuffs can prevent aspiration. Endotracheal tubes with a separate dorsal lumen above the cuff, designed to suction the subglottic secretions continuously, were found able to decrease the rates of early-onset VAP [124, 125] . However, in another randomised trial, no benefit with continous subglottic suction on the overall VAP frequency was found [126] . It can reduce but not eliminate the volume of fluid aspirated into the lungs. The lack of effect on prevention of late-onset pneumonia and the high cost of these tubes restrict the usage of them.\nMicrobial biofilm on the endotracheal tube surface is a reservoir for the pathogens and prevent the microorganisms from the action of antibiotics [35] . Adair and colleagues [127] proposed that high concentrations of antibiotic on the endotracheal luminal surface, achieved either by nebuliser or endotracheal surface modification, would be expected to prevent biofilm formation on the endotracheal tube and may have a role in reducing the incidence of VAP, also minimising patient exposure to systemic antibiotics.\nNasotracheal intubation increases the risk of nosocomial sinusitis, that may predispose VAP by the aspiration of infected secretions from nasal sinuses [128, 129] . There- fore, endotracheal intubation should be preferred to decrease the risk of VAP.\nAs nasotracheal intubation, nasogastric tube can cause orophanryngeal colonization and nosocomial sinusitis. By impairing the function of the upper oesophagus sphincter, it may facilitate the reflux of bacteria from the gut. As a result, it increases the risk of VAP [130, 131] . In a randomized study, gastroesophageal reflux and microaspiration of gastric contents to the lower airways were not influenced by the size of the nasogastric tube. Because of their potential complications (tracheal malposition by coiling and clogging) and high cost, the small-bore nasogastric tubes are not routinely recommended for the prevention of VAP [132] .\nPoor nutritional state and hypoalbuminemia contribute the development of VAP. For this reason, early initiation of enteral nutrition may have preventive effect in mechanically ventilated patients. Moreover, it helps to maintain the gastrointestinal epithelium and reduces the need for stress-bleeding prophylaxis. However, because of using nasogastric tubes and alkalinization of the stomach contents by these feeds; gastric colonization, gastrooesophageal reflux, aspiration and pneumonia might be promoted [29] . In a recent study, postpyloric enteral access placement improved tube-feeding tolerance and reduced the rates of VAP [133] . Heyland et al. [134] used acidified feeds in critically ill patients and demonstrated a dramatic reduction in bacterial growth from aspirates of stomach contents and a lower rate of gram-negative bacterial growth in tracheal secretions in patients receiving acidified feeds, but not a significant reduction in nosocomial pneumonia. They cannot be used in patients with active gastro-intestinal bleeding, acidemia, or renal failure. Furthermore, it was a small size study and further research on the effect of prevention is needed, before it is used in practice.\nIn the recent years, selective decontamination of the digestive tract (SDD) is one of the most extensively studied prevention strategy in VAP. In SDD, topical non-absorbed antimicrobials (usually combining polymyxin, aminoglycoside and amphotericin B) are used to prevent gastrointestinal colonisation by pathogenic microorganisms. It selectively eradicates the potential pathogenic microorganisms (gram-negative aerobic intestinal bacteria, S. aureus and fungi) and does not affect anaerobic flora, as elimination of anaerobic flora leads to increased colonization with gram-negative aerobic flora. Although some investigators used only topical antibiotics applied to the oropharynx and through a nasogastric tube, many of them added systemic therapy with a broad spectrum (e.g. cefotaxime) during the first few days, to prevent early infections with S. pneumoniae, H. influenzae and S. aureus [29] . In a recent meta-analysis that searched 33 randomized controlled trials published from 1984 to 1996, significant reductions in the incidence of respiratory tract infections (65%) and in total mortality (20%) were determined [135] . Also in this meta-analysis and in the other recent prospective, randomized studies, it was mentioned that using only topical antibiotics reduced respiratory infections, but did not influence the survival [135] [136] [137] .\nThe threat of SDD is to lead the selection and overgrowth of antibiotic resistant microorganisms. In a recent study from the Netherlands, where the incidence of MRSA and vancomycin resistant enterococcus (VRE) are very low, a reduction in the frequency of colonization with resistant gram-negative bacteria and no effect on the acquisition of MRSA were reported (138) . But the studies from the ICUs where MRSA was endemic, increased incidence of MRSA was reported (139, 140) . Therefore, in ICUs with high incidence of multi-resistant microorganisms, SDD cannot be used. On the other hand, in trauma and surgical patients, SDD seems more effective than in medical patients, may be due to less colonisation [141] . In conclusion the routine use of SDD in ICUs is not recommended, it should be decided according to the patient population studied and the characteristic of ICU.\nIn addition, the colonization of the oral cavity with pathogens is important risk factor for the develoment of VAP, it is unclear if oral care with chlorhexidine reduces VAP. Also the concern over a chlorhexidine-related increase in colonization of gram negative bacteria should be considered [142] .\nSemirecumbent position (45°) prevents aspiration and the passage of bacteria into the airways, and should be preferred in ICU patients, if there is no contraindication [143] .\n\"Kinetic Beds\" or Continous Lateral Rotational Therapy (CLRT) turn continuously and slowly and change the patient's position. Investigators believe that it helps the drainage of pulmonary secretions. However, these beds are so expensive and their effectiveness are not demonstrated. So, the routine use of these beds is not recommended [97] . Also, chest physiotherapy, to improve the clearance of secretions, for the prevention of VAP is not recommended because of its lack of proven benefits and the associated risks (e.g., arterial oxygen desaturation) [105, 144] .\nStress ulcer prophylaxis is proposed to be a risk factor due to the alkalinization of gastric content. The effect of stress ulcer prophylaxis with H 2 -antagonists or antacids on VAP is still controversial. In some studies [145, 146] , the use of sucralfate was associated with a decreased incidence of VAP, however the other reports did not support this [147, 148] . Also, H 2 -antagonists are more efficient for anti-ulcer prophylaxis than the sucralfate [148] . Therefore, the choice of agent for prophylaxis should be done according to the patient and cost-effectiveness.\nTo reduce the aspiration of oropharyngeal contents, over use of sedatives should be avoided. Kress et al. [149] reported that for reducing over use of sedatives, daily interruption of sedative-drug infusions until the patients were awake decreased the duration of mechanical ventilation and the length of stay in the ICU." }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: Hand, foot and mouth disease (HFMD) is a common acute viral illness that primarily affects infants and young children, and often occurs in clusters or outbreaks. It is characterized by rapid onset of fever and sore throat, accompanied by vesicles and ulcers on the gums, tongue, buccal mucosa and palate. Punctate and usually transient skin lesions appear on the palms, soles and occasionally on the buttocks, knees or other areas. While the fever and rash may subside rapidly, the mouth lesions may last more than a week, and virus may continue to be shed for several weeks [1] . In temperate countries HFMD occurs during the summer but in the tropics HFMD can occur at any time during the year.\nThe major causative agents of HFMD are coxsackievirus A16 (CVA16), human enterovirus 71 (HEV71) and coxsackievirus A10 (CVA10) of the genus Enterovirus in the family Picornaviridae [2] . Other enteroviruses isolated from HFMD cases are the other species A human enteroviruses such as coxsackievirus A (CVA) 4, CVA5, CVA6 and CVA7, and coxsackievirus B (CVB) 1, CVB2, CVB3 and CVB5 [2] [3] [4] . Unlike other aetiological agents of HFMD that normally cause mild disease, HEV71 infection has been reported to cause neurological disease manifesting as aseptic meningitis, encephalitis or poliomyelitis-like acute flaccid paralysis [5] . First isolated from a child suffering from encephalitis in California in 1969, HEV71 was further isolated from 23 cases with severe neurological disease in California during the next three years [3] . Historically, HEV71-associated outbreaks have been reported in Australia in 1972 [6] , Japan in 1973 and 1978 [7] , Bulgaria in 1975 [8] and Hungary in 1978 [9] .\nIn the past decade, countries in the Asia-Pacific region have experienced an increased occurrence of HEV71-associated HFMD outbreaks [10] . HEV71 outbreaks have been reported in Sarawak in 1997, Taiwan in 1998, Perth in 1999, then in Singapore, Korea, Malaysia and Taiwan in 2000 [11] [12] [13] [14] [15] [16] [17] .\nIn an outbreak of HEV71 in 1997 in Sarawak, a state of Malaysia on the island of Borneo, a cluster of unusual paediatric deaths due to encephalitis and cardiac failure was observed [12, 13] . This raised a lot of fear and anxiety and because of the heightened concern about HEV71 in Sarawak, we implemented a sentinel surveillance programme for HFMD beginning in March 1998. This programme was set up as part of the operational functions of the Sarawak Health Department and was approved by the Director of Health. The principles of the Helsinki Declaration were followed throughout the surveillance operation.\nOur aims were to investigate the epidemiology of this common childhood disease in Sarawak, and to determine if there were any differences in the patterns of transmission of HEV71, CVA16 and other aetiological agents of HFMD. It was also the aim of this programme to provide data of practical value for doctors and public health personnel with a view to efficient and effective virological surveillance of HEV71, in particular, to provide an early warning system for HEV71 outbreaks. This paper describes the preliminary observations from our surveillance programme from March 1998 through June 2005.\nIn early 1998 after discussion with a number of community paediatricians, our team set up a protocol for a sentinel surveillance programme for HFMD. The doctors who had consented to actively participate were provided with a standard reporting and specimen collection form, sterile swabs and virus transport medium, a telephone number for obtaining assistance for transport of specimens to the laboratory and a facsimile number to report cases to the Health Department. All sentinel clinic doctors obtained parental consent before swabs were taken. Sentinel clinic doctors were provided with feedback on viruses isolated from their patients and contact was maintained through both outbreak and inter-outbreak periods to assure doctors that the surveillance programme was active and ongoing. Data obtained in this manner were expected to provide accurate information about disease trends and molecular epidemiology of the relevant viruses.\nThree specialist paediatric clinics located in the towns of Kuching and Sibu in the state of Sarawak actively participated in this study from March 1998. In 2000 we included a fourth specialist clinic in Sibu. Two government polyclinics in Kuching and Sibu also participated as sentinel clinics. All children presenting to the sentinel clinics with a history of oral or other skin lesions typical of HFMD were enrolled into the surveillance study, and throat and rectal swabs were obtained from each child enrolled in the first 18 months. Where possible, swabs were also obtained from mouth ulcers, vesicles and other skin lesions. After a preliminary analysis of data from the first 18 months, the protocol was modified to require only throat swabs from sentinel clinics. Rectal swabs were optional and doctors were requested to provide vesicle swabs whenever possible. Specimens were to be transported on ice to the laboratory in 2 ml of viral transport medium (VTM) where they were vortexed, freeze-thawed and aliquoted.\nSince the primary objective of programme was a sentinel surveillance system for HEV71 HFMD, we inoculated specimens into human rhabdomyosarcoma (RD) cells susceptible to both CVA16 and HEV71. It was not a particular objective of this exercise to identify the minor causative agents known to be associated with HFMD and hence we did not include multiple cell lines as part of our virus isolation protocol. RD cell cultures normally showed the characteristic enterovirus CPE in 2 to 10 days and were harvested after the monolayer showed extensive CPE. A blind passage was done with all cultures showing no CPE after 10 to 14 days.\nRNA was extracted from all culture harvests using Tri Reagent LS (Molecular Research Centre, Cincinnati, OH, USA) according to the manufacturer's instructions. The dry RNA pellet was dissolved in 20 μl of sterile ultra high quality RNase-free water and stored at -80°C until use.\nThe presence of enterovirus RNA in culture fluids was determined by a previously described pan-EV RT-PCR method [18] with some modifications. The duration of all the steps in the PCR was reduced to one minute and the final extension was reduced to 5 minutes.\nFrom 1998 through 2002, specimens positive using the pan-EV primers were tested for the presence of HEV71 genome by RT-PCR using the primers 159S and 162A, which anneal to the VP1 gene of HEV71. Dr. Mark Pallansch (Centers for Disease Control, Atlanta) generously made the primer sequences available to us prior to publication [19] . All PCR products were sequenced to confirm the identification. In 2002, we changed our protocol for identification of HEV71 due to problems of misidentification of local strains of CVA16 as HEV71 using the primer set 159S/162A [20] . Currently, HEV71 specific primers designed in-house are used for specific identification of HEV71 [20] . All primers used are listed in Table 1 .\nSequencing reactions were performed using the Big Dye Terminator Cycle Sequencing Kit version 3.0 or 3.1 (Applied Biosystems, Foster City, CA, USA).\nMolecular serotyping of non-HEV71 enteroviruses isolated was carried out using the methods and sequences published by Oberste and colleagues [21] and Chu, Ishiko and colleagues [22, 23] . Prior to 2002, serotyping of selected non-HEV71 enteroviruses was performed exclusively according to Oberste's method. When Ishiko's method [23] was published in 2002, we made a comparison of the methods by serotyping new isolates using both methods. We determined that for human species A enteroviruses circulating in our region, Ishiko's primers gave identical serotype identification to that obtained using Oberste's method [21] . Since there was no discrepancy between the methods, we modified Ishiko's primers to convert the method from a semi-nested to a non-nested method for ease of use. Our modifications were verified and described by Cardosa and colleagues [24] . We then retrospectively retested all non-HEV71 enteroviruses isolated prior to 2002 using the modified method.\nA subset of isolates identified by sequencing of VP4 were subjected to confirmation by using VP1 specific primers and DNA sequencing of the PCR products, as described previously [24] .\nDNA sequences of VP1 and VP4 gene products generated by RT-PCR from isolates were used in this analysis essentially as previously described [24] . The software package ClustalX [25] was used for alignment and to generate a bootstrapped phylogenetic tree using the neighbour joining method according to Saitou and Nei [26] .Primers and DNA sequences TTCCAATACCACCCCTTGGATGA Antisense (1195-1173) HEV71 VP4 gene. 159 [19] ACYATGAAAYTGTGCAAGG Sense (2385-2403) HEV71 VP1 gene. 162 [19] CCRGTAGGKGTRCACGCRAC Antisense (2869-2850) HEV71 VP1 gene. 161 [19] CTGGGACATAGAYATAACWGG Sense (2766-2785) HEV71 VP1 gene. NP1A [19] GCICCICAYTGITGICCRAA Antisense (3355-3336) HEV71 VP1 gene. MAS01S [20] ATAATAGCAYTRGCGGCAGCCCA Sense (2355-2377) partial VP1 gene. MAS02A [20] AGAGGGAGRTCTATCTCYCC Antisense (2731-2712) partial VP1 gene. MD91 [18] CCTCCGGCCCCTGAATGCGGCTAAT Sense (450-474) partial 5UTR. MD90 [18] ATTGTCACCATAAGCAGCCA Antisense (603-584) partial 5UTR.\n*Position relative to the genome of HEV71 strain 7423-MS-87 (GenBank accession number U22522)\nAll primers used in the methods described above are listed in Table 1 . All new DNA sequences used in the phylogenetic analysis but hitherto unpublished have been deposited in GenBank and have the accession numbers AY794032, AY794033, AY794035 to AY794042. All other sequences used are from previous publications by our own as well as other groups [17, 20, [22] [23] [24] 27] . Detailed protocols, sample collection methods and other practical information have been placed in the public domain through our APNET (The Asia-Pacific Enterovirus Surveillance Network) website [28] .\nStatistical analysis was performed using the software package JMP Statistics version 5.01(SAS Institute Inc., USA) and Prism 4 for Macintosh (Graphpad Software, Inc., USA).\nThe first provisional protocol we provided to the sentinel clinics for the collection of specimens required both throat and rectal swabs and vesicle or ulcer swabs where possible. Results from virus isolation studies of specimens obtained from both sentinel clinics as well as hospitals during this period were used to review the protocol that was originally implemented. A total of 579 specimens from 263 children with a clinical diagnosis of HFMD were received during the 18-month period from March 1998 through August 1999. The age of the children ranged from 6 months to 13 years, with 153 (58.2%) males and 110 (41.8%) females.\nAll specimens received were subjected to virus isolation. Fifty specimens from 44 children, of a total of 579 (8.6%) specimens, were too heavily contaminated with bacteria. Twenty four of the contaminated specimens were rectal swabs, 19 were throat swabs and 7 were from various skin lesions. All remaining uncontaminated cell culture harvests were tested for enteroviruses by using the pan-EV set of primers and 235 of the 529 (44.4%) specimens tested yielded an enterovirus, but only 15 of the 235 (6.4%) enteroviruses were HEV71. These specimens were from 259 children and an enterovirus was isolated from 153 (59.1%) children. Only 6 (3.9%) of the children had HEV71.\nFrom this early set of specimens, we were able to isolate an enterovirus from 44% of the throat swabs, 40% of the rectal swabs, 44% of the mouth ulcers and 66% of the vesicle swabs. Clearly vesicle swabs are very useful specimens, but only 18% of the children had had vesicle swabs taken because not all children presented with skin lesions filled with abundant fluid. Since throat swabs provided a reasonably high yield of enterovirus isolates, we made the decision in 2000, to require throat swabs as the primary specimen from the sentinel clinics, with vesicle swabs where possible, while rectal swabs were not required. This served to reduce the laboratory workload during an outbreak.\nOur laboratory received 4290 specimens from 2950 children from March 1998 through June 2005, with a male to female ratio of 1.35:1. The histogram in the top panel of Figure 1 shows the distribution of HFMD cases seen in our sentinel clinics during this period. There have clearly been two large outbreaks of HFMD in 2000 and 2003 (bottom panel of Figure 1 ), with some sporadic activity between these peaks. The dominant enterovirus serotype isolated during both the outbreaks was HEV71 as shown in the middle panel of Figure 1 . CVA16 was always isolated during HEV71 outbreaks as well but was also isolated in interoutbreak periods. Other species A human enteroviruses such as CVA2, CVA4, CVA5, CVA10 and CVA12 were also isolated in inter-outbreak periods.\nPhylogenetic analysis of the HEV71 strains isolated in Sarawak from 1998 to 2005 show that both genogroup B and genogroup C strains circulated in Sarawak during this period (see Figures 2 and 3) . We have used both VP4 and VP1 genes in the phylogenetic analysis in order to be certain that there is no major discrepancy in genotyping associated with using VP4 and VP1 gene regions. Furthermore, we wish to provide both options to other groups who may wish to compare their data with ours since it is known that many groups may still use VP4 sequencing as a first step in molecular identification of human enteroviruses. Although both genogroup B and genogroup C HEV71 strains co-circulated in Sarawak, the predominant genogroup in both the HEV71 outbreaks of 2000 and 2003 was genogroup B. Besides co-circulating with genogroup B strains during outbreaks, genogroup C viruses also appeared sporadically between outbreaks along with other species A human enteroviruses. We never isolated a genogroup B HEV71 in non-outbreak periods. The distribution of genogroup B and genogroup C HEV71 strains during the surveillance period is shown in the bottom panel of Figure 1 .\nThe phylogenetic trees in Figures 2 and 3 \nThe HFMD epidemiological curves for the outbreak years 2000 and 2003 were plotted according to epidemiological week ( Figure 4 ) and show clearly that the first HFMD cases began to be seen early in the year. By week 7 a clear rise in the number of cases was seen. This early rise in cases differs from the summer outbreaks seen in temperate countries, and we suggest that the HEV71 outbreaks in Sarawak preceed the summer outbreaks of countries in the northern hemisphere in each year. In 2000 the HFMD outbreak stretched to the end of the year, peaking between Phylogenetic tree generated from the VP1 gene, showing relationships between HEV71 isolated in different years Figure 1 ). In 2003, the number of HEV71 cases declined sharply by the end of April (by week 18) and were no longer detected by the end of June, coinciding with the last HFMD cases seen that year. Interestingly this outbreak coincided with the SARS outbreak in the region and the public health measures put into place during this time evidently served to control the transmission of enteroviruses as well.\nA detailed analysis was done on data collected from two sentinel clinics, coded S1 and S2, which had sent samples to our laboratory consistently and reliably throughout the seven-year study period. A total of 2570 specimens were collected from 1894 cases during the 7 years. Of the 1894 cases, specimens from 1804 (95%) were subjected to virus isolation. A total of 2272 specimens were subjected to virus isolation, thus ensuring that the majority of specimens from the majority of cases were tested (88.4% of specimens from 95% of cases).\nAn analysis of the proportion of the different types of specimens and the virus yield obtained is shown in Table 2 . More than 2000 specimens from outbreak and nonoutbreak periods were tested from 1998 to 2005. Enteroviruses were grown from 21.6% of those tested. Throat swabs comprised 72.3% of the total number of specimens tested and 25.4% of these yielded enteroviruses. Detailed information about the enterovirus serotypes isolated during this surveillance programme is also provided [see additional file 2] . Although on the whole, the virus isolation success rate was much lower than anticipated from the results for the first 18 months, it remained the case that throat swabs were more useful than the rectal swabs which yielded non-polio enteroviruses in only 5.8% of the samples tested. It should be noted however, that the first 18 months of the study coincided with an inter HEV71 epidemic period, with mostly CVA16 and non-HEV71 species A human enteroviruses causing HFMD. We have compared the virus isolation yields during HEV71 outbreak (2000 and 2003) years with the yields during an HFMD outbreak caused by non-HEV71 enteroviruses (2002) and we found that we successfully isolated virus from 40% of specimens collected in 2002 but only 20% of viruses during the HEV71 outbreak years, suggesting that HEV71 is more difficult to isolate than CVA16 and other species A enteroviruses. The virus isolation rate in the first 18 months (44%) is therefore comparable to that obtained later, when HEV71 was not circulating.\nThere were 491 children from whom a non-polio enterovirus was isolated. Of these, 8 were excluded from the analysis because of missing information on their age at presentation. The children ranged in age from 18 days to 155 months, with a mean of 32.2 months and a median of 27.5 months. There were 3 dominant serotypes of enteroviruses isolated from these 483 children and we asked the question if different serotypes of enteroviruses caused infection in children of different ages. Table 3 shows the mean ages of the children who had CVA16, HEV71 and CVA10 infection. Comparison of means for each pair using an unpaired t test at an alpha of 0.05, showed that there was no significant difference in the mean ages of the children in the different groups (CVA10 versus CVA16: P = 0.0872; CVA10 versus HEV71: P = 0.1800; CVA16 versus HEV71: P = 0.6992).\nFollowing the 1997 outbreak of EV71 associated HFMD in Sarawak, Malaysia, the Health Department installed a sentinel surveillance programme with the expectation that we would be able to study epidemiological trends and begin to predict when to expect outbreaks with sufficient accuracy in order to implement public health interventions to reduce the burden of the disease. Although the surveillance programme is still ongoing in Sarawak, we have sought to glean some preliminary information from the data generated over the first 7 years of the programme. two HEV71 outbreaks. A recent report on a similar surveillance programme in Yamagata Prefecture in Japan (1998) (1999) (2000) (2001) (2002) (2003) suggests that in Yamagata there is frequent importation of HEV71 from surrounding countries seeding the clusters of cases seen annually in this community [10] . The HEV71 strains in this study were isolated from small clusters of cases that tended to be seen in the summer months while in our situation we observed outbreaks of HEV71 every 3 years with cases being seen much earlier in the year, well before the northern summer. In 2003 both Sarawak and Yamagata experienced a large outbreak and in both situations, a genogroup shift from C to B was noted.\nIt is interesting that in Sarawak, of the genogroup C viruses, only genogroup C1 strains have been observed, while genogroup B viruses appear to be changing from outbreak to outbreak, suggesting that it is likely that genogroup B viruses are evolving within Borneo and that the outbreaks we have experienced are being seeded from within rather than from imported viruses. Since the outbreaks in Sarawak typically begin early in the year, it is also possible that genogroup B strains generated in Sarawak may seed HEV71 outbreaks in the region, which typically occur later than the Sarawak outbreaks. This temporal sequence of regional outbreaks is also true of those occurring in Singapore and in Peninsula Malaysia.\nThe data we have obtained through 7 years of our sentinel surveillance programme for HFMD in Sarawak have provided useful clues to understanding the epidemiology of HEV71 in the state. It is clear that the appearance of HEV71 associated HFMD in sentinel clinics signals the start of an outbreak, but the rise in the number of cases is so rapid that this approach is not a suitable early warning system. In 2003 there were only 5 weeks between the time the first HEV71 cases were seen and the peak of the outbreak. Clearly this could be explained by rapid and effective response by the public health teams, but we have no way to know.\nAlternatively, the 3-year cycle of HEV71 outbreaks we have observed could, if verified in the coming years, provide public health officials with the relevant information to plan and to implement their intervention programmes to reduce the disease burden in the years when an HEV71 outbreak is expected. Although this is not expected to prevent the outbreaks entirely, effective public health measures put into place early enough can limit the spread, reduce mortality and reduce the burden on the community and the health system.\nIt is important to note that epidemiological curves showing HFMD alone, without distinguishing the infecting agent for each case, can stretch broadly over many months, with non-HEV71 enteroviruses continuing to be isolated after cessation of HEV71 activity. This was especially evident in 2000, when HEV71 associated fatal cases were reported in neighbouring Singapore in September and October 2000 [29] , and the media attention surrounding these events generated a high index of suspicion in Sarawak as well. No HEV71 was isolated in Sarawak after August that year, but numerous CVA16 continued to be isolated until the end of 2000. Thus even though sociological factors affect the shape of the HFMD epidemiological curves in Sarawak, epidemiological curves specifically showing genogroup B strains of HEV71 were consistently sharp and well defined in 2000 and 2003. The mean and median age of children with HFMD was 36 months and 30 months respectively, but the mean ages did not differ between the groups infected with the different serotypes. It is thus intriguing that HEV71 has caused much larger and sharper outbreaks than either CVA16 or CVA10. This suggests that HEV71 has the capacity to spread rapidly through the susceptible population and then become quiescent in the community. In the third year after any HEV71 outbreak, the whole cohort of children under 3 years of age has not been exposed to HEV71 and all of these children are then susceptible, providing the conditions for another sweeping transmission of HEV71 through the community. The annual birth cohort in Sarawak is 48 to 49 thousand and thus in 3 years there are up to 150,000 susceptible children in the state.\nAccording to the trends we have reported, we expect that the next outbreak of HEV71 in Sarawak will be in 2006. At the time of writing we have already begun to pick up HEV71 cases in our sentinel programme and from past experience, an outbreak in Sarawak is often followed by outbreaks in other countries in the region. We have therefore decided to put our data into the public domain in order that other public health practitioners in the Asia Pacific region may benefit from this experience and prepare for a spread of HEV71 in the region once again in the months to come.\nThe main conclusions arising out of this preliminary report are described below:\na. HEV71 outbreaks have occurred every 3 years in Sarawak starting in 1997. All the 3 outbreaks (1997, 2000 and 2003) have been caused by genogroup B viruses and furthermore, each of the 3 outbreaks has been associated with genogroup B viruses that are genetically distinct from each other.\nb. HEV71 of subgenogroup C1 has been isolated throughout the 7 years of the surveillance programme and are closely related to each other and to genogroup C1 viruses isolated elsewhere. Sarawak has so far not experienced large HFMD outbreaks caused by HEV71 of genogroup C1. Indeed HEV71 of subgenogroup C1 behave much like other species A enteroviruses, occurring sporadically throughout the surveillance period.\nc. In Sarawak, occurrence of HEV71 genogroup B infections is tightly clustered, with cases rising and falling very rapidly." }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: " }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: Infections caused by viruses persistently threaten human health. For example, 40 million, 350 million, and 170 million people in the world are carrying human immunodeficiency virus type 1 (HIV-1), hepatitis B virus (HBV), and hepatitis C virus (HCV), respectively [1] [2] [3] . Annually 5% to 15% of the global population is infected with influenza, resulting in 250,000 to 500,000 deaths [4] . Protection against viral infections may be provided by inoculations with live-virus, killed-virus, or subunit vaccines. Live-virus vaccines offer advantages because they activate both humoral and cellmediated immunity, require only a single boosting, and generally provide longer immune protection than other forms of vaccines. However, they must be adequately attenuated in their growth to minimize the possibility of vaccine-induced pathogenic effects while retaining their immunogenicity. Attenuation of live viruses has traditionally been achieved by serially passaging viruses in tissue or cell culture and adapting them to grow well on new cell types or at elevated or reduced temperatures [5] , a process that tends to reduce their replicative ability and virulence in humans or animals [6] . Such attenuation has historically been a highly empirical process, where its mechanisms are often neither known nor elucidated.\nDuring the last decade the emergence of reverse genetics techniques has created unprecedented opportunities to better control viral attenuation [7] [8] [9] . Reverse genetics enables the production of RNA viruses from cloned cDNA, so specific mutations can be relatively easily introduced into viruses. The challenge to engineering viruses for attenuation then shifts from creating the variants to predicting how specific genetic changes define or correlate with measurable effects on virus growth. Such a challenge can be addressed through the development of quantitative and mechanistic models that map genome-level changes to the dynamics of virus growth under different environmental conditions. Models of intracellular virus growth aim to predict how rapidly a virus-infected cell will produce virus progeny. Inputs to such models include rates of constituent processes such as entry of the virus into the cell, transcription of viral mRNAs, translation of viral proteins, replication of viral genomes, assembly of intermediates, and finally, production and release of viral progeny. Decades of detailed biochemical, biophysical, and genetic studies have, for diverse viruses, contributed toward a level of mechanistic understanding of viral functions and interactions. Various intracellular models of virus growth have been developed for phage Qb [10] , phage T7 [11, 12] , HIV-1 [13] , and influenza A virus [14] .\nHow can a detailed model for the intracellular growth of a virus be used to explore the behavior of mutant viruses that encode alternative designs? As a starting point, one can create alternative designs that reorder or rearrange the wildtype genes or regulatory elements. Such genomic changes can alter the timing and level of expression of different viral genes, and thereby impact growth because the production of viral progeny depends on the dynamic expression of viral genes. Preliminary models of such alternative genome designs can use the ''language'' of the wild-type virus. They retain the parameters that characterize wild-type molecular interactions, wild-type average rates of viral polymerase elongation, and wild-type composition of progeny viruses, but they apply them in a manner that reflects the reordering of wild-type components in the engineered genome. For example, the timing of expression for the genes of phage T7 during infection maps closely to their sequential order on the T7 genome [15, 16] . By relocating an essential early gene, encoding the T7 RNA polymerase to downstream positions, one delays initiation of transcription by the highly efficient T7 RNA polymerase and thereby attenuates phage growth [17] . Preliminary models for the growth of phage carrying the altered genomes, based on wild-type parameters, capture the overall observed trends in attenuation. Here we expand this approach to a mammalian virus of biomedical and agricultural relevance: vesicular stomatitis virus (VSV).\nVSV is a prototype negative-sense single-stranded RNA (Mononegavirales, (-)ssRNA) virus and a member of the family Rhabdoviridae [18, 19] , which includes rabies virus. Each VSV particle has a single copy of an 11-kb genome carrying five genes that encode nucleocapsid (N), phospho (P), matrix (M), envelope (G), and polymerase (L) proteins. VSV is economically important because it can cause symptoms like those of foot-and-mouth disease in livestock [19] . It offers several advantages as a vaccine vector including low seropositivity in humans, a capacity to accommodate foreign genes up to 40% of its own genome size, and an established reverse genetics [18, 20] . Recombinant forms of VSV carrying foreign virus genes that encode immunogenic proteins have been proposed as potential vaccines against HIV, influenza, and respiratory syncytial virus [21] [22] [23] [24] [25] . Less pathogenic but more immunogenic VSV-based vaccines against infection by VSV or other viruses are being sought.\nHere we develop an in silico model of a VSV infection cycle, incorporating known regulatory interactions and mechanisms and relevant quantitative data from the literature of the past 40 years. These interactions and the corresponding equation formulations are described in detail in the model development section of Materials and Methods. Using the model, we first quantitatively analyze how the intracellular growth of wild-type VSV directs host biosynthetic resources toward VSV gene expression, synthesis of progeny genomes, and pathway switching from the synthesis of VSV intermediates to the production of VSV progeny. We then reveal that the model captures experimental results showing progressive attenuation of virus growth associated with moving the N gene downstream from its wild-type position. Finally, we use the model to predict how altering the positions of other VSV genes and promoters may attenuate the growth of VSV while increasing its potential capacity to activate an adaptive immune response.\nUsing our model with the established parameter set (Tables 1 and 2) , we first analyzed quantitatively and systematically how the intracellular growth of VSV is regulated. The improved understanding of the virus infection by this model-based analysis may guide us to identify the key regulatory components to manipulate for developing virus mutants as possible vaccine or vector candidates.\nAttenuation mechanism leads to unequally distributed synthesis of viral mRNAs and proteins. The partial transcription termination mechanism (or attenuation) is common in (-)ssRNA viruses. This mechanism is important to satisfy the different needs of each viral protein during its infection cycle. Five attenuation factors for each intergenic region of the VSV genome (Table 1 and Equation 8 ) were obtained from the literature [18, 19, 26, 27] and incorporated into our model.\nOwing to the step-wise release of polymerases from each gene junction, our simulations estimated the gradual decrease of VSV mRNA synthesis in the order of N . P . M . G . L ( Figure 1A ). Compared with the most abundant N mRNA, L mRNA is very scanty in infected baby hamster kidney (BHK) cells (40 ; 140-fold less). The relative production level of each protein matched the relative availability of the corresponding mRNAs ( Figure 1B ) even though different proteins degrade at different rates ( Table 1 ). Because of the different level of incorporation of each protein into a single virion particle, as defined by the protein stoichiometry (Table 2 , [28, 29] ), the relative levels of free viral proteins in the cytoplasm develop differently from their mRNA levels ( Figure 1C ). P protein is most abundant owing to its low content in the virion, and L and N proteins are least abundant. The persistently low level of N protein is related to its immediate complexation with nascent genomes and antigenomes to make nucleocapsid particles during the replication step. Owing to the cyclic switching between transcription and replication by the encapsidation process, the N protein level is predicted to oscillate as shown in Figure 1C .\nThe engineering of viral genomes provides ways not only to explore viral regulatory mechanisms at a genomic level, but also to produce recombinant viruses that may serve as vaccines, gene delivery vectors, and oncolytic (tumor-killing) agents. However, the complexity of interactions among viral and cellular components involved in the life cycle of a virus can make it challenging to anticipate how altering viral components will influence the overall behavior of the virus. Lim, Lang, Lam, and Yin have developed a computer model that begins to mechanistically account for key virus-cell interactions in its predictions of viral intracellular development. Lim et al.'s model was able to capture experimentally observed effects of gene rearrangements on the levels and timing of viral protein expression and virus progeny production, aspects that are important for the design of live-virus vaccines. Refinement and extension of their approach to current and other virus systems has the potential to advance the application of viruses as therapeutic agents.\nAfter 9 h post-infection, our simulation predicts a significant decrease of free M proteins. This arises from the dominance of the virion assembly process, which depletes M proteins, compared with transcription and replication. In infected murine delayed brain tumor (DBT) cells, similar distributions of viral mRNAs and proteins were obtained (unpublished data).\nHigher demands for genomes are satisfied by the stronger promoter of the anti-genome template relative to that of the genome template. Anti-genome templates are only utilized to amplify genomes, while genome templates are used to amplify both anti-genomes and mRNAs, and they are also incorporated into virion progeny particles. The higher demands for genome by these multiple tasks are satisfied by the stronger promoter of the anti-genome template compared with that of the genome template [30] [31] [32] . More polymerases bind to the stronger promoter of the anti-genome, ultimately enhancing the production of genomes over anti-genomes ( Figure 2 ). In our model the parameter S prom measures the strength of the anti-genomic promoter relative to that of the genomic promoter ( Table 1 ). The production ratio of genomes to anti-genomes was estimated to be dynamically changed [33] , varying from one to 30 (wild-type VSV case (S prom ¼ 5.4), Figure S1 ). Such oscillatory changes in the production ratio shown in Figure S1 follow from the on-off use of the genomes for transcription or replication. They also arise owing to the staggered shifting of dominant templates between genomes and anti-genomes during replication. A large value of S prom favors use of anti-genome templates to replicate genomes. However, as genome templates accumulate in large excess relative to anti-genome templates, they successfully compete for replication resources. Synthesis of anti-genomes then dominates until they accumulate and serve again as the dominant templates.\nThe virion production rate in BHK cells is at maximum 5-10 h post-infection. In infected DBT cells, similar simulation results were obtained except that the synthesis of genomesized viral RNAs continued for longer time (active until 15 h post-infection, Figure 2 ).\nOptimal utilization of genomic nucleocapsids. Genomic nucleocapsids can either be used as templates for RNA synthesis or they may be incorporated into progeny virions. Their fate depends on levels of polymerase and M protein, which respectively favor RNA synthesis or virion production pathways, as well as on the extent to which association of the nucleocapsid with M protein will dominate over association with polymerase, described with the parameter S cond in our model (Table 1) . Because both RNA synthesis and virion production are essential processes of the infection, extreme values of S cond that favor one process over the other will be [49] S pol ¼ 170 nt Strength of anti-genomic promoter relative to that of genomic promoter a S prom ¼ 5.43 Rate constant ratio of the associations of M protein and polymerase with the genomic nucleocapsid a S cond ¼ 6.2 3 10 À5\nFraction of M protein bound to the plasma membrane [51] cond M ¼ 0.1 Attenuation factors [18, 19, 26, 27] / N / / P / / M / / G / / L ¼ 0.0 / 0.25 / 0.25 / 0.25 / 0.95 Degradation rate constants N protein [58] k dp,N ¼ 3.5 3 10 À5 sec À1 P protein [59] k dp,P ¼ 1.4 3 10 À6 sec À1 M protein [58] k dp,M ¼ 1.5 3 10 À4 sec À1 G protein b k dp,G ¼ 5.7 3 10 À5 sec À1 L protein [59] (if concentration P protein ! 10 3 concentration L protein ) k dp,L ¼ 1.2 3 10 À5 sec À1 (if concentration P protein , 10 3 concentration L protein ) k dp,L ¼ 4.3 3 10 À5 sec À1 mRNA [60] k d,mRNA ¼ 1.9 3 10 À4 s À1 nucleocapsid c k d,nc ¼ 1.9 3 10 À5 s À1 Host-dependent parameters Number of ribosomes in host cell [61] nrib ¼ 5 3 10 6 Elongation rate of ribosome [62] k e,rib ¼ 6 aa/s Spacing between neighboring ribosomes a (BHK/DBT) Srib ¼ 238. 5 Owing to their increased stability by encapsidation, nucleocapsids are less degradable than naked mRNAs. It was assumed that the nucleocapsids of VSV are degraded 10-fold slower than its mRNAs, and our simulation results were insensitive to this parameter. DOI: 10.1371/journal.pcbi/0020116.t001 detrimental for growth. For excessively large S cond , newly synthesized genomic nucleocapsids would tend to be prematurely incorporated into virion particles before they could serve as templates for transcription and replication. On the other hand, for extremely small S cond , genomic nucleocapsids would be utilized primarily to produce viral RNA without being packaged into viral progeny. Hence, an intermediate parameter value is expected to be optimal for viral growth. We estimated a possible range for the wild-type value of S cond by fitting our simulation results to previous experimental observations by others (2.5 3 10 À5 to 1.0 3 10 À4 , Figure S4 ). Our simulations further indicate that this range of S cond is near-optimal and optimal for VSV growth on BHK and DBT cells, respectively (unpublished data). Diversion and inhibition of host translation machinery create a time window of opportunity for translation of viral proteins. During the infection cycle, virus actively and passively competes with the host for limited translation resources by inhibiting host transcription and by amplifying viral mRNAs, respectively. Viral leader-mRNA and M protein play key roles in this inhibition [19, 28, [34] [35] [36] . As viral components accumulate in the cytoplasm from the initiation of infection, an ever-increasing fraction of host ribosomes are available for viral mRNAs ( Figure 3A , the fraction of ribosomes associated with viral mRNAs is defined by 1-rib_host in Equation 26 ). However, the inhibition of host macromolecular synthesis causes a failure to supply accessory factors needed for initiation and elongation steps of translation, resulting in a reduction in the fraction of active ribosomes over time ( Figure 3A , as described by f dec in Equation 25 ). These two mechanisms create a time window when active ribosomes are maximally available for viral translation in infected cells ( Figure 3A , refer to the term (1-rib_host)*f dec in Equation 27 ). The abundance of viral mRNAs and the limitation imposed by ribosomal spacing determine the fraction of the active ribosomes involved in translating viral mRNAs (occupied ribosomes in Figure 3B , refer to Equations 11 and 27) . In our model, if the occupied active ribosomes are less than the available ones (in this case the number of free active ribosomes . 0), viral translation is fully supported without any limitation of host machinery. In the early infection stages up to 7 h and 13 h post-infection for BHK and DBT cells, respectively, the host machinery is in excess ( Figure 3B ). However, at later times viral translation becomes limited by the host resources (in this case the number of free active ribosomes ¼ 0). This limitation may cause a transition from replication-dominant to assembly-dominant infection stages because the replication requires the continuous protein synthesis. As shown in Figure 3B , a small fraction of ribosomes as active forms (less than 5% out of 5 3 10 6 ribosomes, Table 1 ) are utilized for viral translation. Experiments and simulations of VSV gene-order mutants. For vaccine applications, one seeks to minimize viral pathogenicity and maximize its immunogenicity. Based on observed correlations between in vitro and in vivo results, we assume here that the pathogenicity and the immunogenicity of a virus are directly linked to the levels of progeny production [20, 37, 38] and G protein expression in infected cells [39, 40] , respectively. In the previous section we have showed that various VSV regulatory mechanisms are involved in maintaining balances, during infection, among viral synthesis processes, which indirectly indicates the importance of such balances for viral growth. Perturbations of such balances by genetic or genomic manipulations could provide ways to obtain viral phenotypes favorable to vaccine applications. We first test the predictive ability of our model by comparing simulated protein expression and growth of several gene-rearranged VSV strains with experimental results. Later we employ the model to predict how various genomic manipulations could attenuate virus growth and increase G protein expression.\nProtein expression rates of gene-rearranged viruses. The stepwise decline in the transcription of genes more distant from the 39-end region promoter highlights how gene order affects gene expression in VSV. Advances in reverse genetics have made it possible to create gene-rearranged virus strains where the transcriptional attenuation mechanism then creates altered levels of gene expression [7, 9, 18, 20] . In one study [18] the three internal genes, P, M, and G, were permuted, and the resulting six possible VSV strains were characterized. Relative rates of viral protein expression in BHK cells were experimentally measured based on their incorporation of [ 35 S]-labeled methionine for a one-hour window at 4 h post-infection [18] . We extended our model to simulate this experiment for mutants representing each geneorder permutation and compared the model prediction with the published results [18] , as shown in Figure 4 . All rates are expressed relative to the synthesis rate of N protein, whose corresponding gene was in position 1, closest to the 39 end of the genome in all strain cases. Expression of gene L, in position 5, was minimal in both simulations and experiments, and the expression of all other genes was above 40%, a feature of the experimental data that the simulation also captured. Most of the points fall close to the parity line, indicating agreement between the simulation and experiment. Noteworthy are two subsets of points. First, the four circled points are exceptions to the general rule that gene order determines the level of gene expression. These were genes in the second position of the genomes that were expressed essentially at the same rate as gene N, in the first position [18] . This result highlights that the expression rate of protein is affected not only by its gene order, or corresponding rate of mRNA production, but also by its length and degradation rate. For a fixed average rate of translational elongation, longer gene products will tend to be produced more slowly. Further, the net rate of protein production will reflect the rates of both protein synthesis and protein degradation. The model accounts for these contributions, and for the circled genes such accounting appears to capture unexpected high translation levels of genes in the second position, which were measured by Ball et al. [18] . The second sets of points, shown in two boxes, indicate mismatches ( Figure 4 ) that, in the most challenging scenario, could reflect unknown strain-specific mechanisms that are not present in our general gene-permutation model. However, one should also note that the experiment is based on labeling and quantifying proteins about 4 h post-infection. This relatively early time point allows one to minimize potentially confounding influences of virion particle assembly and production on cytoplasmic levels of viral proteins, but it also represents a point before the majority of viral proteins have been made ( Figure 1B) .\nGrowth of gene-rearranged viruses. We also employed our model to predict the growth of VSV strains having the N gene first position on the genome grows best, followed by N2, N3, and N4. This result is consistent with the previously suggested hypothesis that relocation of the N gene to 39-distal positions on the genome would be an efficient way to attenuate VSV for vaccine use [20] . The reduction in growth that follows from moving the N gene likely reflects, at least in part, an imbalance between replication and transcription. Insufficient production of N protein would reduce the extent of encapsidation of nascent anti-genome and thereby allow transcription to dominate over genome replication [41] . While the simulation matches the growth ranking, it did not quantitatively match the experimental data. The predicted variation in virion production (N1 ! N4: 4.7-fold decrease) is smaller than the experimentally observed variation (N1 ! N4: 16.7-fold decrease). A potential source of this quantitative difference was our neglect of mass action effects of N proteins on the encapsidation process in our model; encapsidation was simulated as an instantaneous process when free N proteins were available. As shown above, our model could capture the major effects of gene rearrangement on viral growth and protein expression.\nEffects of relative promoter strength on viral growth. The genome and anti-genome of VSV are synthesized in unequal amounts, determined by the differing strengths of their promoters [19, 31, 33, 39] . The stronger promoter of the antigenome allows viral polymerases to produce more genomes to meet their demands as components of virion particles and as templates for transcription and replication. To explore how VSV growth is influenced by differences in the relative strength of the genomic and anti-genomic promoters, we predicted the yield of virus on BHK and DBT cells over a broad range of S prom , as shown in Figure 6A . Small S prom virus cannot grow well because most polymerases would be associated with genomes and tend to synthesize primarily anti-genomes. For example, for S prom equal to 0.1, infected BHK and DBT cells make 5-fold and 26-fold fewer progeny than wild-type VSV infected cells, respectively. However, large S prom virus also cannot grow well because most polymerases would preferentially bind to newly synthesized anti-genomes, producing few of the anti-genomes that are needed as templates to amplify genomes. Our simulations predicted that values of 30 and 50 for S prom would be optimal for VSV growth in BHK and DBT cells, respectively ( Figure 6A ). The estimated wild-type value of S prom of 5.4 gives VSV yields higher than 80% of their maximum yields for both cell types ( Figure 6A) .\nWe speculate that a rational way to attenuate the pathogenicity of the live wild-type virus would be to swap its two promoters, giving an S prom of (5.4) À1 . For this promoter swap we predict virion production would be decreased by 3.3fold and 14.5-fold for infected BHK and DBT cells, respectively, relative to wild-type. However, the extent of growth attenuation by the promoter swapping can be higher than the model prediction because the swapping may also perturb viral transcription and virion assembly and budding processes modulated by the signals encoded in the 39and 59 termini of the genome [42, 43] that are not yet sufficiently defined to be included in the simulation.\nRational vaccine attenuation by double genomic manipulations. Several variant VSV strains, including N1 through N4, have been made by Ball and Wertz [18, 20] . With an aim to generate a potentially broader diversity of growth phenotypes, we created and tested in silico VSV mutants by combining N gene relocations with a range of S prom . This is a computationally simple task, but experimentally nontrivial. For example, the VSV N4 with S prom ¼ 0.1 produced a simulated 38-fold fewer virus progeny than wild-type in BHK Figure 6B ). It is interesting to note that this 38-fold degree of growth attenuation is greater than the product of the constituent attenuations (4.7 3 5.4 ¼ 25) that one calculates by assuming that the effects were uncoupled. Such nonmultiplicative effects of double genomic manipulations on growth would be challenging to predict in the absence of a quantitative model.\nModulation of VSV immunogenicity by gene shuffling. To elicit a systematic immune response, live viral vaccines must present or display neutralizing epitopes, typically through the expression of viral surface proteins. Higher levels of antigen expression have been found to correlate with more rapid and potent induction of anti-viral antibodies [39, 40] . As Flanagan et al. suggested, the gene encoding G may be moved to other positions in the VSV genome to modulate the expression of the VSV surface glycoprotein G [39] . We generated in silico five gene-shuffled VSV strains, having gene orders for the three internal genes, MPG, MGP, PGM, GMP, and GPM, and simulated levels of G protein in BHK cells infected with those strains. Our simulation results were consistent with the idea that the location of G gene affects the production of G protein. The GPM strain gave the highest concentration of G protein in the cytoplasm (almost 2-fold higher than that of wild-type, Figure 7 ). However, in many cases effects of such gene rearrangements can be difficult to anticipate because of the complexity of the involved interactions among viral components. For example, the PGM strain showed only a level of G protein expression similar to those of PMG (wt) and MPG strains even though it has the G gene at an earlier position than the other two strains (Figure 7) .\nFor vaccine use we might aim to maximize the immunogenicity of VSV or a VSV-based vector through the expression increase of VSV G gene or inserted foreign gene while minimizing their potential pathogenicity by growth attenuation. Given such design goals, specifically for a VSV vaccine, we might prefer strain GPM, which showed the highest expression of G protein (Figure 7 ) and the lowest production of virions [18] . Toward such favorable features, Flanagan et al. previously constructed three VSV strains having the following gene orders: 39-G-N-P-M-L-59 (G1N2), 39P-M-G-N-L-59 (G3N4), and 39-G-P-M-N-L-59 (G1N4) [39] . These genome constructions were based on their intuitive idea that translocations of G gene and N gene to earlier and later positions, respectively, compared with wild-type, could not only increase the expression of G protein, but also attenuate virus growth [39] . This idea was supported by their experimental results [39] .\nSeeking a more detailed correlation between locations of the two genes and the viral phenotypes relevant to vaccine application, we simulated in silico the growth of all mutants that retain the gene order P -M -L of the wild-type, but allow G and N to move, criteria that define 20 possible geneorder permutations. The viral growth and the level of G protein in infected BHK cells mainly depend on the locations of N gene and G gene, respectively ( Figure 8A and 8B) , which is consistent with the experimental results of Flanagan et al. [39] . Further, if gene G is fixed, then moving gene N closer to the 39 promoter is predicted to increase protein G expression ( Figure 8B ). Consistent with this prediction, Flanagan et al. also observed a higher G protein expression for the G1N2 strain than for the G1N4 strain [39] . Enhanced replicative ability of VSV strain by locating its N gene at an earlier position in its genome can contribute to increasing the level of G protein. If either gene N or gene G is located at the fifth position, then both levels of virus growth and G protein expression are very low (Figure 8) , because with such genome organizations the stoichiometric amounts of N and G proteins required for replication and assembly (Table 2 , [28, 29] ) cannot be reached. Our simulations with the BHK cell parameters (Table 1 ) overall captured the experimentally established relative growth of the VSV strains in BHK-21 cells, but the growth of the G3N4 strain was significantly overestimated compared with the experimental results [39] ( Table 3 ). The relevant mechanism for such a large discrepancy between the simulation and the experimental results remains to be elucidated.\nThe changes of protein expression levels by gene shuffling can be a rational means to modify the viral features for vaccine use. Robust synthesis of antigen by a highly attenuated strain appears to be an effective vaccine strategy as Flanagan et al. previously suggested. In addition to controlled attenuation of virus growth, a potent vaccine should ideally elicit a strong humoral or cell-mediated immune response.\nIn the era of highly advanced genetic technologies, we have witnessed a turning point for the development of live viral vaccines. Conventional empirical vaccine development processes are now being replaced by more rational reversegenetics-based ones. With this trend, much attention will be focused on mechanism-based design of less pathogenic and more immunogenic virus stains. Mathematical models for intracellular virus growth can support this design process by providing a tool to systematically analyze the viral infection regulatory network, identify critical regulatory mechanisms or components for redirecting viral phenotypes, and reverse engineer desirable phenotypes. One-step infection of cell monolayers. Cells were harvested, resuspended in growth medium, and plated into six-well plates at a concentration of 5 3 10 5 cells per 2 ml per well. Plated cells were returned to the incubator and allowed to grow overnight. The next day, two representative cell monolayers were harvested and counted to give an approximate number of cells per well. Each monolayer was then incubated with 200 ll of virus inoculum (MOI 3) for 1 h to allow virus adsorption. The plates were rocked gently every 20 min to evenly distribute virions on the monolayers during the adsorption step. After the adsorption period, the monolayers were rinsed twice with 1 ml of HBSS and then placed under 2 ml of infection medium for incubation. Medium samples of 200 ll including virion particles were taken from each well at 2, 3, 4, 6, 8, 10, and 20 h postinoculation. Samples were kept frozen at À90 8C until their analysis by the plaque assay.\nPlaque assay. BHK cells were plated into six-well plates and cultured to 90% confluence. Culture medium was removed from each well and replaced with 200 ll of serially diluted viral samples. The inoculated monolayers were returned to the incubator for 1 h to allow virus adsorption. The plates were rocked gently every 20 min. At the end of the adsorption period, the inoculum was removed from each monolayer sample and then replaced with 2 ml of agar overlay. The agar overlay consisted of 0.6% weight/volume (w/v) agar (Agar Nobel, Difco, BD Diagnostic Systems, http://www.bd.com). 5-Bromo-29-deoxyuridine (B5002, Sigma) was added, at 100 lg/ml, to the agar overlay of N3-and N4-infected samples to enhance plaque formation. Following agar addition, the plates were allowed to cool at room temperature for 30 min, returned to the incubator and incubated for 24 h, and then each sample was fixed with 2 ml of fixative for 3 h at room temperature. The fixative consisted of 4% (w/v) paraformaldehyde (VWR) and 5% (w/v) sucrose (Sigma) in 10 mM phosphate buffered saline (PBS, Sigma) of pH 7.4. The agar overlay was then removed, and each sample was rinsed twice with 2 ml of PBS. Gentian violet diluted in methanol (0.01% (w/v), Sigma) was used, at 1 ml each, to stain the samples. Model development. Using algebraic and differential forms of equations, our mathematical model aims to account for established molecular processing steps in the development of VSV. Most model parameters were extracted from the literature. However, five parameters were obtained by fitting our simulation results to experimental data that were from the literature and our own experiments. Key model parameters are given in Table 1 , and detailed descriptions of the model and parameter estimation process are provided below, and in Protocol S1 and Figures S2 and S3 , respectively.\nVirus binding and penetration. As shown in Figure 9A , VSV initiates an infection by binding to a receptor such as phosphatidyl serine, a lipid component in the plasma membrane [19, 44] . After the binding step, the VSV particle is endocytosed via a clathrin-coated pit, and then penetrates intracellular vesicles such as endosomes by membrane fusion [19, [45] [46] [47] . The penetration leads to the release of the encapsidated negative-sense viral genome and virus proteins into the cytoplasm of the host cell. By assuming the binding step is ratedetermining [48] , we lump these early steps from the binding to the penetration into a first-order expression:\nwhere V b and V ex are the concentrations of bound and extracellular virus particles, respectively, t is time, and k b is the apparent rate constant for virus binding. After binding, we assume the bound virus is immediately endocytosed and fused, and its genome and protein components are instantaneously released into the cytoplasm at the expense of the fused virus particle. The protein stoichiometry of a single VSV particle and the lengths of each viral gene and protein are summarized in Table 2 , [28, 29] . Population distribution of polymerases and nucleocapsids. Following the release of the encapsidated genome and proteins into the cytoplasm, VSV transcription is initiated. The viral transcription was assumed to be independent of host-cell functions such as replication [33] . Instead, the viral complex of L and P proteins, with a stoichiometry of 1-3.6, was taken to function as polymerase in transcription and replication [49] . In the absence of P protein, L protein cannot bind to the genome or anti-genome [50] . After binding to the 39 promoter regions of the genomic and anti-genomic templates, the viral polymerase starts to synthesize its own RNA transcription and replication products by elongating along the templates. During transcription a fraction of elongating polymerases terminate transcription by dissociating from the templates as they encounter regulatory signals at intergenic regions [19, 26] . In addition to the regulated polymerase dissociation, time-dependent concentration changes of the polymerases and the viral templates in the cytoplasm influence the distribution of polymerases on the viral templates during transcription and replication. Hence, the distribution of polymerases continuously varies over the viral templates, ultimately determining the relative synthesis levels of mRNAs and genome-size RNAs.\nWe simulate the transcription and replication processes by considering the spatial-temporal distribution of template-associated polymerases. We first partition the viral genome and anti-genome templates into 40 segments, excluding their 39 and 59 end regions, which are the leader (Le) and trailer regions (Tr) for the genome, and the complementary trailer (Trc) and complementary leader regions (Lec) for the anti-genome, respectively ( Figure 9B ). For the genome template that is used for transcription as well as for replication, we specially grouped the segments into five genes ( Figure 9B ). We chose 40 as a minimum number for total segments which allows each gene to be split into a specific integer number of segments, proportional to the length of the gene. By considering the mechanisms for the interactions between polymerase and the intergenic regulatory sequences of the templates, as described below in the Transcription section, we simulated the polymerase flux into each segment over the time elapsed from the initiation of transcription on each template. Then we correlated the level of polymerase occupying each geneencoding section of the template with the synthesis rate of each corresponding viral mRNA. In a similar way, the distribution of polymerases on the replication templates was correlated with the synthesis rate of viral genome-sized RNA. Such explicit treatment of polymerase spatial distributions on the viral genome and antigenome templates was central to modeling the growth of wild-type and gene-rearranged virus strains. This treatment systematically accounts for polymerization-associated time delays and the polymerase fluxes into each template segment.\nBefore estimating the polymerase flux, we need to figure out how the polymerase complex and M protein compete with each other for binding to the genomic nucleocapsids as well as how the polymerases bound to nucleocapsids are subsequently distributed to one of three possible tasks: transcription, replication of genome, or replication of anti-genome. In our model we assume that the genomic templates (negative-sense nucleocapsids) whose promoters (leader regions) are free of polymerases are available for association with free polymerase or M protein. We further assume that the associations of the free genomic templates by M proteins or polymerases take place instantaneously:\nwhere (-)nc, (-)nc M,new , and (-)nc pol,new are the concentrations of total genomic nucleocapsids and subsets of genomic nucleocapsids whose promoters are newly occupied by M protein and polymerase, respectively. S pol is the spacing between neighboring polymerases on the genomic or anti-genomic template, pol l is the concentration of polymerases bound to the promoter region (Le) of the genomic template, and l l is the length of the promoter region. Specifically, the second term in the left-hand side of the equation denotes the concentration of the genomic templates whose promoters are currently occupied by polymerases. In our model the concentration of the genomic nucleocapsids whose promoters are bound to polymerases and the concentration of the polymerases bound to the promoters of the genomic nucleocapsids are interchangeable with each other by the factors (l l /S pol ) and (S pol /l l ), respectively. The binding of M protein or polymerase initiates reactions leading to virion assembly or RNA synthesis, respectively. Because the initiation of RNA synthesis by the polymerase requires a finite time, a space between adjacent polymerases on the template (S pol ) would be maintained during infection, assuming a fixed elongation rate. With these considerations, one may expect that at any time the concentration of nucleocapsids available for the new binding of the free proteins is inversely proportional to the concentration of polymerases currently bound to the leader region of the genomic nucleocapsids (pol l ) and the polymerase spacing (S pol ) as shown in the second term of Equation 2.\nThe ratio of (-)nc M,new to (-)nc pol,new is determined by the ratio of the association rates of M protein and polymerase with the genomic nucleocapsid, which is further a function of the rate constants and relative amounts of the corresponding free components in the cytosol:\nwhere r asso,M and r asso,pol are the rates of the associations of M protein and polymerase with the genomic nucleocapsid, respectively, and k M and k pol are the rate constants for each association reaction, respectively. S cond denotes the ratio of the two rate constants (¼k M / k pol ). Unlike L protein, 10% of synthesized M proteins are associated with the plasma membrane [51] . In Equation 3, cond M is the fraction of M proteins associated with the plasma membrane, trans is the fraction of L proteins satisfying the polymerase stoichiometry with P protein, pol total is the total concentration of polymerases associated at the time with nucleocapsids, and M and L are the total concentrations of M and L proteins not assembled into viral progeny. If the concentration of P protein (P) is larger than 3.6-fold concentration of L protein, then trans is equal to 1. Otherwise, trans is equal to P/(3.6L). In our model, M and L proteins compete for free genomic nucleocapsids, and the condensed nucleocapsids, owing to their association with M proteins, cannot be utilized for transcription or replication [19] . From Equations 2 and 3, the newly occupied nucleocapsids by polymerases ((-)nc pol,new ) can be calculated:\nIn the same way, given S pol , the concentration of positive-sense antigenomic nucleocapsids available for binding to polymerases would be ((þ)nc -pol trc (S pol /l trc )), where (þ)nc is the total concentration of antigenomic nucleocapsids, pol trc is the concentration of the polymerases bound to the promoter region (Trc) of the anti-genomes, and l trc is the length of the promoter region. Because the anti-genome has a stronger promoter than the genome [19, 31] , which is quantified by S prom in our model, S prom -fold, more polymerases bind to the promoter of the anti-genome than to that of the genome. Under the limitation of free polymerase complex, the concentration of the polymerases newly binding to the promoters of the genomes or the anti-genomes (pol term new ) could be described as follows: \nwhere pol trc new is the concentration of the polymerases newly binding to the complementary trailer region (promoter) of the anti-genome. The polymerases newly binding or already bound to the promoters of the genomes and anti-genomes start viral RNA synthesis as transcription or replication process.\nTranscription. The viral polymerase on the leader region of the genome starts either transcription or replication. If there are sufficient N proteins, transcription is inhibited by the encapsidation of nascent positive-sense RNAs by N proteins; then replication dominates transcription [41, 52, 53] . In contrast, if there are insufficient free N proteins, then transcription dominates replication. In the model we correlate the extent of transcription dominance with the availability of N proteins by introducing a factor, encap. This factor is defined as the ratio of the number of free N proteins to the number required to encapsidate all available nascent genome-sized viral RNAs. Only nocap (¼ 1 À encap) of the polymerases bound to the genomic promoters can start the transcription: dpol N;1 dt ¼ k e;pol ðð1 À / N Þ nocap pol l l l À n sec;N l mRNA;N pol N;1 Þ ð 7Þ\nwhere pol N,1 is the concentration of the polymerases located at the first segment of the N gene ( Figure 9C) , k e,pol is the elongation rate of polymerase, / N is the attenuation factor for N gene, n sec,N is the total number of the segments of N gene, and l mRNA,N is the length of N mRNA (Table 2 , [28, 29] ). The genome segments are continuously charged with incoming polymerases and discharged with outgoing polymerases with a rate of k e,pol (Equations 7-9). If the polymerase input to the leader region of the genome is decreased owing to a lack of free polymerases, then the polymerase concentrations downstream of the leader region will be subsequently reduced ( Figure 9C ). There are conserved intergenic sequences involved in letting a fraction of viral polymerases release from the genome template at intergenic sections during transcription, which is so-called partial transcription termination or attenuation ( Figure 9C ) [18, 19] . Because the transcription is initiated from the 39 end promoter, the attenuation mechanism causes genes more proximal to the 39 end to be more highly expressed, which ultimately leads to an unequal concentration distribution of viral mRNAs. The extents of partial transcription termination are quantified by the attenuation factors, / i , in our model ( Figure 9C ). These are 0/0.25/0.25/0.25/0.05 for leader-N/N-P/ P-M/M-G/G-L intergenic regions, respectively [18, 19, 26, 27] . / i fraction of polymerases are released at intergenic region i. With Equations 7-9, we simulate the polymerase flux into each gene segment, which is proportional to the elongation rate of polymerase, but inversely proportional to the extent of attenuation: dpol i;j dt ¼ k e;pol ðð1 À / i Þ n sec;iÀ1 l mRNA;iÀ1 pol iÀ1;nsec;iÀ1 À n sec;i l mRNA;i pol i; j Þ j ¼ 1; i ¼ P; M; G; L ð8Þ dpol i;j dt ¼ k e;pol n sec;i l mRNA;i ðpol i; jÀ1 À pol i; j Þ j 6 ¼ 1;\nwhere pol i,j is the concentration of the polymerases located at the jth segment of gene i, and iÀ1 indicates the prior gene of gene i. The amount of newly synthesized mRNAs for each gene is determined by the concentration of polymerases occupying each gene section on the genome template and the decay rates of the mRNAs:\nwhere mRNA i is concentration of mRNAs for gene i, k d,mRNA is the decay rate constant of mRNA that is the same for all five viral mRNAs [54] , and pol t,i is the total concentration of the polymerases occupying on the ith gene. Our formulation for transcription assumes that the synthesis of viral mRNAs is rate-controlled by the transcription initiation as well as the elongation of polymerase. Transcription initiation rate is parameterized by the spacing between neighboring polymerases in our model. At a given polymerase elongation rate, the larger polymerase spacing indicates the lower rate of transcription initiation. Transcription initiation modulates the input of polymerases to the leader region of the genome.\nTranslation. We consider that both translation initiation and polypeptide chain elongation contribute to the rate of viral protein synthesis. The translation initiation rate is parameterized by the ribosomal spacing. In our model we first calculated the number of ribosomes involved in viral translation by considering the maximum concentration of the ribosomes bound to viral mRNAs at a fixed ribosomal spacing:\nwhere rib and n rib,avail are the concentrations of the ribosomes actually involved in viral translation and the ribosomes available for viral translation, respectively, and S rib is the spacing between neighboring ribosomes.\nThe ribosomes involved in viral translation (rib) are allocated to the five types of viral mRNAs according to their length and abundance, assuming that each viral mRNA has the same efficiency of translation initiation [55] :\nwhere rib i is the concentration of the ribosomes assigned to mRNA i. The synthesis rate of each viral protein depends on the elongation rate of the ribosome, linear density of ribosomes on its corresponding mRNA, and its first-order decay rate: dp i dt ¼ k e;rib l p;i rib i À k dp;i p i i ¼ P; M; G; L ð13Þ\nwhere p i is the concentration of protein i, k e,rib is the elongation rate of ribosome, l p,i is the length of protein i, and k dp,i is the decay rate constant of protein i. We also accounted for the consumption of free N proteins during the encapsidation of genome-length nascent RNAs and assumed that the degradation of nucleocapsids yielded intact N proteins: dp i dt ¼ k e;rib l p;i rib i À k dp;i p i À n i ðencap Á k e;pol pol tr l tr þ pol lec l lec\nwhere n N is the stoichiometry of N protein in a single nucleocapsid or virion progeny (Table 2 , [28, 29] ), pol tr and pol lec are the concentrations of the polymerases located on the trailer and complementary leader regions of the genomes and the anti-genomes, respectively, l lec (¼ l l ) is the length of the complementary leader region, and k d,nc is the decay rate constant of nucleocapsid. As progeny virions are assembled, the concentration of each protein is reduced by the amount corresponding to its stoichiometry in a single virion particle.\nReplication. We assumed that N protein regulates the switch of the role of polymerase between transcription and replication by encapsidating the newly synthesized RNAs [41, 52] . The polymerase that starts the replication at the leader region of the genome requires further supply of N proteins to skip the attenuation signals at each gene junction and thereby to complete each round of replication. Depending on the availability of N proteins, nocap(¼1-encap) fraction of polymerases terminate the replication at each gene junction in our model: dpol r;n;N;1 dt ¼ k e;pol ðencap pol l l l À n sec;N l mRNA;N pol r;n;N;1 Þ ð 15Þ\nwhere pol r,n,N,1 is the concentration of the replicating polymerases on the first segment of the N gene section in the negative-sense genomic nucleocapsid. dpol r;n;i;1 dt ¼ k e;pol ðencap n sec;iÀ1 l mRNA;iÀ1 pol r;n;iÀ1;nsec;iÀ1 À n sec;i l mRNA;i pol r;n;i;1 Þ i ¼ P; M; G; L ð16Þ\nwhere pol r,n,i,1 and pol r,n,i-1,nsec,i-1 are the concentrations of the replicating polymerases on the first segment of gene i, and on the last segment of gene iÀ1, respectively. The level of polymerases that scan through the whole genome (pol tr ) determines the amount of newly synthesized anti-genomic nucleocapsids, (þ)nc: dpol tr dt ¼ k e;pol n sec;i l mRNA;i pol r;n;i; j À pol tr l tr i ¼ L; j ¼ n sec;L ð17Þ dðþÞnc dt ¼ k e;pol encap pol tr l tr À k d;nc ðþÞnc ð18Þ\nwhere l tr (¼ l trc ) is the length of the trailer region of the genome. We also considered the first-order kinetics for the decay of anti-genomic nucleocapsid.\nThe synthesis and decay of genomic nucleocapsids are described in the same way as for those of the anti-genomic nucleocapsids except that the polymerases on the anti-genomic templates are not released at intergenic regions: dpol r;p;j dt ¼ k e;pol ðencap pol trc l trc À n sec l À l trc À l lec pol r;p; j Þ j ¼ 1 ð19Þ\nwhere pol r,p,j is the concentration of the replicating polymerases on the jth segment of the positive-sense anti-genomic nucleocapsids, l is the total length of the genome, and n sec is the total number of segments of the genome ( Figure 9B ). dpol r;p; j dt ¼ k e;pol n sec l À l trc À l lec ðpol r;p; j À 1 À pol r;p; j Þ j ¼ 2; . . . n sec ð20Þ dpol lec dt ¼ k e;pol n sec l À l trc À l lec pol r;p;nsec À pol lec l lec ð21Þ dðÀÞnc dt ¼ k e;pole ncap Á pol lec l lec À k d;nc ðÀÞnc ð22Þ\nIn our model, non-encapsidated nascent genome and anti-genome fragments are released from polymerases and immediately degraded.\nAs polymerases leave the promoter regions by moving toward the downstream sequences, the concentration of polymerases on the promoters will decrease. The dynamic changes of the polymerase concentrations on the promoters of the genomic and the antigenomic templates are finally described, respectively: pol l;nþ1 ¼ pol l;n þ pol new l;n À pol lÀleave;n ð23Þ\nwhere pol l-leave is the concentration of the polymerases leaving the genomic promoters, O ,n and O ,nþ1 are the concentrations of a component (O) at time n and time nþ1 (in our numerical integration, time nþ1 À time n ¼ Dt), respectively. pol trc;nþ1 ¼ pol trc;n þ pol new trc;n À pol trcÀleave;n ð24Þ\nwhere pol trc-leave is the concentration of the polymerases leaving the anti-genomic promoters. Assembly and budding. We assume that the condensation of negative-sense nucleocapsid by M protein initiates the virion assembly and the condensed nucleocapsids are not degraded in the same manner as virion progeny. Whenever the requirement for the stoichiometric amounts of proteins is satisfied, progeny virions are instantaneously assembled and released to the extracellular space. The time required for the condensation of the negative-sense nucleocapsid, the assembly, and the budding of progeny virion was assumed to be negligible relative to the preceding steps.\nHost cell. In our model, the host cell provides unlimited building blocks such as nucleoside triphosphates and amino acids for the growth of virus. However, as viral components accumulate during the course of infection, some key host components for translation such as initiation and elongation factors may be depleted [28, 63, 56] . Two main viral products, leader-mRNA and M protein, contribute to the deficiency by inhibiting the synthesis of host macromolecules at the transcription level [19, 28, 35, 36] . Because leader-mRNA starts to accumulate soon after the initiation of infection, and a small amount of the component is enough to trigger the inhibition [28, 35] , the pool of host factors is continuously reduced from the onset of infection. We quantify this reduction with a single decay rate constant specific to the type of host cell:\nwhere f dec is the level of host translation factors at time t, relative to that of the initial state of the cell before infection (at t ¼ 0), and k d,host is the decay rate constant. The inhibition by the leader mRNA causes a first-order decay of the host factors, resulting in a shortage of the ribosomes equipped with the accessory factors for viral translation in the late infection stage in our model. Unlike viral transcription and replication, viral translation is directly affected by the decay of host factors since it depends entirely on host machinery. In the early infection, host mRNAs outnumber viral mRNAs and thereby successfully compete for the host translation machinery. However, the newly synthesized M proteins inhibit the host transcription initiation and the export of host mRNAs from the nucleus to the cytoplasm [57] , thereby causing a gradual shift in translation from host mRNAs to viral mRNAs. For our model we assumed that the potency of the inhibition by the M protein was independent of the type of cell and its differentiation state [36] , and we developed an empirical formula using available experimental data from the literature [36] to account for the competition between host and viral mRNAs for ribosomes. Lyles at al. cotransfected the host cells with VSV M mRNA and chloramphenicol acetyl transferase (CAT) plasmid DNA, and then they quantified the expression of CAT based on its activity, as a function of the expression of VSV M protein [36] . In their experiment the gene expression of CAT was more reduced at higher M protein expression levels. We assume that the decrease of the expression of CAT (or its activity decrease) is proportional to the decrease of the occupancy of host mRNAs by the translation machinery. Using their experimental data, the occupancy of host mRNAs by the translation machinery is correlated with the number of newly synthesized M proteins in the cytoplasm:\nwhere rib_host is the fraction of the translation machinery associated with host mRNAs, and M cell is the total number of newly synthesized M proteins per cell. Considering the decay of host factors and the competition between host and viral mRNAs, we could derive a formula to quantify the number of the fully functional ribosomes that are available for the viral protein synthesis over time post-infection (nrib avail ):\nwhere nrib denotes the total concentration of ribosomes whether or not they incorporate all the required accessory factors for their translation function. Although the ribosomes distribute into membrane-bound and cytoplasmic forms, each class supporting the syntheses of the viral G protein and the other four viral proteins (N, P, M, and L), respectively, we treated the ribosomes in our model as one population.\nInitial condition for simulation. The initial condition for our simulation is set by a fixed number of infectious extracellular virus particles per cell (V ex (0)). At time zero (t ¼ 0), the number of bound virus particles and the level of all viral components within cells are zero. In our model, binding of extracellular virus particles to cells reduces their level (Equation 1), and an encapsidated genome and stoichiometric amounts of viral proteins (Table 2 , [28, 29] ) are then immediately released from each bound virus particle to the cytoplasm. Specifically, we assume that all N proteins from a bound virus particle are released as a form of encapsidated genome complex. Downstream processes, beginning with transcription, are then initiated. In our simulation, viral infection starts with rib_host ¼ 0.99 (with rib_host ¼ 0.9, ; 0.9995 simulations showed the same results). Other key model parameters for simulation are summarized in Table 1 . In addition, a nomenclature list is shown in Table 4 . Figure S1 . Ratio of Genome to Anti-Genome During Simulated VSV Infection Depends on Relative Promoter Strengths The relative strength of the anti-genomic promoter relative to the genomic promoter is given by S prom . PI stands for time post-infection. Figure S4 . Productivity Ranking of Six Gene-Shuffled Viruses Provides Parameter Constraint For each value of S cond and its corresponding host parameter values (k d,host and S rib ), the virus productivity of each gene-shuffled virus in BHK cells was determined and normalized by the highest productivity. Simulated rankings of six strains matched experimentally observed rankings [9] over a narrow range of S cond , indicated by the bar. Initial number of infectious extracellular virus particles per cell was three. Found at DOI: 10.1371/journal.pcbi/0020116.sg004 (872 KB TIF).\nProtocol S1. Parameter Estimation" }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: Altered expression levels of certain genes play a pivotal role in several pathological conditions. For example, in many cancers the upregulation of certain growth factors or growth factor receptors, or the deregulation of intracellular signal transduction pathways, represents key elements in the process of malignant transformation and progression of normal cells towards tumor cells leading to uncontrolled proliferation and decreased apoptosis. Since these processes may result in the direct, autocrine stimulation of the tumor cell itself as well as the paracrine stimulation of other cells, including the stimulation of tumor-angiogenesis, many novel therapeutic strategies focus on the reversal of this effect, that is, the inhibition of these proteins or the downregulation of their expression. Likewise, several other diseases have been firmly linked to the (over-)expression of endogenous wildtype or mutated genes. Taken together, in addition to strategies based on the inhibition of target proteins, for example, by low molecular weight inhibitors or inhibitory antibodies, this opens an avenue to gene-targeting approaches aiming at decreased expression of the respective gene.\nThe first method to be introduced for the specific inhibition of gene expression was the use of antisense oligonucleotides in the late 1970s [1, 2] . Upon their introduction into a cell, antisense ODNs are able to hybridize to their target RNA leading to the degradation of the RNA-DNA hybrid double strands through RNAase H, to the inhibition of the translation of the target mRNA due to a steric or conformational obstacle for protein translation and/or to the inhibition of correct splicing. In the early 1980s, the discovery of ribozymes, that is, catalytically active RNAs which are able to sequence-specifically cleave a target mRNA, further expanded gene-targeting strategies [3] [4] [5] . Subsequently, both methods were extensively studied and further developed with regard to the optimization of targeting efficacies and antisense-ODN/ribozyme delivery strategies in vitro and in vivo.\nMost recently, another naturally occurring biological strategy for gene silencing has been discovered and termed RNA interference (RNAi). Since RNAi represents a particularly powerful method for specific gene silencing and is able to provide the relatively easy ablation of the expression of any given target gene, it is now commonly used as a tool in biological and biomedical research. This includes the RNAimediated targeting in vitro and in vivo for functional studies of various genes whose expression is known to be upregulated as well as the development of novel therapeutic approaches based on gene targeting. double-stranded RNA molecules as described first in C elegans by Fire et al [6] who then introduced the name RNA interference. These findings also explained earlier observations in petunias which turned white rather than purple upon the introduction of the \"purple gene\" in form of dsRNA [7] , and on gene silencing by antisense oligonucleotides as well as by sense oligonucleotides in C elegans [8] . Subsequent studies demonstrated that RNAi, while described under different names (posttranscriptional gene silencing (PTGS), cosuppression, quelling), is present in most eukaryotic organisms with the response to dsRNA, however, being more complicated in higher organisms.\nRNAi relies on a multistep intracellular pathway which can be roughly divided into two phases, that is, the initiation phase and the effector phase. In the initiation phase, double-stranded RNA molecules from endogenous or exogenous origin present in the cell are processed through the cleavage activity of a ribonuclease III-type protein [9] [10] [11] [12] into short 21-23 nucleotide fragments termed siRNAs. These effector siRNAs, which contain a symmetric 2 nt overhang at the 3 -end as well as a 5 -phosphate and a 3hydroxy group, are then in the effector phase incorporated into a nuclease-containing multiprotein complex called RISC (RNA-induced silencing complex) [13] . Several structural and biochemical studies have shed light on the processing of double-stranded RNA and the formation of the RISC complex (see, eg, [14] for a recent review). Through unwinding of the siRNA duplex by an RNA helicase activity [15] , this complex becomes activated with the single-stranded siRNA guiding the RISC complex to its complementary target RNA. Upon the binding of the siRNA through hybridization to its target mRNA, the RISC complex catalyses the endonucleolytical cleavage of the mRNA strand within the target site, which, due to the generation of unprotected RNA ends, results in the rapid degradation of the mRNA molecule. With the RISC complex being recovered for further binding and cleavage cycles, the whole process translates into a net reduction of the specific mRNA levels and hence into the decreased expression of the corresponding gene. For an overview of the RNAi pathway, see Figure 1 .\nWhile from this mechanism it becomes obvious that siRNA molecules complementary to the target mRNA and thus being able to serve as a guide sequence for the RISC complex play a pivotal role in this process, they need not be derived from long double-stranded precursor molecules. Rather, omitting the initiation phase, they can be delivered directly into the target cell ( Figure 1 , upper right arrow).\nSeveral studies have led to the development of guidelines for the generation of siRNAs which are optimal in terms of efficacy and specificity [12, 16] . This includes the initial definition of the preferable length (19-25 bp) combined with a low G/C content in the range between 36% and 52% and the requirement of symmetric 2 nt overhangs at the 3 -end [16] [17] [18] . Later studies on synthetic siRNA molecules, however, revealed an up to 100-fold higher targeting efficacy in the case of even longer duplexes (25-30 nucleotides) which act as a substrate for Dicer and which therefore allow the direct incorporation of the newly produced siRNAs into the RISC complex [19] . As to be expected, intramolecular foldback structures which can result from internal repeats or palindrome sequences decrease the numbers of functional siRNA molecules with silencing capability [20] . Additional silencing-enhancing criteria include an A in position 3 and a G at position 13 of the sense strand, the absence of a C or G at position 19 and, most importantly, a U in position 10 of the sense strand. Since nucleotides 10-11 represent the site of the RISC-mediated cleavage of the target mRNA, this indicates that RISC is comparable to most other endonucleases in preferentially cleaving 3 of U rather than any other nucleotide [20, 21] . Furthermore, it was shown more generally that the thermodynamic flexibility of the positions 15-19 of the sense strand correlates with the silencing efficacy and that the presence of at least one A/U base pair in this region improves siRNA-mediated silencing efficacy due to a decreased internal stability of its 3 -end [20] .\nStill, different siRNA sequences may display differing efficacies, which suggest additional still unknown criteria for optimal siRNA selection and emphasize the influence of target mRNA accessibility. In fact, several studies also correlate the siRNA efficacy with the mRNA secondary structure [18, [22] [23] [24] [25] [26] [27] .\nIn conclusion, apart from the selection criteria defined above, the individual screening of different siRNAs for highly efficient and specific duplexes, or the pooling of multiple siRNAs, is the most effective approach to increase siRNAmediated targeting efficacy.\nFor the design of effective siRNAs, several algorithms on publicly accessible web sites are available (see [28] for review). To reduce the risk of nonspecific (\"off-target\") effects of the siRNAs, a homology search of the targeting sequence against a gene database is necessary and already incorporated in some of these web sites. Nevertheless, it has also been shown that siRNAs may cross-react with targets of limited sequence similarity when regions of partial sequence identity between the target mRNA and the siRNA exist. In fact, in some cases regions comprising of only 11-15 contiguous nucleotides of sequence identity were sufficient to induce gene silencing [29] . The prediction of these off-target activities is difficult so far.\nAn additional mechanism that may lead to nonspecific effects in vivo relies on the interferon system [30] [31] [32] [33] which is induced when double-stranded RNA molecules enter a cell activating a multi-component signalling complex. This effect is particularly true for long dsRNA molecules and essentially prevents them from being used as inducers of RNA interference in mammalian systems. The development of synthetic siRNAs [10, 12, 33, 34] largely circumvents this problem since they seem to be too small. However, some synthetic siR-NAs do induce components of the interferon system which seems to be dependent on their sequence [31, 32, 35] as well as, in the case of in vitro transcribed siRNAs, on the 5 initiating triphosphate [36] . Thus, strategies to avoid as far as possible the unwanted interferon response upon application of siRNAs in vivo will include a design of siRNAs without known interferon-stimulating sequences, the use of the lowest possible siRNA dose to still achieve the desired effect and optimized siRNA delivery methods.\nBased on the known mechanisms of antisense technology, ribozyme-targeting or RNAi, small oligonucleotides or plasmid-based expression vectors can be used to specifically downregulate the expression of a given gene of interest or of pathological relevance in vitro. In principle, this also applies to the in vivo situation leading to novel, potentially relevant therapeutic approaches.\nFor the delivery of therapeutic nucleic acids, viral vectors have been used which have the advantage of high transfection efficacy due to the inherent ability of viruses to transport genetic material into cells. On the other hand, however, viral systems show a limited loading capacity regarding that the genetic material are rather difficult to produce in a larger scale and, most importantly, pose severe safety risks due to their oncogenic potential and their inflammatory and immunogenic effects which prevent them from repeated administration [37] [38] [39] [40] .\nIn the light of these problems, concerns, and limitations, nonviral systems have emerged as a promising alternative for gene delivery. Main requirements are the protection of their nucleic acid \"load\" as well as their efficient uptake into the target cells with subsequent release of the DNA or RNA molecules and, if necessary, their transfer into the nucleus. Several strategies can be distinguished, mainly lipofection and polyfection relying on cationic lipids or polymers, respectively (see, eg, [41] [42] [43] ).\nThe efficient protection against enzymatic or nonenzymatic degradation is particularly important for RNA molecules including siRNAs. In fact, while the therapeutic potential of siRNAs for the treatment of various diseases is in principle very promising, limitations of transfer vectors may turn out to be rate-limiting in the development of RNAi-based therapeutic strategies. One approach to solve this problem is the use of DNA expression plasmids which encode palindromic hairpin loops with the desired sequence. Upon transcription and folding of the RNA, the doublestranded short hairpin RNAs (shRNAs) are recognized by Dicer and cleaved into the desired siRNAs. Additionally, an in vitro method has been described recently which is based on the expression of shRNAs in E coli and their delivery via bacterial invasion [44] . While all these different DNAbased systems offer the advantage of siRNA expression with a longer duration and a probably higher level of gene silencing, they still rely on (viral or nonviral) delivery of DNA molecules and again raise safety issues in vivo. Hence, the direct delivery of siRNAs molecules, derived from in vitro transcription or chemically synthesized, offers advantages over DNA-based strategies and may be preferable for in vivo therapeutic use.\nIn the last years, a large body of studies has been published which describe different strategies for the systemic or local application of siRNAs in vivo. Tables 1-3 give an overview. The probably largest number of papers focuses the use of unmodified siRNAs (Table 1 ) whose administration is often performed IV by hydrodynamic transfection (high pressure tail vein injection). While this method is widely used and in some cases led to efficient target gene inhibition in the liver and, to a lesser extent, in lung, spleen, pancreas, and kidney, it may suffer from certain technical and practical limitations at least in a therapeutical setting since it relies on the rapid IV injection of a comparably large volume (>= 1 ml/mouse/injection, in theory equivalent to a ∼ 3 l IV bolus injection in man). Alternative strategies for the application of naked siRNAs include various delivery routes which, however, often provide an only local administration or rely on an administration at least close to the target tissue or target organ, thus restricting the number of target organs which may not be relevant for certain diseases. It should also be noted that several studies described here and below use rather large amounts of siRNAs and that upon intravenous injection of siRNAs the liver is the primary site of siRNA uptake. As an alternative approach for the application of siR-NAs in vivo, their delivery by liposomes/cationic lipids has been described. For liposome-based siRNA formulations, a wide variety of modes of application allowing local or systemic delivery has been used (Table 2) . Finally, several other strategies for local or systemic siRNA administration have been explored, including chemical modifications of siRNA molecules, electropulsation, polyamine, or other basic complexes, atelocollagen, virosomes, and certain protein preparations (Table 3 ).\nAn alternative approach relies on the complexation of unmodified siRNA molecules with a cationic polymer, polyethylenimine (PEI).\nPolyethylenimines (PEIs) are synthetic polymers available in branched or linear forms (Figure 2 , upper panels) and in a broad range of molecular weights from < 1000 Da to > 1000 kd. Commercial PEI preparations, although labelled with a defined molecular weight, consist of PEI molecules with a broad molecular weight distribution [45] [46] [47] . PEIs possess a high cationic charge density due to a protonable amino group in every third position [48, 49] . Since no quarternary amino groups are present, the cationic charges are generated by protonation of the amino groups and hence are dependent on the pH in the environment (eg, 20% at pH 7.4, see [50] for review). Due to its ability to condense and compact the DNA into complexes, which form small colloidal particles allowing efficient cellular uptake through endocytosis, PEI has been introduced as a potent DNA transfection reagent in a variety of cell lines and in animals for DNA delivery (for review, see [51, 52] and references therein). In fact, in several studies PEI has been shown to be able to deliver large DNA molecules such as 2.3 Mb yeast artificial chromosomes (YACs) [53] as well as plasmids or small oligonucleotides [48, [54] [55] [56] into mammalian cells in vitro and in vivo. The N/P ratio, which indicates the ratio of the nitrogen atoms of PEI to DNA phosphates in the complex and thus describes the amount of PEI used for complex formation independent of its molecular weight, influences the efficiency of DNA delivery. A positive net charge of the complexes, resulting from high N/P ratios, inhibits due to electrostatic repulsion their aggregation and improves their solubility in aqueous solutions as well as their interaction with the negatively charged extracellular matrix components and thus their cellular uptake [57] . Additionally, the strong buffer capacity, described by the \"proton sponge hypothesis\" which postulates enhanced transgene delivery by cationic polymer-DNA complexes (polyplexes) containing H + buffering polyamines due to enhanced endosomal Cl − accumulation and osmotic swelling/lysis [48] , seems to be responsible for the fact that PEI-based delivery does not require endosome disruptive agents for lysosomal escape. This tight condensation of the DNA molecules as well as the buffering capacity of PEI in certain cellular compartments like endosomes and lysosomes also protects DNA from degradation [48, 49, 58, 59] . PEIs have been successfully used for nonviral gene delivery in vitro and in vivo. While initial publications showed increased transfection efficacies when using high molecular weight PEIs [45] , more recent studies demonstrated the advantages of certain low molecular weight PEIs [47, 60, 61] . The higher transfection efficacy of low molecular weight PEIs may be due to a more efficient uptake of the resulting PEI/DNA complexes, a better intracellular release of the DNA and/or lower in vitro cytotoxicity as compared to high molecular weight PEI [60] [61] [62] [63] . In fact, a decrease in the molecular weight of the PEI leads to an increase in complex size which may be favourable at least for in vitro use [64, 65] . On the other hand, other PEIs with very low molecular weight (< 2 kd) display little or no transfection efficacy even at very high N/P ratios which may be attributed to the fact that a decrease in the molecular weight of PEI has been shown to translate into an increasingly lower ability to form small complexes [63] . Therefore, low molecular weight PEIs require higher N/P ratios for optimal transfection efficacies as compared to higher molecular weight PEIs since higher N/P ratios lead to an increase in compaction with reduced complex sizes and a reduced tendency of the complexes to aggregate due to hydrophobic interactions [61, 63, 64] . Nevertheless, while several parameters have been extensively studied, some precise determinants for transfection efficacy remain to be elucidated (see [50, 66] for review). Also, the mechanism of the cytotoxic effects of PEI complexes is only poorly understood. It may rely on the formation of large aggregates in the range of up to 2 μm which, when formed on the cell surface, impairs membrane functions finally leading to cell necrosis [60] . Clearly, there is a trend towards low molecular weight PEIs as rather nontoxic delivery reagents in vitro and in vivo, which combine high biocompatibility and reduced side-effects thus also allowing to employ larger PEI/DNA complex amounts without significant cytotoxicity. More recently, the use of polyethylenimines has been extended towards the complexation and delivery of RNA molecules, especially small RNA molecules like 37 nt all-RNA ribozymes [67] [68] [69] and siRNAs [70] (Figure 2 ). While chemically unmodified RNA molecules are very instable and prone to rapid degradation, the PEI complexation has been shown to lead to an almost complete protection against enzymatic or nonenzymatic degradation. In fact, PEI-complexed siR-NAs, which are [ 32 P]-labeled for better detection, remain intact in vitro for several hours even in the presence of RNase A or fetal calf serum at 37 • C, while non-complexed siRNAs are rapidly degraded (Figure 3(a) ). This indicates that siRNA molecules are efficiently condensed and thus fully covered and protected by PEI. Indeed, the analysis of PEI/siRNA complexes by atomic force microscopy showed the absence of free siRNAs or siRNA molecule ends and thus confirms these findings regarding an efficient complexation (Grzelinski et al, submitted). However, while the complex stability seems to be sufficient for siRNA protection with all PEIs tested (Werth et al, in press; Aigner et al, unpublished data), several of these complexes do not show any targeting efficacy at all. In fact, only when using certain polyethylenimines, PEI/siRNA complexes are efficiently delivered into target cells in vitro, where siRNAs are released and display bioactivity (Figures 1 and 2) . In general and as seen before for PEI/DNA complexes (see above), the transfection efficacy is dependent on the PEI used, also indicating that the siRNA targeting efficiency mainly depends on the endocytotic uptake of the complex and/or its intracellular decomposition rather than on the in vitro complex stability. Good results were obtained with commercially available JetPEI [70] while the in vivo JetPEI from the same supplier showed only poor siRNA delivery efficacies [71] . Likewise, a novel low molecular weight PEI based on the fractionation of a commercially available polyethylenimine demonstrates high siRNA protection and delivery efficacies in vitro (Werth et al, in press). Under certain conditions, the PEI/RNA (siRNA or ribozyme) complexes retain their physical stability and biological activity also after lyophilization ( [72] and Werth et al, in press). Although the PEI transfection is only transient, data from our lab show that PEI/siRNA effects are stable for at least 7 days (Urban-Klein and Aigner, unpublished results). Finally, another study has explored the use of siRNA nanoplexes comprising of PEI that is PEGylated with an RGD peptide ligand attached at the distal end of the PEI. Again, siRNA nanoplexes protect siRNAs against serum degradation and show in vitro activity [73] .\nThe ultimate goal is the application of siRNAs in vivo which has been explored in some studies in different mouse models. Ge et al showed that PEI-complexed siRNAs targeting conserved regions of influenza virus genes are able to prevent and treat influenza virus infection in mice. Upon IV injection, PEI promoted the delivery of siRNAs into the lungs where, either given before or after virus infection, siRNA reduced influenza virus production in the lungs [74] .\nMost biological effects of the systemic application of PEIcomplexed siRNAs, however, have been determined in different mouse tumor models and by targeting different proteins which have been shown previously to be tumor-relevant. This includes the epidermal growth factor receptor HER-2 (c-erbB-2/neu), the growth factor pleiotrophin (PTN), and vascular endothelial growth factor (VEGF) and its receptor (VEGF R2), and the fibroblast growth factor-binding protein FGF-BP.\nThe in vivo administration of PEI complexed, but not of naked siRNAs, through IP or subcutaneous injection resulted in the detection of intact siRNAs even hours after injection (Figure 3(b) ). Radiolabeled siRNA molecules were found in several organs including subcutaneous tumors, muscle liver, kidney and, to a smaller extent, lung and brain. It is important to note that the siRNAs were actually internalized by the tissues as indicated by the fact that blood was negative for siRNAs ( Figure 3(b) ).\nOverexpression of the HER-2 receptor has been observed in a wide variety of human cancers and cancer cell lines. Since HER-2 displays strong cell growth-stimulating and antiapoptotic effects especially through heterodimer formation with other members of the EGFR family, its overexpression has been established as a negative prognostic factor and linked to a more aggressive malignant behaviour of tumors (eg, [75] ). Consequently, HER-2 qualifies as an attractive target molecule for antitumoral treatment strategies including anti-HER-2 antibodies, low molecular weight inhibitors, or HER-2-specific gene-targeting approaches. In fact, the relevance of HER-2 (over-)expression in tumor growth has been established in several in vitro HER-2 targeting studies including the use of ribozymes [76, 78, 79] or siRNAs [80, 81] . proposed mechanism of PEI-mediated siRNA transfer. Due to electrostatic interactions, PEI is able to complex negatively charged siRNAs leading to a compaction and the formation of small colloidal particles which are endocytosed. The \"proton sponge effect\" exhibited by PEI complexes leads to osmotic swelling and ultimately to the disruption of the endosomes. siRNAs are protected from degradation due to their tight condensation in the complex and the buffering capacity of PEI. Upon their release from the PEI-based complex, intact siRNAs are incorporated into the RISC complex and induce RNAi (see Figure 1 ).\nIt was demonstrated that HER-2 reduction in vitro leads, among others, to the inhibition of cell proliferation and increased apoptosis.\nThe systemic treatment of athymic nude mice bearing subcutaneous SKOV-3 ovarian carcinoma tumor xenografts through IP injection of PEI-complexed HER-2-specific siRNAs led to marked antitumoral effects as seen by a significant reduction tumor growth (Figure 4 ) [70] . PEIcomplexed nonspecific siRNAs or HER-2-specific, naked siR-NAs had no effects. This was paralleled by the detection of intact HER-2-specific siRNAs in the tumors of the specific treatment group already 30 min after administration and for at least 4 h, and by the downregulation of HER-2 on mRNA and protein levels [70] .\nAnother receptor, VEGF R2, was targeted in a study employing self-assembling nanoparticles based on siRNAs complexed PEI which is PEGylated with an RGD peptide ligand attached at the distal end of PEG. While the PEGylation allows steric stabilization and reduces nonspecific interactions of the complexes, the RGD motif provided tumor selectivity due to their ability to target integrins expressed on activated endothelial cells in the tumor vasculature. Upon IV administration into mice bearing subcutaneous N2A neuroblastoma tumor xenografts, a selective tumor uptake and a VEGF R2 downregulation were observed, resulting in decreased tumor growth and tumor angiogenesis [73] .\nThe receptor ligand, VEGF, is a mitogenic and angiogenic growth factor stimulating tumor growth and angiogenesis in several tumors including prostate carcinoma. Thus, it may represent attractive target molecule for RNAi-based genetargeting strategies also bearing in mind the double antitumoral effect due to reduction of tumor cell proliferation as well as tumor angiogenesis. The subcutaneous or intraperitoneal injection of VEGF-specific siRNAs complexed with a novel PEI obtained through fractionation of a commercially available PEI (Werth et al, in press) resulted in the reduction of tumor growth due to decreased VEGF expression levels (Höbel and Aigner, unpublished results). The same was true for PEI/siRNA-mediated targeting of FGF-BP (Dai and Aigner, unpublished results), which has been established Figure 3 : Protection and in vivo delivery of siRNAs upon PEI complexation. In [70] (a) in vitro protection of siRNAs against nucleolytic degradation. [ 32 P] end-labeled siRNAs, complexed (upper panel) or not complexed (lower panel) with PEI, were subjected to treatment with 1 % fetal calf serum at 37 • C. At the time points indicated, the samples were analysed by agarose gel electrophoresis, blotting, and autoradiography. The bands represent full-length siRNA molecules indicating that PEI complexation leads to the efficient protection of siRNAs while noncomplexed siRNAs are rapidly degraded. (b,c) In vivo delivery of intact siRNAs upon PEI complexation. [ 32 P]-labeled siRNAs, complexed (+) or not complexed (−) with PEI, were injected IP into mice bearing subcutaneous SKOV-3 ovarian carcinoma cell tumor xenografts, and after 30 min (b) or 4 h (b) total RNA from various organ and tissue homogenates was prepared and subjected to agarose gel electrophoresis prior to blotting and autoradiography. The bands represent intact [ 32 P]-labeled siRNA molecules which for several hours are mainly found in tumor and muscle as well as in liver and, time-dependently, in kidney. Only little siRNA amounts are detected in the lung and traces in the brain.\npreviously as \"rate-limiting\" for tumor growth and angiogenesis in several tumors ( [82, 83] , see [84] for review).\nFinally, PEI/siRNA-mediated targeting of pleiotrophin (PTN) exerted strong antitumoral effects. PTN is a secreted growth factor which shows mitogenic, chemotactic, angiogenic and transforming activity [85] [86] [87] [88] [89] [90] [91] [92] [93] and which is markedly upregulated in several human tumors including cancer of the breast, testis, prostate, pancreas, and lung as well as in melanomas, meningiomas, neuroblastomas, and glioblastomas. The in vivo treatment of nude mice through systemic subcutaneous or IP application of PEI-complexed PTN siRNAs led to the delivery of intact siRNAs into subcutaneous tumor xenografts and a significant inhibition of tumor growth. Likewise, in a clinically more relevant orthotopic mouse glioblastoma model with U87 cells growing intracranially, the injection of PEI-complexed PTN siRNAs into the CNS exerted antitumoral effects. This establishes, also in a complex and relevant orthotopic tumor model, the potential of PEI/siRNA-mediated PTN gene targeting as a novel therapeutic option in GBM, and further extends the modes of delivery of PEI/siRNA complexes intrathecal strategies as employed in the therapy of glioblastomas with antisense oligonucleotides.\nOnly a few years after their discovery, siRNAs are catching up with ribozymes and antisense oligonucleotides as efficient tools for gene targeting in vitro and, more recently, also in vivo. This includes the exploration of their potential as therapeutics which will lead to the development of siRNA-based therapeutic strategies. Their ultimate success, however, will Figure 4 : Systemic treatment of mice with PEI-complexed HER-2-specific siRNAs leads to reduced growth of subcutaneous SKOV-3 tumor xenografts due to decreased HER-2 expression. In [70] athymic nude mice bearing subcutaneous tumor xenografts were injected IP with 0.6 nmoles HER-2-specific naked (open circles) or PEI-complexed (closed circles) siRNAs 2-3 times per week and tumor sizes were evaluated daily from the product of the perpendicular diameters of the tumors. Mean +/-standard error of the mean (SEM) is depicted and Student's unpaired t test was used for comparisons between data sets ( * * P < .03, * * * P < .01). Differences in tumor growth reach significance at day 5 indicating the antitumoral effects of the PEI-complexed HER-2-specific siRNAs. strongly depend on the development of powerful and feasible siRNA delivery strategies which need to address several issues including the stability/stabilization of siRNA molecules while preserving their efficacy and maintaining their genesilencing activity, an efficient delivery into the target organ(s) as well as a sufficiently long siRNA half life in the organism and particularly in the target organ. Thus, siRNA delivery strategies must provide siRNA protection and transfection efficacy, the absence of toxic and nonspecific effects, they must be efficacious also when using small amounts of siRNAs and must be applicable in various treatment regimens and in various diseases even when this requires to overcome biological barriers after their administration to reach their target tissue or target organ. The research done on DNA-based gene delivery, ribozyme-targeting, and antisense technology will facilitate this process since it already provides a basis of established technologies. This is also true for the complexation of siRNAs with polyethylenimine, which may represent a promising avenue for siRNA applications in vivo. This may eventually lead to novel therapeutic strategies. \nThe work of A. Aigner is supported by the Deutsche Forschungsgemeinschaft (AI 24/5-1) and by the Deutsche Krebshilfe. The author would like to apologize to the authors whose primary works have not been cited due to length considerations." }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: " }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: In fish, as in other lower vertebrates, the post-vitellogenic period is very important for the completion of the oogenetic process. During this step, the follicle-enclosed postvitellogenic oocyte undergoes several key events such as the final acquisition of the ability to resume meiosis in response to the maturation-inducing steroid (MIS), the resumption of the meiotic process and, finally, its release from the surrounding follicular layers. In addition, the whole follicle (oocyte and surrounding follicular cells) undergoes a progressive differentiation ultimately leading to the release of a metaphase 2 oocyte. The key hormonal and molecular events involved in the control of meiosis resumption have been thoroughly studied and many studies have been dedicated to the action of gonadotropins, the regulation of steroidogenenic events and the action of the MIS (see [1] [2] [3] [4] [5] [6] for review). However, the associated follicular or extra-follicular events involved in concomitant processes such as oocyte-follicular cells cross talk and ovulationmechanisms have received far less attention. Nevertheless, several researchgroups have studied the periovulatory ovarian physiology using classical biochemical or histological tools and, later, molecular approaches. Thus, several studies have dealt with ovarian proteases in their participation in the ovulatory process [7] [8] [9] . Differential display PCR and suppressive subtractive hybridization (SSH) approaches have also been developed in order to identify new differentially regulated genes in the fish periovulatory ovary [10] [11] [12] [13] . In addition, numerous candidate gene studies have also been performed in the fish periovulatory ovary. Apart from genes related to hormonal controls, these studies were mostly dedicated to some specific gene families such as TGF beta family [14, 15] or connexins [16, 17] . Finally, fewer studies have simultaneously analyzed the expression profiles of several genes belonging to different families [18, 19] . However, in contrast to other biological processes, such as immune response [20] , the post-vitellogenic period has never benefited from genome-wide transcriptomic studies that could provide a global view of the molecular events occurring in the post-vitellogenic ovary undergoing oocyte maturation. In this context, the present study aimed at performing a transcriptomic analysis of the postvitellogenic rainbow (Oncorhynchus mykiss) trout ovary. In order to do so, 9152-gene rainbow trout cDNA microarrays were hybridized using RNA samples originating from rainbow trout ovarian tissue collected during late vitellogenesis, post-vitellogenesis and oocyte maturation. A statistical analysis was performed in order to identify all the genes exhibiting a differential expression over this period. In addition, a supervised clustering analysis was performed using only the differentially expressed genes in order to identify groups (or clusters) of genes exhibiting similar expression profiles. Furthermore, as a first step in a long-term transcriptomic analysis of the rainbow trout post-vitellogenic ovary, we deliberately chose to focus on 3 gene clusters exhibiting the most remarkable expression patterns. Finally, specific genes were selected in each cluster based on the novelty of their putative identity and/or function. For each gene, a real-time PCR analysis of their ovarian expression profiles was performed using additional ovarian RNA samples.\nInvestigations were conducted according to the guiding principles for the use and care of laboratory animals and in compliance with French and European regulations on animal welfare. Two year old female rainbow trout (Oncorhynchus mykiss) were obtained during their first reproductive season from our experimental fish farm (Sizun, France) and held under natural photoperiod in a re-circulated water system in INRA experimental facilities (Rennes, France). The water temperature was kept constant at 12°C. Ovaries were sampled from individual females during late vitellogenesis (N = 6), post-vitellogenesis (N = 6) and during oocyte maturation (N = 6). Oocyte developmental stage was assessed under binocular microscope according to previously described criteria [21, 22] . Late vitellogenic samples were collected at the end of the vitellogenic process, approximately 3-4 weeks before expected ovulation. At this stage, germinal vesicle is not visible and no polarized cytoplasm area can be observed. Post-vitellogenic samples were collected 2-3 weeks later but before any noticeable morphological changes in yolk structure due to the process of meiosis resumption. At this stage, oocytes can display a subperipheral or peripheral germinal vesicle. When germinal vesicle is not visible, a dark mass of polarized cytoplasm can be observed. Oocyte maturation samples were collected after meiosis resumption. Those samples were thus collected after yolk clarification and around the time of germinal vesicle breakdown (GVBD). For tissue collection, trout were deeply anesthetized in 2-phenoxyethanol, killed by a blow on the head and bled by gill arch section. Ovaries were then dissected out of the body cavity under sterile conditions. Ovarian aliquots were frozen in liquid nitrogen and stored at -80°C until RNA extraction.\nLow oligonucleotide signals (lower than three times the background level) were excluded from the analysis. After this filtering step, signal processing was performed using the vector oligonucleotide data to correct each spot signal by the actual amount of DNA present in each spot. After correction, signal was normalized by dividing each gene expression value by the median value of the array.\nA statistical analysis was performed in order to identify differentially expressed genes between late vitellogenic, post-vitellogenic and maturing groups using SAM software [28] . Three 2-by-2 statistical analyses were performed in order to compare each group with the two other ones. In addition, a comparison was performed between samples taken prior to meiosis resumption (from late and post-vitellogenic females, N = 7) and during oocyte maturation (N = 6). For each comparison, the lowest false discovery rate (FDR) was used to identify differentially abundant genes. All genes identified in at least one of the above comparisons were kept for clustering analysis in order to characterize the expression profiles of statistically relevant genes. For supervised clustering analysis [29] , data was log transformed, median-centered and an average linkage clustering was performed using CLUSTER software [29] . Clusters were visualized using TREEVIEW software [29] .\nRainbow trout sequences originating from INRA Agenae [24] and USDA [30] EST sequencing programs were used to generate publicly available contigs [31] . The 8th version (Om.8, released January 2006) was used for BlastX [32] comparison against the Swiss-Prot database (January 2006) [33] . The score of each alignment was retrieved after performing a BlastX comparison. In addition, for each EST spotted onto the membrane, the accession number of the corresponding rainbow trout cluster (Unigene Trout, January 2006), if any, was retrieved from the UniGene database [34] .\nReal-time PCR was performed using all RNA (N = 18) samples including those used for microarray analysis. Several over and under expressed clones belonging to three selected remarkable clusters, were selected according to their putative identity and/or function for analysis. Reverse transcription and real time PCR were performed as previously described [19] . Briefly, 3 μg of total RNA were reverse transcribed using 200 units of Moloney murine Leukemia virus (MMLV) reverse transcriptase (Promega, Madison, WI) and 0.5 μg random hexamers (Promega) per μg of total RNA according to manufacturer's instruction. RNA and dNTPs were denatured for 6 min at 70°C, then chilled on ice for 5 min before the reverse transcription master mix was added. Reverse transcription was performed at 37°C for 1 hour and 15 min followed by a 15 min incubation step at 70°C. Control reactions were run without MMLV reverse transcriptase and used as negative controls in the real-time PCR study. Real-time PCR experiments were conducted using an I-Cycler IQ (Biorad, Hercules, CA). Reverse transcription products were diluted to 1/25, and 5 μl were used for each real-time PCR reaction. Triplicates were run for each RT product. Real-time PCR was performed using a real-time PCR kit provided with a SYBR Green fluorophore (Euro-gentec, Belgium) according to the manufacturer's instructions and using 600 nM of each primer. After a 2 min incubation step at 50°C and a 10 min incubation step at 95°C, the amplification was performed using the following cycle: 95°C, 20 sec; 60°C, 1 min, 40 times. For all primer pairs, the relative abundance of target cDNA within sample set was calculated from a serially diluted ovarian cDNA pool using the I-Cycler IQ software. This dilution curve was used to ensure that PCR efficiency was within an 80-100% range and that amplification was linear within sample set. After amplification, a fusion curve was obtained using the following protocol: 10 sec holding followed by a 0.5°C increase, repeated 80 times and starting at 55°C. The level of 18S RNA in each sample was measured and used for target genes abundance normalization within sample set. In addition to the genes identified from the transcriptomic analysis, a widely used standard gene, elongation factor 1 alpha (ef1α), was monitored using the same sample set to validate the normalization procedure. GenBank accession number and primer sequences are shown in table 1. Statistical analyses were performed using Statistica 7.0 software (Statsoft, Tulsa, OK). Differences between ovarian developments stages were analyzed using non parametric U tests.\nAfter signal processing, 8263 clones out of 9152 were kept for further analysis. From the statistical analysis, 310 clones were found to exhibit a differential abundance between at least 2 of the studied ovarian stages (late vitellogenesis, post-vitellogenesis and oocyte maturation). For all SAM analyses performed, the false discovery rate (FDR) was always lower than 0.7%. Among the 310 identified clones, 90 were up-regulated during oocyte maturation while 220 exhibited an opposite pattern. A clustering analysis was performed using only expression data of the 310 identified clones in order to characterize the expression profiles of those genes. The clustering analysis clearly separated the over from the under expressed genes ( Figure 1 ). The number of each clone in the clustering analysis ( Figure 1 ) was kept in subsequent tables 1, 2, 3, 4 and in the text. Within down-regulated genes, a cluster of 32 genes (cluster 1, Figure 1 ) was characterized by high expression levels during late vitellogenesis, low levels during oocyte maturation and intermediate or variable levels during post-vitellogenesis ( Figure 1 ). Within up-regulated clones, a cluster of 44 genes (cluster 2, Figure 1 ) was characterized by a strong over expression at the time of meiosis resumption while a cluster of 14 genes (cluster 3, Figure 1 ) exhibited a very low expression during late and post-vitellogenesis and an up-regulation before meiosis resumption ( Figure 1 ). \nThe rainbow trout (Oncorhynchus mykiss) genome has not been sequenced and the number of characterized rainbow trout proteins and mRNAs is limited. The identity of studied transcripts was therefore based on the most significant hit obtained after performing a BlastX search against the SwissProt database. For the clones belonging to cluster 1-3, the results of this blast search is presented in tables 2, 3, 4. For each clone, the identity of the best hit in SwissProt and the score value of the BlastX comparison are given. However, this similarity search was performed using all EST sequences available in public databases and not using fully characterized cDNAs displaying the full coding sequence of the transcript. For some of the clones spotted on the trout array, the corresponding mRNA was previously characterized and made available in public databases. The identity of those clones is therefore unambiguous. In contrast, for some other clones, the best hit in SwissProt only gives significant, but incomplete, information. This is especially true for protein family members for which only a phylogenetic analysis will allow a more relevant identification of the gene. However, the name of the best hit was used in the text for clarity reasons.\nThis large cluster of 32 clones (# 189-220) was characterized by a clear under expression at the time of oocyte maturation. Among those 32 clones, 29 belonged to a UniGene cluster and 30 had a significant hit in Swiss-Prot ( (Table 3 ). In addition, 39 clones exhibited a significant hit in SwissProt while 5 clones had no significant sequence similarities with known genes (Table 3) . Within this cluster, several genes exhibited inflammation or ovulation-related functions. Thus some of the clones exhibited sequence similarities with human chemokine cxcl14 (clone # 250), clawed frog adam22 (clone # 258) and coagulation factor V (cf5) (clone # 235). In addition, one clone (# 245) exhibited strong sequence similarity with human angiotensin-converting enzyme 2 precursor (ace2). Two clones (# 238 and 239) exhibited strong sequence similarity with salmon (Oncorhynchus keta) vasotocin-neurophysin (avt) and isotocin-neurophysin respectively. Finally, cluster 3 also contained clones exhibiting sequence similarity with, human Forkhead box protein O3A and human pendrin, also know as solute carrier family 26 member 4 (slc26) (clone # 236). Within cluster 2, cxcl14, adam22, slc26, avt, ace2 and cf5 genes were kept for real-time PCR analysis.\nThis small cluster of 14 clones (# 296-309) was characterized by an over expression occurring earlier than for the genes belonging to cluster 3. Among those 14 clones, 12 belonged to a UniGene cluster and 11 had a significant hit in SwissProt (Table 4 ). Two clones (# 305 and 306) were most similar to rat and human aquaporin 4 (aqp4) respectively. These 2 clones belonged to the same UniGene cluster (Omy.23866). In addition, one clone (# 296) was most similar to mouse serine protease 23 (sp23). Within cluster 3, aqp4 and sp23 genes were kept for real-time PCR analysis. \nFor all the genes selected for the real-time PCR analysis, a similar up or down regulation was observed between microarray and real-time PCR experiments.\nWe observed a dramatic under expression of aromatase (cyp19a1, clones # 196 and 198) in the ovary during the preovulatory period ( Figure 2 ). The mRNA abundance of cyp19a gene during oocyte maturation was more than 200 times lower than during late vitellogenesis. In addition, successive decreases of cyp19a gene expression levels were observed during post-vitellogenesis and during oocyte maturation ( Figure 2 ). The mRNA abundance of vitamin K-dependent protein S precursor gene (clones # 199 and 200) was lower during oocyte maturation than during late or post-vitellogenesis. In contrast, no significant differences were observed between late and post-vitellogenesis ( Figure 2) . A similar expression profile was observed for Cytidine monophosphate-N-acetylneuraminic acid hydroxylase (cmah) gene ( Figure 2 ).\nWe observed a strong over expression of aquaporin 4 (aqp4) gene during post-vitellogenesis and at the time of oocyte maturation (Figure 3) . The mRNA abundance of aqp4 gene exhibited a 6-fold increase during post-vitellogenesis and a further 12-fold increase during oocyte maturation. In addition, the mRNA abundance of pendrin (slc26) gene exhibited a 1500-fold increase during oocyte maturation while no significant differences were observed between late and post-vitellogenesis. Similarly, vasotocin (avt) mRNA abundance exhibited a 500-fold increase at the time of oocyte maturation ( Figure 3 ). Angiotensinconverting enzyme 2 (ace2) gene expression levels exhibited a 215-fold increase between late vitellogenesis and oocyte maturation (Figure 3) . A similar profile was observed for the chemokine cxcl14 gene. The mRNA abundance of this gene exhibited a 35-fold increase between late vitellogenesis and oocyte maturation ( Figure 3 ). The mRNA abundance of coagulation factor V (cf5) gene exhibited a 177-fold increase between late or postvitellogenesis and oocyte maturation while adam22 mRNA abundance exhibited a 6-fold increase between late or post-vitellogenesis and oocyte maturation ( Figure 3) . Finally, the mRNA abundance serine protease 23 (sp23) gene monitored during oocyte maturation was higher than in the late vitellogenic ovary. However, this difference was not significantly different (p = 0.078).\nThe mRNA abundance of elongation factor 1 alpha (ef1α), a translation regulatory protein commonly used as a stable reference, did not exhibit any significant difference over the preovulatory period ( Figure 3 ).\nThe hybridization of radiolabeled cDNAs with cDNAs deposited on nylon membranes has been used for several decades. However, the use of nylon cDNA microarrays is not very common in comparison to glass slide microarray technology. Nevertheless, this technology has successfully been used for several years [27, 35] . In the present study we used similar cDNA manufacturing and hybridization protocols. While most of the 9152 clones used to generate the microarray putatively correspond to distinct genes, a small proportion of genes are represented by 2 distinct clones (e.g clones belonging to the same UniGene cluster). In our data, it is noteworthy that those clones are usually found in the same gene clusters (e.g clones #196 and 198, #199 and 200, #305 and 306). Since the position of clones in the clustering analysis is based on the correlation between their profiles, this indicates that they display very similar expression profiles. In addition, for all genes selected for real-time PCR analysis, the over or under expression observed was always consistent with microarray data. Furthermore, the expression of ef1α, a widely used reference gene, was stable over the preovulatory period. Together, these observations suggest that our overall microarray analysis is extremely robust and reliable.\nIn the present study, we identified 310 genes exhibiting a differential expression during the preovulatory period. Among them, 220 were down-regulated during oocyte maturation while 90 exhibited an opposite pattern. However, because we decided, as a first step, to focus our anal-ysis on the genes exhibiting the most differential regulation in the periovulatory period, we only present the identity of the 90 genes belonging to 3 specific clusters exhibiting the most remarkable patterns. Among those 90 transcripts we have chosen to discuss the most informative or novel genes based on their identities and/or putative involvement in the rainbow trout preovulatory ovarian functions.\nAmong the 32 clones belonging to cluster 1, two clones correspond to rainbow trout ovarian aromatase (cyp19a1). The real-time PCR study confirmed that cyp19a1 was dramatically under expressed during the preovulatory period. This observation is in total agreement with existing data on aromatase expression during this period [19, 36] . In addition, a clone putatively encoding for a NADPH-cytochrome P450 reductase (EC 1.6.2.4) was also located in cluster 1. The aromatase enzyme complex is formed from 2 principal protein components. CYP19a1 contains the catalytic domain that binds C19 steroid substrates in the proximity of the heme prosthetic group critical in the activation of molecular oxygen and subsequent substrate hydroxylation. The other essential component is the redox partner flavoprotein, NADPH cytochrome P450 reductase. Interestingly, present data show that both transcripts exhibited an under expression during the rainbow trout preovulatory period, although it should be confirmed that the identified clone is coding for the oxydoreductase protein involved in the aromatase complex.\nOther cytochrome P450 genes Two other cytochrome P450 genes, exhibiting similar expression profiles were found in the same cluster. One clone (# 194) was most similar to rat cytochrome P450 2J3 while the other one (# 202) putatively corresponded to rainbow trout cytochrome P450 1A3 (cyp1a3). Cytochrome P450 1A proteins are ubiquitous proteins that have been associated with the detoxification of several organic compounds such as PCB (polychlorinated biphenyl), PAH (polyaromatic hydrocarbons), and dioxin [37] . In fish, these compounds are able to induce cyp1a gene expression in a variety of tissues. In the rainbow trout immature ovary, a constitutive expression of CYP1A protein was previously reported [38] . Together, previous and present observations suggest that a CYP1A-related detoxification activity in the rainbow trout ovary. From the under expression of cyp1a3 gene observed in the ovary immediately prior to ovulation we could speculate that a decrease of the detoxification activity of the ovary is required before the beginning of the ovulation process. In addition, it was previously shown in rat C6 glioma cells that epoxygenases could inhibit prostaglandin E2 production [39] . Interestingly, C6 cells express epoxygenase mRNAs, CYP1A1, CYP2B1 and CYP2J3, which convert arachidonic acid to epoxyeicosatrienoic acids; those epoxyeicosatrienoic acid being able to inhibit the activity of cyclooxygenase [39] . The role of prostaglandins in the ovulatory process has been thoroughly studied (see [40] for review). Thus, in rainbow trout, prostaglandin F2α was able to induce in vitro ovulation [21, 41] . Therefore, the observed down-regulation of cyp1a1 and cyp2j3 genes in the ovary prior to ovulation is therefore totally consistent with available data on the participation of prostaglandins in the ovulatory process.\nIn the present transcriptomic analysis, two aquaporin 4 (aqp4) clones were found in cluster 3. Real-time PCR data confirmed that rainbow trout aqp4 gene exhibited a strong over expression in the preovulatory ovary. In mammals, AQP4 is also known as mercurial insensitive water channel (MIWC). It was previously shown that water permeability was strongly increased in African clawed frog oocytes expressing MIWC [42] . In marine fish, a strong oocyte hydration occurs during oocyte maturation [43, 44] . In addition, it was recently shown that this oocyte hydration involves an aquaporin1-like protein in seabream [45] . In freshwater species, data on oocyte hydration is more controversial. However, a limited but\nOvarian expression profiles of aromatase (cyp19a1), vitamin K dependent protein S (proteinS) and cytidine monophosphate-N-acetylneuraminic acid hydroxylase (cmah) genes during rainbow trout late oogenesis (mean ± SEM) Figure 2 Ovarian expression profiles of aromatase (cyp19a1), vitamin K dependent protein S (proteinS) and cytidine monophosphate-Nacetylneuraminic acid hydroxylase (cmah) genes during rainbow trout late oogenesis (mean ± SEM). Ovaries were sampled from separate females during late vitellogenesis (LV, N = 6), post-vitellogenesis (PV, N = 6) and oocyte maturation (MAT, N = 6). The mRNA abundance of each gene was determined by real-time PCR and normalized to the abundance of 18S. Abundance was arbitrarily set to 1 for LV stage and data are expressed as a percentage of the transcript abundance at this stage. Bars sharing the same letter(s) are not significantly different (p < 0.05).\nRelative mRNA abundance Ovarian expression profiles of angiotensin-converting enzyme 2 (ace2), coagulation factor V (cf5), CXC chemokine L14 (cxcl14), aquaporin 4 (aqp4), pendrin (slc26), vasotocin (avt), serine protease 23 (sp23), ADAM22 (adam22), and elongation fac-tor 1 alpha (ef1α) genes during rainbow trout late oogenesis (mean ± SEM) Figure 3 Ovarian expression profiles of angiotensin-converting enzyme 2 (ace2), coagulation factor V (cf5), CXC chemokine L14 (cxcl14), aquaporin 4 (aqp4), pendrin (slc26), vasotocin (avt), serine protease 23 (sp23), ADAM22 (adam22), and elongation factor 1 alpha (ef1α) genes during rainbow trout late oogenesis (mean ± SEM). Ovaries were sampled from separate females during late vitellogenesis (LV, N = 6), post-vitellogenesis (PV, N = 6) and oocyte maturation (MAT, N = 6). The mRNA abundance of each gene was determined by real-time PCR and normalized to the abundance of 18S. Abundance was arbitrarily set to 1 for LV stage and data are expressed as a percentage of the transcript abundance at this stage. Bars sharing the same letter(s) are not significantly different (p < 0.05). significant hydration was also observed in several freshwater species including rainbow trout [46] . Our data suggest that, similarly to marine species, the oocyte hydration occurring during oocyte maturation could also be aquaporin-mediated in freshwater species such as rainbow trout. In addition to aqp4 gene, we also observed a dramatic over expression of slc26 gene at the time of meiosis resumption. Solute carrier family 26 member 4 (slc26) is also known as sodium-independent chloride/ iodide transporters or pendrin. The over expression of slc26 gene at the time of oocyte maturation is dramatic, as demonstrated by real-time PCR. Together, the strong upregulation of aqp4 and slc26 genes at the time of meiosis resumption stresses the importance of water and ion transports in the rainbow trout preovulatory ovarian functions. In marine species, the major oocyte hydration occurring before ovulation is probably important for adjusting egg buoyancy. In contrast, in freshwater species laying demersal eggs such as rainbow trout, it has been hypothesized that the limited (25%) oocyte hydration occuring before ovulation could be necessary for the completion of the ovulation process [46] . Thus, the increase of oocyte volume could facilitate the rupture of the follicular walls and subsequently, the release of the oocyte from its follicular layers.\nThe neurophysial hormones arginine vasotocin (AVT) and isotocin (IT) are the fish counterparts of argininevasopressin and oxytocin respectively. Vasotocin precursor and isotocin precursor cDNAs were previously cloned in several fish species including chum salmon [47, 48] . In fish, AVT is involved in several physiological processes including water conservation and excretion of electrolytes [49] . However, existing data in fish correspond to the local effect, in various tissues, of circulating AVT [49] . Surprisingly, we observed that AVT precursor (avt) mRNA is expressed in the rainbow trout preovulatory ovary. To the best of our knowledge, there is no evidence of non-neural expression of avt mRNA in fish. In addition, it is noteworthy that we also observed a similar over expression of isotocin mRNA precursor in the ovary at the time of oocyte maturation. Further investigations are needed to elucidate the role of AVT and IT in the trout preovulatory ovarian functions.\nOvulation is a complex process resulting in the release of the oocyte from surrounding follicular layers. Since the early eighties, the similarities between ovulatory and inflammatory processes have been thoroughly discussed [50] [51] [52] and it is now well accepted that mammalian ovulation is an inflammatory-like reaction. In fish, despite numerous studies on the hormonal control of spawning, the ovulatory process has been far less documented.\nIn mammals, ovulation is accompanied by broad-spectrum proteolysis and the implication of several classes of proteases is well documented (see [53] for review). In salmonid fish, several proteases have been identified in the periovulatory ovary [54] . In mammals, there is evidence that mature ovarian follicles contain proteolytic enzymes, including serine proteases. Indeed, serine proteases have been implicated in both ovulatory and inflammatory reactions (see [50] for review). In the present study, serine protease 23 (sp23) gene appears progressively up-regulated during the preovulatory period. To our knowledge, sp23 gene expression was never reported in the periovulatory ovary of any vertebrate species. However, we could speculate that this protease participates in the rainbow trout ovulatory process. Interestingly, our data showed that adam22 metalloprotease-disintegrin gene was sharply up-regulated at the time of oocyte maturation. The metalloprotease-disintegrin protein family (also known as ADAMs: A Disintegrin And Metalloproteinases) is thought to function in cell-cell interactions and in the proteolysis of luminal or extracellular protein domains. In mammals, several ADAMs family members are involved in the ovulatory process. In brook trout (Salvelinus fontinalis), metalloprotease activity increases in the ovary prior to ovulation [8, 9] . Together, these observations also suggest that adam22 also participates in the rainbow trout ovulatory process.\nMammalian CXC chemokines, named after a conserved pattern of conserved cysteine residues, have been initially identified as potent mediators of neutrophil chemotaxis [55, 56] and are also involved in chemotaxis of monocytes and lymphocytes. They have also been implicated in angiogenesis and, later, in a large variety of functions [57, 58] . In mammals, 16 CXC have been described. In Fish, however, several CXC have been identified but only CXCL12 and CXCL14 exhibit unambiguous orthologues [59] . In the present study, we showed that cxcl14 gene expression strongly increases during the preovulatory period. In catfish, RT-PCR data showed that cxcl14 gene was expressed in a wide variety of tissues, including the ovary [60] . In carp, quantitative PCR data showed that cxcl14 was predominantly expressed in the brain [61] . Despite its good conservation throughout vertebrate evolution [59] , the number of studies addressing the in vivo role(s) of CXCL14 is limited. As a consequence, a lot of information is still unavailable in fish. In a murine model used to study Crohn's disease, cxcl14 expression is induced during inflammation [62] . Together, these observations suggest that cxcl14 gene expression induction contributes to the inflammatory-like events occurring in the rainbow trout at the time of ovulation. To date the participation of this gene in preovulatory ovarian functions was unsuspected.\nIn mammals, coagulation factor V participates in the coagulation process. In zebrafish, a coagulation factor V (cf5) cDNA was previously characterized [63] . According to these authors, several lines of evidences including biochemical and phylogenetic analyses suggest that the modern coagulation pathways found in mammals could also be functional in fish. Furthermore, it was previously shown that cultured rabbit macrophages were able to generate factor V procoagulant activity [64] . In the present study, we observed a dramatic increase of cf5 gene expression in the ovary during oocyte maturation. However, no significant difference was observed between late and postvitellogenesis. From these observations we could speculate that, immediately prior to ovulation, the trout ovary secretes coagulation factors in order to prevent bleeding from ruptured ovarian follicles at the time of ovulation. Interestingly, the transcriptomic analysis showed that a transcript exhibiting sequence similarity with clotting factor C (Clone # 251, Table 3 ) was also over expressed immediately prior to ovulation.\nAngiotensin-converting enzyme (ACE) cleaves Angiotensin I (Ang I) to form Angiotensin II (Ang II). Angiotensin-converting enzyme 2 (ACE2) is a recently described ACE homolog [65] . Both ACE and ACE2 are zinc-dependent peptidases of the M2-metalloprotease family. Within the renin-angiotensin system (RAS), ACE2 competes with ACE because it is capable of hydrolyzing Ang I into the nonapeptide Ang(1-9) [65] . In humans, ace2 gene expression was predominantly detected by Northern blot analysis in kidney, heart and testis [65, 66] . In addition, a moderate expression was also observed in several other tissues including the ovary [66] . Using semiquantitative RT PCR, a wide distribution was observed in rat tissues [67] . In mammals, previous observations suggested that the renin-angiotensin system was functional in the ovary. In cattle, a greater expression of Ang II was observed in large follicles. In addition, several lines of evidence supported the idea of Ang II in blocking the inhibitory effect of theca cells on meiosis resumption of bovine oocytes [68] . In brook trout (Salvelinus fontinalis) salmon Ang I and human Ang II were both able to increase the level of in vitro spontaneous ovulation [69] . In the present transcriptomic study, we observed a dramatic increase of ace2 gene expression during the preovulatory period. This observation was confirmed by real-time PCR data. Together, these observations suggest that the dramatic upregulation of ace2 gene immediately prior to ovulation is important for the ovulatory process. In mammals, little is know about the role of ACE2 in the ovary. However, it is known in mammals that ACE2 can function as an Ang II degrading enzyme, forming the vasodilatator peptide Ang(1-7) [70, 71] . Interestingly, a local vasodilatation is a key characteristics of the inflammatory response that is also observed during the mammalian ovulatory process (see [50] for review). Therefore, it can be hypothesized that the observed increase of ace2 gene expression in the trout preovulatory participates in the vascular dynamics changes that are putatively occurring during the ovulatory process.\nGenes involved in the synthesis of egg components Cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) is the key enzyme for the synthesis of N-glycolylneuraminic acid. In salmonid eggs, cortical alveoli contain polysialoglycoproteins (PSGP). In rainbow trout, it was previously shown that those PSGP contain N-glycolylneuraminic acid residues [72] . In the present study we observed a significant decrease of cmah gene expression at the time of oocyte maturation. While the presence of cmah gene expression in the ovary is totally consistent with the presence of N-glycolylneuraminic acid in rainbow trout cortical alveoli content, it seems however difficult to speculate on the dynamics of PSGP accumulation in the oocytes.\nOur observations further confirmed that a progressive shut down of estrogen synthesis genes expression occurs in the ovary prior to meiosis resumption. In addition to already well studied genes such as aromatase, the present work shows that other genes exhibit a similar down-regulation, thus suggesting their participation in the preovulatory decrease of circulating estrogen levels.\nIn addition, we observed a strong up-regulation of ion/ water transport genes in the preovulatory ovary. The identity of those genes is consistent with the recent identification of aquaporin mediated mechanisms in the fish oocyte hydration process and further supports the recent description of a limited but significant oocyte hydration occurring in the rainbow trout preovulatory ovary.\nFinally, in addition to oocyte hydration-related genes, we also observed a strong over expression of several genes such proinflammatory factors, coagulation/clotting factors, vasodilatation factors and proteases in the ovary immediately prior to ovulation. Together, these observations suggest that, similarly to the theory developed in mammals, fish ovulation could also be compared ton an inflammatory-like reaction. In addition, the identification of those genes will allow specific studies leading to a better understanding of the ovulatory process in fish.\nIn the future, a global analysis of differentially regulated genes, based on their ontologies, is needed to satisfyingly describe preovulatory ovarian mechanisms. In addition, a cellular localization of gene expression will contribute in the understanding of their respective roles in the preovulaory ovarian physiology. Nevertheless, the present study clearly demonstrates that distinct (i.e. steroidogenic, proteolytic, proinflammatory) but concomitant events occur in the preovulatory ovary. Together, all those events concur to achieve the same goal which is the release, at the time of ovulation, of a fully competent oocyte, ready to be fertilized." }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: The most commonly used measure in public health, R 0 , is estimated from historical data and derived from SIS/SIR type models (and descendents) for forward projection [14, 15] R 0 is the basic reproductive ratio for how many individuals each infected person is going to infect [16] R 0 is often used on its own in public health as an indicator of epidemic probability; if R 0 < 1 then an epidemic is not generally considered possible, for R 0 > 1, the larger the value, the more likely an epidemic is to occur. R 0 is a composite value describing the behavior of an infectious agent. Hence, R 0 can be decomposed classically, for example, as: p d c, where p is probability of infection occurring for a contact, d is duration of infectiousness, and c is number of contacts [17] .\nHowever, R 0 in the classical decomposition above, while it is one of the best tools we have, does not account for age segregation of response, existing immunity in population, network topology of infectious contacts and other factors.\nThese observations were significant in the motivation for developing EpiFlex.\nThe EpiFlex model was designed to create a system that could incorporate as much realism as possible in an epidemic model so as to enable emerging disease events to be simulated. There are limitations, described below in a separate section, but the model is quite effective as it stands. In most cases, the limitations of EpiFlex are shared by other modeling systems.\nThere are a variety of methods used for mathematical modeling of diseases. The most common of these are the SIR (susceptible, infected, recovered) of Kermack and McKendrick [15] , SIS (susceptible, infected, susceptible), SEIR (susceptible, exposed, infected, recovered), and SIRP (susceptible, infected, recovered, partially immune) as developed by Hyman et al. [18] and further developed by Hyman and LaForce [19] . The SIRP model was used as the starting point for development of the object model of Epi-Flex. In SIRP, the SIR model is extended to include partial immunity (denoted by P) and the progressive decline of partial immunity to allow influenza to be modeled more accurately. (See Appendix.)\nThere is a need for experimentation in more realistic discrete modeling, since the lattice type of discrete modeling is understood to skew in favor of propagation, as discussed by Rhodes and Anderson [20] and Haraguchi and Sasaki [21] . Others such as Eames and Keeling [22] and Edmunds et al. [12] have explored the use of networks to model interactions between infectable entities, and Ferguson et al. [23] and others have called for more balance in realism for epidemiology models. Since EpiFlex was completed, Lloyd-Smith et al. [17] have shown the importance of superspreading in disease transmission for the SARS epidemic. EpiFlex is designed to take these issues into account.\nThere are known weaknesses in SIS-descended models, some of which are discussed by Hyman and LaForce [14] . They suggested that a model dealing with demographics and their subgroups would be useful and described a start Theoretical Biology and Medical Modelling 2006, 3:32 http://www.tbiomed.com/content/3/1/32 toward conceiving such a model, creating a matrix of SIRP flows for each demographic group within a \"city\" and modeling contacts between these groups. Thus, the possibility of building an entirely discrete model using the object-oriented approach, essentially setting the granularity of the Hyman-LaForce concept at the level of the individual, together with Monte Carlo method, was attractive. The object method of design seemed to be a good fit, since object-oriented programming was invented for discrete simulations [24] . An Object-Oriented (OO) design defines as its primitive elements \"black box\" subunits that have defined ways of interacting with each other [25] .\nThe OO language concept originally was conceived for the Simula languages [24] for the purpose of verifiable simulation. Enforcement of explicit connections between objects is fundamental to OO design, whereas procedural languages such as FORTRAN and COBOL do not because data areas can be freely accessed by the whole program. OO languages wrap data in methods for accessing the data. If each \"black box\" (i.e. object) has a set of specified behaviors, without the possibility of invisible, unnoticed interactions between them, then the simulation can potentially be validated by logical proof in addition to testing. (It would take an entire course to introduce OO languages and concepts, and there is not space to do so here. Interested readers are suggested to start with an implementation of Smalltalk. There are excellent free versions downloadable. Smalltalk also has an enthusiastic and quite friendly user community. See: http:// www.smalltalk.org/main/.)\nThe design of EpiFlex is described more completely in the appendix. Design proceeded by establishing the definition of a disease organism as the cornerstone, then defining practical structures and objects for simulating the movement of a disease through populations. The disease object was assigned a set of definitions drawn from literature that would allow a wide spectrum of disease-producing organisms to be specified. The aim was to minimize the number of configuration parameters that require understanding of mathematical models.\nThe hosts that are infected became the second primary object. A host lives and works in some area, where hosts are members of some demographic group, which together determine what of n types of contacts they might have to spread an infectious disease. The hosts move about the area in which they live between locations at which they interact. In EpiFlex, an area contains some configured number of locations, and locations are containers for temporary groups of hosts. Since people travel between metro areas, the model supports linkages between areas to move people randomly drawn from a configurable set of demographic groups.\nThe remainder of this section presents the disease model adopted, an overview of each component, an overview of program flow, and a description of the core methods. This is followed by discussion of results from the EpiFlex software system.\nThis model has up to four stages during the infection cycle: the Incubation, Prodromal, Manifestation, and Chronic stages; to this is added a fatality phase. I have named this 'extended-SIRP'. Fig. 1 shows a diagram of this model.\nThe model of Fig. 1 allows us to track the different phases of the disease process separately, and to define variable infectiousness, symptoms, fatality, recovery and transition to chronic disease at each stage as appropriate. This allows us to model the progress of a disease in an individual more realistically. For diseases that have no identifiable occurrence of a particular stage, this stage can be set to length zero to bypass it entirely.\nThe 8 contact types designed into EpiFlex are drawn from literature in an attempt to model spread of infection more accurately. These contact types are: blood contact by needle stick, blood to mucosal contact, sexual intercourse, skin contact, close airborne, casual airborne, surface to hand to mucosa, and food contact. The probability of infection for a contact type is input by the user as estimated from literature or based on hypothetical organism characteristics.\nDurations of disease stages are chosen uniformly at random from a user-specified interval [R low , R high ]. Random numbers, denoted by ξ, on [0, 1] are used to seed the determination of the infected disease stage periods (denoted I Incubation , I Prodromal , I Manifestation , I Chronic ). R low and R high are taken from medical literature and describe a range of days for each stage of an illness. These calculations are simply: (ξ × (R high -R low )) + R low = D, where D is days for a particular stage. (This may be extended in the future to include ability to define a graph to determine the flatness of distribution and the normative peak. This will make a significant difference in modeling of diseases such as rabies, which can, under unusual circumstances, have very long incubations.)\nOne of the following three equations describing immunity decay is chosen; L is the current level of partial immunity, P is the level of partial immunity specified as existing Random values on [0, 1] are then used to decide whether an infection occurs during the partial immunity phase P shown in the chart above. This decision uses the output of the immunity level algorithm, L, which is a number on [0, 1], as is the random value ξ:\nEpiFlex uses a dynamic network to model the interactions between hosts at a particular location based on the skew provided and the demographic segments movement cycles. The networks of contacts generated in this version\nExtended-SIRP disease model of Epiflex Figure 1 Extended-SIRP disease model of Epiflex. S: susceptible I: Infected R: recovered P: partially immune F: fatality Extended SIRP breaks the infected stage I into 4: I Incubation , I Prodroma l, I Manifestation , I Chronic , and adds a fatality terminating stage.\nof EpiFlex are not made visible externally; they can only be observed in their effects. (See: Limitations of EpiFlex modeling.) Their algorithms were carefully designed and tested at small scales, observing each element.\nA location describes a place, the activities that occur there, and the demographic groups that may be drawn there automatically. A location can have a certain number of cells, which are used to specify N identically behaving locations concurrently. This acts as a location repetition count within an area when the location is defined. The user sets an average number of hosts inhabiting each cell, and a maximum. There is also a cell exchange fraction specifiable to model hosts moving from cell to cell. The algorithm for allocating hosts in cells is semi-random. It randomly puts hosts into cells in the location. If a cell hits the average, then it does another random draw of a cell. If all locations are at maximum, then it overloads cells.\nInteractions are within the cell. So a host must be exchanged to another cell in order to be infective. See the appendix for 'Location component', and also with an open model look at how hospitals were defined. Households are modeled at this time using a cell configuration.\nEpiFlex is implemented with a Monte Carlo algorithm such that each host in a location is assigned a certain number of interactions according to the Cauchy distribution parameter setting for that location. This distribution describes a curve with the y axis specifying the fraction of the maximum interactions for the location and x axis specifying the fractional ordinal within the list of hosts in the location. The distribution can be made nearly flat, or severely skewed with only a few actors providing nearly all contacts, as desired by the user of EpiFlex. Note that the structure of the network formed also depends on what locations are defined, what demographic groups are defined for the population, and how demographic groups are moved between locations. Each location has a maximum number of interactions specified per person, which is used as the base input. Initially, a Gaussian equation was used, but it was discarded in favor of a Cauchy function since this better fits the needs of the skew function and computes faster. The algorithm iterates for each infectious host, and selects other hosts to expose to the infected party in the location, by a Monte Carlo function. This results in a dynamically allocated network of interactions within each location.\nThe exposure cycle also makes use of Monte Carlo inputs. Each location has a list of contact types that can take place at a particular location, and a maximum frequency of interactions. This interaction frequency determines how many times contacts that can spread a disease will be made, and the contact specification defines the fractional efficacy of infection by any specific route. Modeling the effect of different types of contacts has been discussed in the literature, e.g. Song et al. [26] . EpiFlex attempts to make a more generalized version.\nFor each host infection source, target hosts are drawn at random from the location queue. A contact connection is established with the target as long as the contact allocation of that target has not been used up already. Contact connections made to each target are kept track of within the location to prevent over-allocation of contacts to any target. Thus, for each randomly established connection, a value is set on both ends for the maximum number of connections that can be supported. Once the maximum for either end of the link is reached, the algorithm will search for a different connection.\nThe location algorithm is described below in more detail. The user specifies the maximum number of connections for a location; the σ output from a Cauchy distribution function determines how many connections an individual will have. This allows variations in the degree of skewness for superspreading in a population to be modeled, which has been shown to be of critical importance by Lloyd-Smith et al. [17] .\nIf p = position in queue, q = number of hosts in queue for location: X = p/q, where X denotes the proportional fraction of queue for position.\nIf K is a constant chosen for the location to express skew distribution, the Cauchy distribution function is:\nIf κ is the number of contacts for a particular host and κ max is maximum number of contacts for any given host in the location:\nWhen hosts move from one location to another within the model, they tend to maintain a rough order of ordinal position. Consequently, when there is a high σ for a location, the high connection host in one location tends to be a high connection host in another. This reflects real-world situations, (though not perfectly) and corresponds better than persistently maintaining high connection individuals from location to location, since host behavior changes from place to place.\nThe Cauchy distribution function is fairly fast in execution. The function can be used to approximate the often radical variations seen in epidemiology studies; as an extreme example, one active super-spreader individual might infect large numbers, when one or even zero is typical [17] . This type of scale-free network interaction has been explored by Chowell and Chavez [27] . The Cauchy function allows networks to be generated dynamically within each type of location in a very flexible manner, such as corresponding to super-spreader dynamics [17] . In addition to the specification of skew within a location, the network of contacts is also defined by (a) what locations are present and (b) the movement cycles defined for each demographic group within the model.\nProcessing time increases with population. This slowing is an expected characteristic of an object modeling system and is the price paid for the discrete detail of the EpiFlex model. The primary source of this increase in processing time is the sum of series of possible infectious events that are modeled for each iteration. It therefore scales as a series sum not as a log, based on the contagiousness of the disease and the number of potential hosts in a location with an infected host. This is minimized by only processing infectious host contacts. The increase stems from the characteristics of networks in which each node has n connections to other nodes. When iteration is done for a location containing infectable hosts, it is the number of infected hosts that creates an element of the series. The infected hosts are put into a list, and each one interacts randomly with other hosts (including other infected ones) in the location. Thus, considered as a network with m nodes, each of the m nodes is a host. A temporary connection to another host is made to n other nodes where n <m, and n<k. The value of k is determined by a randomized input that returns the number of contacts of this infected host in this location. Consequently, the series consists of all the temporary connections made for contact modeling for each cycle.\nIn the interest of completeness, the limitations of the Epi-Flex model are described here. The plan is to address these elements for implementation in future versions.\nOnly one infectious disease can be occurring at a time. Thus, competitive inhibition [28] and synergistic effects will not be seen. Hosts do not reproduce Hosts do not reproduce within a model. Removal and addition rates are defined for the population as a whole, and the basis is US Census data. To meet the specifications for removal and addition within the model, hosts are removed from randomly chosen locations, and similarly added to randomly chosen locations. Demographic group is also randomly assigned. For long-term modeling, and modeling of alternative short-lived hosts, a reproduction cycle is desirable. However, EpiFlex is a practical way of modeling periods of a few years.\nThere is no explicit definition of an age distribution for the host population, which can be quite significant [29] . To a degree, age is taken into account through the demographic segmentation of populations. A demographic can be defined with a fraction or multiple of baseline susceptibility. However, hosts do not age, nor do they move from one demographic to another as they age.\nNo provision is made to define a previous exposure profile for hosts [30, 31] . In real populations previous exposures can have significant effects on the spread of a disease and dramatic effects on mortality where infection does occur [32] . Proper implementation of previous exposure profiles is intertwined with age definition.\nThere is no implementation of mutation rate for diseases. Mutation rates vary considerably by type, particularly for viruses [33] . Decay of immunity is modeled, and immunity decay can act as a fair surrogate for antigenic change.\nPass-through events must be defined as part of surface contacts For efficiency, EpiFlex eliminates pass-through infection events from being modeled: for example, an infected A shakes the hand of a non-infected B, who then shakes the hand of another non-infected C, but B washes hands and does not become infected while C does. Therefore, the model definition must account for this through \"Surface to hand to mucosa\" contacts, where a person can also be a surface.\nThe model does not at this time record the contact network that is dynamically created except in the log file at this time. Those that are logged are only potential infectious contacts. To get at that data requires looking at the log file and writing an extract program. Making the network visible is an item for the future.\nCurrently, EpiFlex has no way of accounting for seasonal damping. Similarity of results is due to the settings of the rate at which immunity declines. Addition of a seasonal damping function would be expected to cause EpiFlex results to synchronize with a yearly cycle. Seasonal damping would result in loss of interesting epidemic behavior with an overriding function that would virtually be guaranteed to drown out other behaviors.\nPublic health response to epidemics is not optimally modeled A public health response definition component is present in EpiFlex. Testing of this component, and more thorough review of literature, indicate that the method used is not optimal. Current public health responses are centered on contact tracing, ring vaccination and quarantine [34] , with mass vaccination as a backup when it is available. Closures of schools, daycare and travel restrictions are also used. These methods are not modeled in EpiFlex' response component. Their importance has recently been underscored by Lloyd-Smith et al. [17] . As a consequence, results from the current system that defines across-theboard cuts in the probability of infection should be considered in this light. It is not clear whether any other object system can model all current techniques properly.\nDiseases have ranges of times for each stage that can be drawn from literature. Probability of a specific disease stage time period for an infected host being chosen within the range is equal. This is reasonably adequate for most diseases where times are measured in a few days, however, some, such as rabies have a quite unequal distribution, and their very long tail makes a difference in modeling.\nThe discussion is presented in three parts: (1) a brief set of examples of native EpiFlex displays to develop a better feel for the system; (2) comparisons of EpiFlex results with real world data; (3) a set of examples of observations made using EpiFlex. The purpose of these examples is to serve as a guide to others who may want to experiment and analyze results.\nDifferent views of the epidemic data for simulated influenza in two different populations are shown in Figure 2 . Figures 2a and 2b show graphs of the second and third epidemics in the population. These graphs show the kinds of commonly-seen deviations from a smooth curve that occur in real world data [35] . In the EpiFlex model, this is attributed to less synchronization of immunity combined with the formation of small world networks among demographic groups as they move from location to location. Figures 2c and 2d are alternate views of a simple influenza epidemic occurring within a naïve population (Figure 3 ).\nComparison of EpiFlex with WHO/NREVSS surveillance Comparing EpiFlex with surveillance data, we see that WHO/NREVSS surveillance data [36] have a qualitatively similar graph form to EpiFlex for influenza, as shown in Figures 4 and 5. The width of the primary curves per season for EpiFlex is 3.5 to 4.5 months while that of the NREVSS data is approximately 5 to 7 months, which can be explained by the NREVSS data being collected nationally from surveillance centers, whereas the EpiFlex data shown are for a single area. EpiFlex runs executed with multiple cities connected by transport, such as the 3.5 million population 35 city model, have a combined graph for all cities showing self-similarity to the graphs for individual areas, becoming wider, matching the NREVSS data graph formation.\nThe NREVSS data consist of diagnostics of samples sent in by physicians. Comparisons of absolute numbers in terms of quantity are therefore not applicable. A percentage of population comparison is done below.\nIn Table 1 , EpiFlex indicates that roughly 48% of the population has been infected before herd immunity stops the epidemic, though this depends on population size. Total morbidity is obtainable from EpiFlex by adding maximum immune level to deaths, although deaths contribute such a small amount to influenza morbidity that for practical purposes the immune level is used as a proxy for morbidity. Moreover, true morbidity itself is relatively prone to inaccuracy, whereas better measures of immune fractions for influenza are available. The California state average for 2000-2003 is 25.4% infected in a range from 12.7 to 44.6 depending on county [37] . Thus, EpiFlex is above the high end of the state of California estimated morbidity range.\nDowdle [32] gives serological influenza data categorized by age. For influenza A/Swine/15/30 H1, seroprevalence ranges from roughly 25% to over 95%. For influenza A/ Hong Kong/68 H3, the range is from 5% to 99%. EpiFlex figures fall within this latter pair of ranges, and EpiFlex immune fraction is more properly comparable. \nWhat is most notable in Figure 7 is the relationship between the rough sine wave form of the classically derived SIRP [19] once the initial startup period is over for influenza, a repeating wave develops that is similar in overall shape and variability to real world data such as those for Milwaukee, at a roughly similar scale. These two graphs refer to populations that differ in size by about 1 order of magnitude (i.e. Milwaukee is 9 times the size of the model run shown). We can also see a similar number of peaks. Owing to the need to compare these two graphs natively, these two figures are not optimum. However, they show essential features.\nTotal morbidity rate linked to population size The smaller a population over the range 1,000 to ~3.5 million, the higher the total morbidity rate, given identical organisms ( Figure 6 ). It is intuitively expected that population size will affect morbidity since, for any given network of contacts connecting individuals in populations, the chance of the epidemic spreading during the window prior to the development of immunity in parts of the population increases as the population size decreases. This This is the simulation that was imported for comparison Figure 3 This is the simulation that was imported for comparison. Vertical scale 1000 per demarcation. Horizontal scale one year per demarcation. Upper white line is total population with standard removal rate. effect is most striking when very small populations in the order of 1,000 are examined. Literature data regarding this in real world populations are sparse. However, there are indications from historical accounts of small populations in the new world that a link between population size and morbidity is observable in real world populations [38] [39] [40] [41] [42] [43] . The most recent such account is from Heyerdahl in the Pacific in the mid 20 th century [44] .\nIn the graphs of Figure 6 , the immune fraction at completion is used as a proxy for total morbidity on a log scale of population. The longer an epidemic takes to progress within an enclosed population, the greater the number of potentially infectious contacts that hit a dead end because the host is already immune. Since very small populations will mostly function within the window when there is no host immunity, the infection will spread to a larger fraction. This effect has public health implications because, clearly, the structure of the network is highly significant in determining the likelihood that an infected host will contact naïve hosts. Essentially what this EpiFlex result indi-cates is that during the period prior to the development of an immune subpopulation, a disease has a functionally higher R 0 . (i.e. R 0 is variable through the course of an epidemic.)\nIn the Figure 6 graphs, EpiFlex is also suggesting that there are more asymptomatic infected spreaders of influenza in our populations than surveillance data estimate. This is also suggested by the discussion above regarding comparison with seroprevalence.\nA minor experimental result is that for a repeating illness such as influenza, when a continuously active initiating disease vector tries to infect 3 people per day, it will develop higher peaks after the initial event than a vector that tries to infect 30 people a day (where both are randomly distributed through the population.) This makes intuitive sense, because there is lower probability that a subpopulation of susceptible hosts will become large when there are more attempts to infect them. Similarly, in a system of cities interconnected by transport linkages, later peaks tend to be smaller and more variable than earlier ones. This is due to two things. First, a degree of lowgrade infection linking back through the system provides a higher total level of infection events in the whole system than the formally defined initiating disease vector. Second, as time passes, the mix of immune versus susceptible becomes unsynchronized for the population as a whole, since hosts that escaped infection during one epidemic may be infected during the next, and some whose immunity has declined may also become infected. Thus, it is expected that we would see the development of a complex non-repeating waveform with some similarity. This type of waveform is what EpiFlex shows with longer simulations in large populations, as illustrated in Figure 8 .\nA variety of results is obtained when one or just a few index cases are provided to seed a single city's susceptible population when those index cases are not repeated. This is expected, owing to random interactions that break the chain of contacts in some percentage of cases. This effect would be expected to increase with a higher skew on super-spreaders. The significance of this for modeled epidemics, particularly in the light of recent work [17] , is that in some cases (the proportion would be expected to vary in rough accordance with R 0 ) the infection dies out owing to random chance. Thus, a Monte-Carlo model such as EpiFlex, when used in multiple trials, clearly reveals the potential range of variation in epidemics given apparently identical conditions.\nEpiFlex shows wave propagation of epidemics through its transport network that are similar to real world epidemic studies such as those of Viboud et al. [45] . Viboud et al. state a mean duration of 5.2 weeks to spread across the United States, with a range from 2.7 to 8.4 weeks. The Epi-Flex results shown using a simplified city configuration of 35 major airline hub cities, with a total 3.5 million population among all 35 cities, shows a propagation wave of 1.8 weeks. While this is shorter than real world data, several factors account for the difference. First, in the current EpiFlex \"vanilla\" configuration, the transport network is flat in terms of the numbers of persons moved from city to city. Second, each city contains the same population of only 100,000 due to practical limitations. For the propagation histogram of Figure 9a , 1000 manifesting cases or more was used as the data point. Please note, however, this flatness of transport and population is purely a matter of the configuration of the specific model used. The Epi-Flex system allows separate specification of all parameters for each city, and any kind of transport level between any two cities that is desired. Figure 9a shows a histogram of cities in which 1,000 manifesting cases are first occurring. Figure 9b shows a histogram of cities in which the first occurrences of at least 10 incubating cases of influenza appear. Note that in Figure Upper graph shows graphed points for population versus total morbidity as estimated from immunity Figure 6 Upper graph shows graphed points for population versus total morbidity as estimated from immunity. Lower two graphs show sample graphs for 3500 (lower left) and 35000 (lower right). The green line in the lower two graphs shows immunity. One vertical demarcation on the x axis is one month in the lower two graphs. \nEpiFlex is useful for doing in-silico experiments with epidemic behavior and easy to configure. It can run on an ordinary computer without special configuration. Data can be imported from it into other tools and worked with there. It is effective for showing factors that are invisible or difficult to access under normal conditions, such as visibility of incubation and prodromal stages, true morbidity and estimates of immunity. EpiFlex has capabilities that were not discussed owing to time and space constraints. This system is effective in duplicating the kind of multimodality and apparent stochastic variation [13] that are seen in real populations. EpiFlex results can provoke thought about the nature of epidemics and infectious disease spread in interesting ways by providing an experimental test environment that is not as abstracted from its subject as most mathematical models are.\nItems in bold below are entities that are objects in the Epi-Flex system. They are the \"nouns\", each of which became a class when the program was written in C++. Items in italics are actions, the \"verbs\" that represent interactions between objects.\nHosts go through the infectious stages, are members of Demographic groups and move between locations\nLocations contain hosts (For example a hospital, home or workplace.)\nAreas contain locations (A city, typically) and contain special locations that can move hosts to locations in other areas.\nDisease Vectors introduce disease into host populations at some location in some area, in either a limited way or on a regular cycle.\nEpidemic responses modify the spread of an infection. They represent actions taken to respond to the epidemic that will damp the spread. \nSIRP is a simple flow chart, which is then elaborated into a multi-city system. Three graphics are reproduced in Figure 10 concluding with output of the model, because the SIRP paper is in a specialty publication and the work was important to the conception of EpiFlex.\nAs seen in the \"SIRP diagram\" of Figure 10 , a host begins as susceptible (S) in the upper left. They can become infected (I), and then they either recover (R) or are removed (removal not shown). After recovery (R), they have an immunity level that starts at or near complete as in SIR. This immunity declines, in the case of influenza, over a period of roughly 2 years, with a steep drop-off beginning after the first year, and hence is termed partial immunity(P). (It is not actually the case that the immune system's response to influenza antigens degrades so rapidly; rather, the virus mutates rapidly, and host immunity declines. The net result is similar.) This decline in immunity models the real world antigenic mutation of influ-enza viruses. In a more perfect model, the influenza virus would change and multiple viruses would be modeled. At some point the immunity declines so that it is effectively zero in the model (which corresponds to large antigenic change) and the person is returned to the fully susceptible(S) population. A person can become reinfected (diagonal arrow), though their probability of reinfection is lower, during this period of declining immunity.\nIn the \"Multi-city SIRP\" flows of Figure 10 center, m [x,y] denote movement between the cities in a four city example. Moving further to the right of Figure 10 and looking at the \"Actual versus model graph\", one can see how a sinusoid curve, with some general correlation with period, results from the conventional model.\nThere are four files used or generated by EpiFlex. All three generated files have the name of the configuration from A 5.5 year, 9 city simulation linked by transport corridors. 100,000 people per city Comparison of distribution of days when 1,000 manifesting cases or more first appear in a city (upper graph -9a) versus distri-bution of days when 10 or more incubating cases first appear in a city (lower graph -9b) Figure 9 Comparison of distribution of days when 1,000 manifesting cases or more first appear in a city (upper graph -9a) versus distribution of days when 10 or more incubating cases first appear in a city (lower graph -9b). \nModel files end in .EPDM. These files are an XML format. This is the file edited by Epiflex using the various panels available from the menu. An EPDM file can also be edited directly in WordPad or a good XML editor by more sophisticated users. Reading through them should be selfexplanatory to most users who familiarize themselves with the software.\nRun record files end in .RPX. These files are in text format, comma, and semicolon delimited. This format is intended to be as easy to import as possible. RPX files can be imported into Excel, read by SAS, SPSS, etcetera. At the top of this file is a descriptor of the fields. The most common problem is to import using spaces as a delimiter and have spaces in area names define new columns.\nLog files end in .LOG. Everything that happens in a model is logged. These files can get fairly large. Potentially, they could be parsed by a secondary piece of software to match them up with RPX files. However, that is an exercise for the user at this time. There is a viewer for log files under the View menu.\nSnapshot files end in .SNAP. Whenever a run completes, a SNAP file is written that is read only. This file is a duplicate of the model that was used to run -as that model existed in memory at completion of the run. This is done as a \"lab notebook\" aid because it was recognized early on that remembering exactly what was contained in a model was well nigh impossible and a lot of work to document.\nNote that if your model does not complete its run, the RPX file will still be there, but the SNAP file will not be. In that case it is up to the experimenter to make a record of the state of the model.\nThe SNAP file can be copied outside of Epiflex, manually changed from read-only and the extension renamed to .EPDM. The resulting model can then be used and run just like any other.\nSoftware engineering details such as internal class definitions and structure of components will not be presented since the UI does a better job of educating, and is much more compact.\nDisease component Looking at screen shots of the current UI in Figure 11 , one can see what the functional parametric elements of a disease specification are. This particular example is selected for influenza. The parameters set reproduce the known characteristics of the disease.\nThe disease stages of Figure 11 correspond to the model diagram shown in Figure 1 . One can see that this definition is superior in several ways to what is available with SIRP modeling as shown in the previous section. (See Figure 10 ). In addition to multiple stages, one can define the level of infectiousness for each type of contact that an infected host might have. On the lower right, the partial immunity stage is specified. Here, one is able to define a wide variety of immune responses. Asymptotic decline seems to fit available data and theory on immune system (from left) SIRP Diagram, Multi-city SIRP, Actual versus model Figure 10 (from left) SIRP Diagram, Multi-city SIRP, Actual versus model.\nfunction the best for many diseases. Use of the slider allows one to \"eyeball\" the asymptotic curve to approximate what seems reasonable. In this case, immunity is set to begin dropping after approximately half the 730 day period has passed. It makes sense to do this because data in this area are fairly sparse, and one may want to perform multiple runs with slightly different immune drop characteristics. Keep in mind that the width of the graph is for the number of days entered on this panel.\nA host is implicitly one of the number in the population of an area. In the EpiFlex system, there is one object defining each disease. A host infected by a disease receives a pointer to this archetypal disease object, and stores their disease stages internally as the disease progresses. Most of the functionality of hosts consists of an implementation of disease stage transition. Figure 12 is a screen shot of the UI for a set of demographic definitions selected from US Census data for the nation as a whole. In the resulting model, one can over-ride the default fractions later when using them in populations for specific areas. No fractions are represented that could not be derived from recent census data.\nEach demographic group can be set up to specify variations in overall susceptibility to disease, or modify it by specific type of contact if that is desired. In the example, no fractional modifiers have been created for this demographic group. There is no necessity to stick to the demographic groups defined here. One can add new ones as desired.\nA location describes a place, the activities that occur there, and the demographic groups that may be drawn there automatically. A location can have a certain number of cells, which are used to specify N identically behaving locations concurrently. This acts as a location repetition count within an area when the location is defined.\nA technical point with homes as cells is that the model cannot yet be set to create a number of homes based on population size with a distribution of household sizes. Figure 11 Disease definition panel.\nConsequently, one must, at present, define the number of home cells appropriate to hold the population for each area. If a distribution function were available to specify household sizes and households were definable by population size, it would then be practical to provide canned profiles of household sizes based on US census data.\nThe types of contacts that are available among hosts in the location are defined, together with the average number of such contacts per host during one cycle of the model; see Figure 13 . To the right of the contact definitions is a skew function that enables one to specify the degree to which contacts vary. Within any host subpopulation, there are high contact individuals and lower contact individuals. The example shown sets the difference rather high to describe the way that service workers interact more strongly than customers. When running the simulation, during determination of which hosts become infected in this cycle, the position of the host in the queue is used together with the contact frequency to decide how many contacts an individual will get in a particular cycle. Think of this adjustable skew graph as the histogram of contact counts.\nFor each demographic group, a cycle of movement between locations in an area is defined as illustrated in Figure 14 . In the model used as an example, days are divided into three cycles. So a sequence of 21 movements defines a 7 day week. Once a cycle completes, it restarts at the top. This is how hosts in the model are moved from one location to the next.\nAn area is made up of a list of locations that it contains, as shown in Figure 15 . An area specifies a particular population level, and the population fractions for each demographic group making up the area are allocated at runtime. Demographic population, removals and additions can be overridden at the area level.\nAn area also has linkages to other areas, through special locations that are eligible as links. In the example of Fig- Group demographic population definition panel Figure 12 Group demographic population definition panel.\nure 16, the Anchorage airport is going to send 2% of its population to Chicago, Illinois, each cycle. In each cycle, 70% of the population of the Anchorage airport location is going to be shipped off somewhere.\nA 'Vector' is defined in EpiFlex as n infection events to attempt at a specific location. A vector can repeat each cycle, or have a limited input. A vector can represent an arthropod or snail, or it can represent people arriving on aircraft from Hong Kong. The vector shown in Figure 17 is a startup vector for influenza used for these modeling runs. This initiating disease vector will operate for the first 3 cycles, then stop, attempting to produce 9 cases at a location in Chicago. An initiation disease vector can have delay parameters, run continuously, or run once. It can force the infection of a specific number of people or infect them at a particular rate.\nWhen a disease occurs, the medical system will respond to it in some way. Some illnesses, such as influenza A, will result in very little intervention. Others, such as SARS or smallpox, would result in a great deal of intervention very rapidly. Consequently, a reasonable modeling system will allow response to an epidemic occurrence to be modeled.\nIn the example shown in Figure 18 , we see the response specification for an emergent illness. A response has an alert trigger, which in this case is the appearance of fatalities. This results in a period of heightened awareness of the disease and its symptoms, which is set in this example to 100 days. The probability of noticing the triggering event is set, to unity in this example, which may be overly optimistic. This specification says that after three fatalities, an alert would be triggered. Since this alert trigger is for fatalities, not symptoms or instruments, the detection fraction for each disease stage is not applicable.\nOnce the alert has been triggered, the detection method for the illness switches primarily to symptoms. We can then specify what fraction of each occurrence will be diagnosed and at what stage. If we detect an event, then we can mitigate it. In this implementation, one has to specify the degree of infectiousness remaining after intervention is in process. Some methods, for some diseases, can stop disease spread completely. This is not true for all diseases, so it must be specifiable. Figure 13 Location definition panel.\nNote that this component is the one with which the author is least comfortable. None of the examples for this paper have used responses as a consequence. The primary reason is that the way response is modeled is over-simplified and does not conform well to the response that happens in the real world; see: Limitations of EpiFlex modeling in the introduction.\nEpiFlex is now composed of 83 major classes, of which 45 are core internal model functionality, the rest being infrastructure and UI. A functional walkthrough of how the system operates is presented below.\nA model starts running by verifying the model data. It looks for any references to things that were deleted and definitions that are impossible. It writes an error log file with this information that can be audited after the run. If errors are found, you will be informed of their severity and given the option to cancel. Many of these errors are non-fatal, and may be modified with a warning, but they may change the results of a run.\nThe EpiFlex system iterates through all areas in a model and allocates hosts, putting them in their initial locations, per the movement definitions for the demographic group.\nEach group steps through its location movement list to determine whether the area to which it is attached has the locations the group needs. If it does not, EpiFlex will increment the pointer when it comes to this location, but it will leave the hosts in their previous real location until a good one is found. This was done for ease of practical use, allowing the same demographic to be used when a location may not exist in an area.\nStep 1 -disease stage pass. For each infected host, it will update the stage of the illness for that host. This is also where the system checks to see if disease response conditions have been met yet.\nStep 2 -vector pass. EpiFlex will iterate through each vector and apply the disease to the locations in each area in the vector according to the rules defined.\nStep 3 -infection pass. Iterate through each location in each area, and figure out the contacts between infected and uninfected hosts. For each contact between an Group movement cycle definition panel Figure 14 Group movement cycle definition panel.\ninfected host and a host not infected with the disease, a probabilistic determination will decide whether or not this illness is communicated.\nStep 4 -iterate areas looking for locations that have group draw factors. Randomly pull hosts from the groups specified, and move them to the group draw location. This feature is used to simulate various things; generally an airport will have this type of specification.\nStep 5 -iterate all areas looking at area links. Area links push population from one location to another area's location as specified.\nStep 6 -Areas will be iterated, and each location in each area will be iterated using the random exchange factor to move hosts assigned to cells of a location between cells in that location.\nStep 7 -Normal addition and removal of population groups is applied to model. This allows the user to model normal birth and death rate plus immigration and emigration across the outer boundary of the model.\nStep 8 -iterate all areas, and iterate through each location in each area. For each location, it will iterate each host attached to the location. Each host has a location pointer for its group that indicates where it is in its movement cycle. It increments the pointer, and if the location name is different from the one it is in, it will find a location of that name in the area to put itself into. If the location has N cells, it will put itself into the cells in a semi-randomized fashion according to the parameters defined. Note that currently the hosts do not remember which cell they are put into from one iteration to another. So they will not return to the same cell next time they come back around in their movement cycle. This is a limitation at present, which would require greatly increased space allocations to change.\nStep 9 -An audit is done to verify that all hosts occur only once in a location.\nStep 10 -Areas are iterated, locations are iterated, to total results. Results are then either written to the log file and/ or displayed on the main window in a graph form depending on how parameters were specified. Figure 15 Area definition panel. Area linkages panel Figure 16 Area linkages panel.\nInitiating disease vector definition panel Figure 17 Initiating disease vector definition panel." }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: Few multiplexed technologies with sufficient specificity to identify small changes within the human genome are available for clinical use. Line probe or linear array technology uses nitrocellulose paper strips as the support matrix (1 ) . A benefit of the line probe is that expensive instrumentation is not required. A technology termed \"oligonucleotide ligation assay\" uses target-specific ligation to generate mutationspecific products that are analyzed by gel electrophoresis (2 ) . Other genetic analysis systems are coupled to microparticle flow cytometry (3) (4) (5) (6) (7) (8) . Limitations of these methods include complicated procedures that require extensive training (washings, centrifugations, and transfers), long completion times, expensive instrumentation, and technician-dependent result analysis. In addition, these technologies are not easily amenable to automation. Many hospitals and clinics currently send molecular diagnostics tests to large reference laboratories. This is in part attributable to the limitations described. Because there are benefits in having local laboratories perform diagnostic tests (9 -12 ) , there appears to be a need for molecular testing methods that circumvent the limitations posed by current testing methods.\nWe have constructed a three-step platform technology called MultiCode ® PLx that eliminates the most technically challenging steps involved in multiplexed genetic analysis and compared it with the line probe assay in a clinical setting. MultiCode PLx uses one additional nucleobase pair constructed from the complementary nucleobases 2Ј-deoxy-isoguanosine (diG) 5 and 5-Me-iso-cytosine (iC). These nucleobases specifically recognize each other based on a different pattern of hydrogen bondings. We chose this pair because no other such pair is available commercially and their chemistries have been well explored. For example, iG and iC have been successfully used for both molecular recognition (13) (14) (15) and for site-specific incorporation (16 -18 ) . In addition, because iG:iC recognition is \"orthogonal\" to the naturally occurring nucleobase pairs, a strand of DNA that contains several iC/iG components can be constructed so that it will not hybridize to natural DNA. In complex reactions in which high concentrations of natural DNAs of known or unknown sequence exist, this orthogonal attribute allows specific molecular recognition to take place without interference. These additional nucleobases are used in each step of the PLx process: PCR, extension labeling, and liquid decoding. PCR primers are first designed to be target-specific and contain single iCs. The amplicons act as labeling templates for the target-specific extension (TSE) step. During that step, labels attached to diG triphosphate (diGTP) are incorporated site specifically into coded target-specific extenders (Fig. 1 ). The tags are short sequences (typically 10 nucleotides in length) assembled by use of a mixture of natural and nonnatural bases. The tags are designed to hybridize only to their perfect complements encoded on color-addressed microspheres. In the final step, decoding of the extension reactions is accomplished at room temperature by capture of the coded extenders on the addressed microspheres and reading of the reporter signal on a Luminex 100 instrument. All steps are carried out in the same reaction vessel without transfers or washings.\nTo demonstrate the approach, we developed a screening test for carriers of cystic fibrosis (CF) that tests for all 25 mutations and the reflex targets in the widely used panel (19 ) and also tests for 394delTT and 3199del6. 394delTT is the second most prevalent mutation in populations with Nordic heritage and may be important in Wisconsin, where testing was performed. 3199del6 was added because several lines of evidence indicate that 3199del6 is a disease-causing mutation and may replace I148T (20 ) .\nThe Wisconsin State Laboratory of Hygiene, in conjunction with the University of Wisconsin, was chosen as the major testing site because of its long history of screening newborns for CF (21 ) and study of screening methods (22) (23) (24) (25) . In March 2002, DNA testing of the top 4% of the daily immunoreactive trypsinogen results expanded to include all 25 mutations recommended by the American College of Medical Genetics, the American College of Obstetricians and Gynecologists, and the NIH.\nIn this report, we first present MultiCode PLx testing data on a variety of genomic DNAs with various genotypes prepared from several sample types for the presence or absence of mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Finally, data are presented on a prospective study comparing testing results obtained for freshly prepared blood spots by the PLx assay and the commercially available CF Gold Linear Array TM system (Roche Molecular). ) . DNA was extracted from whole blood and dried blood spots on Guthrie cards. The dried blood spots were stored at room temperature for 2-3 days after receipt until routine testing was completed. For long-term storage, dried blood spots were stored at 4°C. DNA extraction from Guthrie cards was performed with the Generation Capture Card Kit (Gentra Systems) according to the manufacturer's guidelines for DNA purification and elution. DNA was extracted from whole blood by use of the Puregene DNA Isolation Kit (Gentra Systems) or the Whatman FTA system (Whatman Inc., Clifton, NJ). All genomic DNAs from these preparations were diluted 1:10 in water, and 1L was added to each PCR reaction (1-50 ng of total DNA). As a cross-reference and in the prospective study, genotypes were also determined by CF Gold linear array or DNA sequencing if discrepancies were detected.\nPCR and TSE oligonucleotides were manufactured inhouse with a 48-column DNA Synthesizer (Northwest Engineering; now the 3900 from Applied Biosystems Inc.), using standard ␤-cyanoethyl phosphoramidite chemistry (for sequence information, see Tables 1, 2, and 3 in the Data Supplement accompanying the online version of this article at http://www.clinchem.org/content/vol50/ issue11/). Synthesis of sequences that contain iC and iG can also be purchased from IDT, Eurogentec, or EraGen. A demonstration assay encompassing this specific system can be purchased from EraGen Biosciences. Amino-labeled EraCode oligonucleotides were manufactured with an ABI 394 DNA Synthesizer (Applied Biosystems) using 3Ј-PT-Amino-Modifier C6 CPG (Glen Research). Target-specific extender primers were manufactured with the spacer phosphoramidite C3 (Glen Research) placed between the EraCode complement and the target-specific region. The isoG and isoC phosphora-\n, respectively] were coupled and deprotected under the conditions used for the natural base phosphoramidites. Postsynthesis work-up consisted of ammonium hydroxide deprotection followed by ethanol precipitation (PCR and TSE oligonucleotides) or 2-propanol precipitation (EraCode oligonucleotides).\nEraCode bead coupling For each conjugation reaction, a mixture (400 L) containing ϳ5 ϫ 10 6 carboxylated microspheres (Luminex Corporation) was centrifuged at 8000g for 1 min. The supernatant was removed, and the beads were resuspended in 50 L of 0.1 mol/L MES, pH 4.5. After resuspension, 3.3 L of 300 mol/L 5Ј-amine-modified EraCode DNA oligonucleotide and 5.0 L of freshly prepared 10 g/L EDC (Pierce Chemical) were added. The reaction was mixed thoroughly and incubated at room temperature in the dark for 30 min. A second 5.0 L of 10 g/L EDC was added, and the mixture was incubated for 30 min. Era-Code microspheres were washed by the addition of 1.0 mL of 10 mmol/L Tris (pH 8.0) containing 0.2 mL/L Tween 20 and pelleted by centrifugation at 8000g for 1 min; the supernatant was then removed. EraCode microspheres were washed a second time with 1 g/L sodium dodecyl sulfate in 10 mmol/L Tris (pH 8.0) and resuspended in 100 L of bead buffer (10 mmol/L MOPS, pH 7.5; 1 mmol/L EDTA, 0.5 g/L sodium dodecyl sulfate, 200 mmol/L sodium chloride, and 0.1 g/L sonicated herring sperm DNA). EraCoded microspheres were combined and diluted in bead buffer to generate a 50-code mixture at a concentration of 800 microspheres/L for each class of microsphere.\nCapture at room temperature of a tag to its specific complementary EraCode was tested by use of a model extension system where one target with a 5Ј-iC was constructed and 50 tags were interchanged onto a targetspecific extender sequence. EraCodes were constructed by use of 9 -10 bases comprising 2-3 iCs. In an effort to minimize interference from naturally occurring sequences, EraCodes were designed to have no more than four naturally occurring bases in a row. In an effort to minimize code-to-code cross-hybridization, no two codes were allowed to contain the same series of more than four nucleobases in a row.\nFifty EraCodes were validated by use of 50 extenders, all containing the identical 3Ј target-specific sequence. The extenders were individually labeled using the target, standard concentrations of all four naturally occurring deoxynucleotide triphosphates, TiTaq, and diGTP-biotin at 1.5 mol/L. Each EraCode sequence was separately coupled to a specific color-coded fluorescent microsphere type (Luminex Corp). Each microsphere type contains a unique color or signature that can be determined by the Luminex 100 . After coupling, each EraCode bead conjugate was pooled to create an equalized bead master mixture (bead array: 50 different EraCode-labeled beads). The bead array was distributed into 50 separate reaction vessels. To each of the vessels, a specific extension reaction from above was added at room temperature along with streptavidin-phycoerythrin and was read on the Luminex 100 .\nIn a 10-L extension reaction, an excess of a template oligonucleotide (5Ј-iC-CGTATGGGCTCACCTCGCTGTG-3Ј) at 150 nmol/L was combined with a control extension sequence at 100 nmol/L in EraGen MC TSE solution 35 (prod. no. PN1236; EraGen Biosciences, Inc.) with 100 mol/L each deoxynucleotide triphosphate, 1.5 mol/L diGTP-biotin, and 1ϫ Titanium Taq (Clontech). Each control extension oligonucleotide was composed of a 5Ј sequence complementary to each of the EraCode sequences, a three-carbon spacer, and a 3Ј sequence complementary to the template oligonucleotide (5Ј-CACAGC-GAGGTGAGCCCA-3Ј). Reactions were subjected to the following thermal cycling protocol: 30 s at 95°C and 1 min at 65°C. Each 10-L extension reaction was then hybridized to the EraCode bead mixture according to the described hybridization protocol. In addition, to mimic MultiCode conditions, unextended TSEs to the other EraCodes were also added.\nHybridization of extension products in each 10-L reaction was performed by mixing 40 L of EraGen MC Hyb Solution A (prod. no. PN1237; EraGen Biosciences) and 0.25 L of EraCoded bead mixture. To each hybridization reaction, 0.5 L of 2 g/L streptavidin-R-phycoerythrin conjugate (Prozyme) plus 39.5 L of sheath fluid (Luminex Corp.) were added, and 65 L of the final reaction was injected into the Luminex 100 . pcr PCR primers were designed using Primer 3 (http:// www.broad.mit.edu/cgi-bin/primer/primer3_www.cgi) or Hyther (http://ozone2.chem.wayne.edu). Primer pairs were designed to flank the polymorphism(s) of interest and have a melting temperature of 53-57°C (see Table 2 in the online Data Supplement). A single iC was added to the 5Ј end of the PCR primers.\nPCR reactions were performed in a final volume of 8.0 L; each reaction contained EraGen MC PCR solution 35 (prod. no. PN1235 or PN1305 for the 579T system; Clinical Chemistry 50, No. 11, 2004 EraGen), 0.4 L of 20ϫ EraGen CFTR PCR primer mixture, 1ϫ Titanium Taq, and 1 L of diluted genomic target DNA. Reactions were cycled according to the following profile: 1 cycle of 30 s at 95°C, and 40 cycles of 1 s at 95°C, 10 s at 55°C, and 30 s at 72°C. Each of the primer mixtures contained forward and reverse primers for a subset of the mutations at concentrations of 100 -200 nM.\ntse TSE was used to determine the exact mutations present. TSE requires TiTaq, diGTP-biotin, and extenders that contain 3Ј target-specific bases and 5Ј codes specific for the EraCode beads described above. During TSE, TiTaq incorporates labels on extenders only when specific targets having 5Ј iCs are present. The nonnatural base pair formed by iG and iC has been successfully used for both molecular recognition (13) (14) (15) and for site-specific enzymatic incorporation (16 -18 ) . To date, we have covalently coupled a series of different reporter groups to diGTP, using succinimidyl ester chemistry and confirmed enzymatic incorporation into nucleic acids by use of several commercially available polymerases (data beyond the scope of this report). In many cases, single amplicons spanning regions that incorporated multiple mutations were used as templates for multiple extenders. After TSE, EraCode-labeled beads and streptavidin-phycoerythrin were added, and the reactions were analyzed on the Luminex 100 . Data are reported in median fluorescence intensity (MFI).\nFor each polymorphism, pairs of TSE oligonucleotide primers were designed by use of Primer 3 and Hyther (see Table 3 in the online Data Supplement). Pairs were designed to have balanced melting temperatures ranging from 63 to 67°C. Each TSE oligonucleotide was composed of three components: a 5Ј sequence tag complementary to one of the EraCodes; a three-carbon spacer; and a 3Ј target-specific sequence fully complementary to the allele of interest. The polymorphic sequence for each pair of TSE oligonucleotides was the 3Ј base.\nExtension reactions were performed by bringing the 8-L PCR reactions to 10 L with addition of 2.0 L of EraGen MC TSE solution 35 (prod. no. PN1236; EraGen Biosciences) including primers for the 579T system (prod. no. PN1310), and 0.4 L of 20ϫ EraGen CFTR TSE primer mixture; final concentrations of TSEs were 25-100 nmol/L (see Table 3 in the online Data Supplement). Reactions were cycled according to the following profile: 1 cycle of 30 s at 95°C; 5 cycles of 1 s at 95°C and 2 min at 65°C.\nFor this report, an assignment is defined as a genotype for a given target within the CFTR gene made for any given sample. Each sample tested will have 30 possible assignments because 30 targets for each sample are analyzed. To rapidly analyze the data obtained, we developed software to present the raw data from the instrument. The software directly imports the Luminex 100 data and organizes them by target mutation. Fractions of wild-type to wild-typeplus-mutant and raw MFI signals can be visualized graphically (Fig. 2) . The program sets default fraction windows at 1.0 -0.8 for wild-type samples, 0.6 -0.4 for heterozygous samples, and 0.0 -0.2 for homozygous mutant samples. Default windows can be changed by the end user. The area between the windows is designated as the no-call window. The program also sets no-amplification windows where wild-type and mutant MFIs are Ͻ200.\nTemplate set-up within the Luminex LabMap software was required, and importation to analysis software was achieved by dragging the Luminex file folder into the Analysis Software. Data files were parsed, and the resulting raw MFI values were organized by target and sample. After data acquisition from all 225 clinical samples tested, default cutoff windows for each target were empirically determined and set in a blinded fashion. Once determinations were made, reports were generated for offline analysis. The analysis software was written in Java and is compatible with Windows, Mac OS X, and Linux operating systems. Data importation is achievable with Luminex LabMap 100 (software versions 1.7, 2.1, and 2.2) and Bio-Rad BioPlex 3.0. The software displays scatter plots and fraction plots of each given target for all samples.\nautomation Automation was performed on a Tecan Genesis Model 200 robotic workstation (TRP) starting from either the PCR amplicon formation or from isolated genomic DNA and continuing through data analysis. Samples used were a subset of the 225 used in the initial manual testing. Instructions for the automation program were written with Gemini software (Tecan). Initialization and launch of the bead-reading protocol on the Luminex 100 was performed automatically by use of a custom image-recognition and command-scripting program written in Java. After the read, the results were remotely analyzed by MultiCode Analysis software. Genomic DNA was purified manually before automation as described above. The TRP was fitted with the low-volume eight-channel dispense pipette system, TeStack, to supply disposable tips, a MJ Research thermocycler equipped with remote Alpha docks with Power Bonnet, and a robotic manipulator arm. Liquid reagents and disposable tips were also placed on the deck before runs were started. Reactions and additions for the automated process were the same as those described for the manual method unless noted. Reactions were performed in low-profile hard-skirted polypropylene well microplates, which allowed the robotic arm to grip and move the plates as needed. Each reaction was overlaid with 15 L of light mineral oil (Sigma) to prevent evaporation during thermal cycling. The robotic manipulator was used to transfer the plates to and from the thermocycler block and the Luminex 100 mounted off deck. Initialization and launch of the bead-reading protocol on the Luminex 100 were performed automatically by use of a custom image-recognition and command-scripting pro-gram written in Java. After bead-reading of each reaction, the results were analyzed by the MultiCode Analysis software as described above.\nA schematic diagram of the MultiCode system is shown in Fig. 3 . The MultiCode process has three steps. For the first step, regions of the target of interest are amplified by PCR using primers that contain a single 5Ј-iC. In the second step, the amplicons from the first step are used as templates for the TSEs, and iC directs specific diGTP-biotin incorporation. The TSEs are coded on their 5Ј ends with sequences that specifically recognized short complementary sequences (EraCodes) attached to an addressed solid matrix. The molecular recognition of any given code with its EraCode is specific enough so that increased temperatures, incubation steps, or washings are not required (typical requirements for hybridization capture using only naturally occurring base chemistries). During this second step, labels are placed site specifically on the TSEs. This process requires the presence of iC-containing amplicons that are complementary to the TSE. When the correct target is available, the extension reaction continues to the 5Ј end of the amplicon, where the diGTP-biotin is used to insert the biotin into the TSE. Specificity and signal consistency arise when coded TSEs are covalently attached to a single biotin reporter in the presence of the correct target. In the final step, the TSEs are captured on the solid matrix and detected. The presence of the target is then determined by where the reporters are located spatially on the liquid matrix (created by the differentially colored microspheres).\nBecause each EraCode should be specific only for its complementary tag, only the bead that contains the specific EraCode should report a high MFI. Results show that for the correct complementary code, specific signal was between 2130 and 4357 MFI, with a mean (SD) MFI of 3040 (532; see Table 4 in the online Supplemental Data). Signals generated on microspheres that contained Era-Codes that were not completely complementary varied from 0 to 320 MFI with a mean (SD) MFI of 92 (28) . This set gave signal-to-noise ratios that ranged from 32:1 and 10:1 for the worst cases (i.e., where the specific signal was compared with the nonspecific signal from the microsphere presenting the highest noise). To our understanding, there have been no reports of a genetic-based molecular recognition system that was successfully implemented at room temperature without washing. mutation analysis using the manual cf multicode system PCR amplicons were generated for 17 regions within the CFTR gene, and 63 tagged extenders were used to analyze 27 mutations and 4 reflex targets. To improve the robustness of the overall results, the test was divided into four reactions with one additional test targeting the 5T7T9T reflex target. Two additional targets were included late in the development phase to include 394delTT and 3199del6. The test was transferred to the Wisconsin State Laboratory of Hygiene, where newborn CFTR genetic screening has been ongoing since 1994 and multimutation testing used since 2002.\nFor the CFTR-specific PLx assay, positive signal intensities varied between 2000 and 5000 MFI and background signal varied from 50 to 1500 MFI. The variation in signal depended strictly on the target sequence. We observed that determination windows varied from 0.99 to 0.7 for wild-type samples, from 0.4 to 0.6 for heterozygous samples, and from 0.0 to 0.2 for homozygous mutant samples.\nThe calling results are summarized in Table 1 [also see Table 4 in the online Data Supplement for additional details]. The 225 samples included 65 wild-type samples; 93 heterozygous samples representing 22 types; 36 compound heterozygous samples for 9 types; and 31 homozygous mutant samples for 6 types. Of the 225 samples tested manually, 203 (90.2%) samples reported all correct assignments. The failure rate of 10% was most likely a Step 2), tagged target-specific extenders are site-specifically labeled with diGTP-label when the correct amplicon is present. For this study, the label was biotin, and streptavidin-phycoerythrin was used for signaling purposes. ( Step 3), the tags are captured on EraCode-modified Luminex microspheres. The captured reactions are analyzed with the Luminex 100 system. product of DNA quality and not assay sensitivity. As stated in the Materials and Methods, the DNA samples were not fresh, and signal intensities indicated gross failure consistent with PCR failure. There were no discrepancies for genotype classification. For these 225 samples, there were a total of 6750 potential assignments (225 ϫ 27 mutations ϩ 3 reflex tests; 5T7T9T was not included). Correct assignments were made 99.3% (6704 of 6750) of the time. For the separate 5T7T9T-specific reflex test, 194 samples were tested and included at least 10 separate samples from each of the six possibilities (see Table 4 and Fig. 1 in the online Data Supplement). Test results showed that there were zero incorrect determinations and one no amplification failure.\nmutation analysis using the automated cf multicode system\nThe final automated processes for the CFTR gene were conducted on a total of 86 samples. The results are reported from two separate processes: (1) PCR through data acquisition (20 samples) and (2) TSE through data acquisition (66 samples; Table 1 ; also see Table 5 in the online Data Supplement for further sample and testing information). Because workstation space is limited on the Tecan Genesis Model 200, tip usage is high when disposable tips are implemented, and amplicon carryover may be a concern. Process 1 may be applied for higher throughput scenarios where the PCR step could be implemented on yet another workstation or manually. For process 1, 20 (100%) samples reported calls correct. For process 2, 64 (97%) samples reported all correct assignments. There were no incorrect determinations.\nAfter assay validation, which included the testing of various mutations and sample types, the PLx assay for CFTR gene analysis was directly compared with the CF Gold linear array method. Using freshly obtained blood cards from newborns that had immunoreactive trypsinogen scores in the upper 4%, we used both tests to analyze CFTR for the recommended core panel of mutations and reflex targets. The testing was performed on 419 samples, and 100% concordance was recorded (no incorrect assignments; Table 1 , column B). Of the samples tested, there were 21 samples (5%) in which the complete lists of calls could not be obtained with the PLx system. The failure rate of 5% was documented as errors resulting from pipetting errors. These samples were retested, and correct assignments were made. Of the possible 12 570 potential assignments, 12 521 (99.6%) were correct. Run time after sample preparation for the CF Gold test was ϳ7 h compared with Ͻ3 h for the PLx system. Total hands-on time for the CF Gold test was ϳ3.5 h compared with 0.5 h for the PLx system. The data output for the CF Gold test was visual inspection of the nitrocellulose strips, whereas the PLx system has a computerized calling system as described.\nWe have described a novel high-throughput platform that incorporates nucleic acid chemistries that simplify genetic analysis. PLx uses one additional base pair not found in nature in combination with microsphere flow cytometry technology to eliminate the most cumbersome steps commonly found in established multiplexed systems. The EraCode molecular recognition system described is particularly suited for the Luminex 100 instrumentation because the solid support can be dispensed into the complex reaction and decoding can be done at room temperature without washing, creating a true \"Liquid Chip\" type of system. Our study indicates that the chemistry is transferable to a clinical setting. In addition, although not required, the process can be performed with a robotic workstation for higher throughput.\nWe demonstrated the principles of the chemistry by use of an essential target and selected mutations found within the CFTR gene recommended by leading medical organizations. Data from the Wisconsin State Laboratory of Hygiene confirmed that the chemistry is accurate. For example, there were no incorrect determinations, and the percentage of correct determinations was high. The data presented here were tabulated by use of a broad sample set that included every mutation possible within the panel targeted with the exception of the 3199del6. For 24 samples, the entire manual process takes ϳ3 h to obtain final results. An upgraded prototype version of the CFTR test where all mutations are analyzed in a single well takes ϳ2.5 h to complete analysis of 96 patient samples (validation data not yet available). To demonstrate the system in a fully automated mode, we elected to use the Tecan genesis liquid-handling system. The entire automated process also takes ϳ3 h from prepared sample to final read for an entire 96-well plate or 24 samples. We presented data from two possibilities (when automation started at the PCR step and when it started at the TSE step) because there may be a need to perform PCR offline.\nWe believe that the expanded genetic alphabet enabled us to eliminate many of the steps that are required in technologies based on just the two base pairs provided by nature. The steps eliminated include solid support washings, transfers, aspirations, and high-temperature hybridizations. Specifically, we believe that there are many reasons that the extra base pair is beneficial. For example, we used short code sequences in the capture step to decode the complex extension mixtures. This may be possible with code sets constructed from only A, G, C, or T, but optimization would surely be more difficult because of cross-hybridization with sequence complements or close complements found in nature. The additional informational content that iC:iG builds into DNA also allows for greater diversity when compared with their all-natural counterparts. Specifically, if the four naturally occurring bases are used to make a library of 10mers, there are 4 10 , or 10 6 possibilities. In contrast, a six-letter system generates 6 10 , or 10 8 unique 10mers. As demon-Clinical Chemistry 50, No. 11, 2004 strated here, specific assembly can occur at conditions such as room temperature, which is in effect more amenable to and simplifies molecular diagnostics.\nWe also demonstrated for the first time that biotin labels attached to diGTP could be site specifically incorporated into extension primers. More generally, we demonstrated that labeled diGTP can be incorporated enzymatically into DNA. This could allow for broad use of such compounds in molecular biology. Whenever a reporter group is required at a specific site within an amplicon, transcript, or extension product, the use of an additional base pair may be helpful.\nRecently we reported that site-specific enzymatic incorporation of a quencher attached to diGTP can be used to create a new and highly sensitive real-time PCR system (26 ) . Polymerase incorporation of labels only at sites where expanded genetic alphabet bases are positioned will also have other uses, such as 3Ј end labeling. Polymerase incorporation of EraCodes within PCR amplicons has now also been reported (27 ) . The EraCode system that uses additional nonnatural base pairing may have advantages over other DNA-based molecular recognition systems (28 -30 ) . These short DNA codes are simple to produce, specifically hybridize at room temperature, do not require wash steps, can be amplified with standard polymerases, and do not cross-hybridize with natural DNA. Taken together, we believe that the expansion of the genetic alphabet is a true paradigm shift to the ways molecular diagnostics will be developed in the future." }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: The standard triplet readout of the genetic code can be reprogrammed by signals in the mRNA to induce ribosomal frameshifting [reviewed in (1) (2) (3) ]. Generally, the resulting trans-frame protein product is functional and may in some cases be expressed in equal amounts to the product of standard translation. This elaboration of the genetic code (4, 5) demonstrates versatility in decoding.\nRequirements for eukaryotic ribosomal frameshifting include a shift-prone sequence at the decoding site and often a downstream secondary structure in mRNA. The majority of À1 programmed frameshift sites consist of a heptanucleotide sequence X XXY YYZ [where X can be A, G, C or U; Y can be A or U; and Z can be any nucleotide (6) ].\nIn this configuration, the P-and A-site tRNAs can re-pair with at least 2 out of 3 nt when shifted 1 nt towards the 5 0 end of the mRNA. Similarly, for +1 frameshift sites, the identity of the codons in the P-and A-sites of the ribosome is critical for efficient frameshifting. One factor affecting +1 frameshift efficiency is the initial stability of the P-site tRNA-mRNA interaction in the 0 frame (7) . High-efficiency frameshifting occurs when the P-site tRNA does not form standard codon-anticodon interactions (8) . In some studies, a correlation between +1 frameshift efficiency and the final stability of the P-site tRNA-mRNA interaction in the +1 frame has been shown previously (9, 10) . However, in other systems there appears to be little correlation (11) . In addition, competition between decoding of the 0 frame and +1 frame codons in the A-site may affect frameshifting efficiency (7) . Slow to decode 0 frame codons such as stop codons or those decoded by low abundance tRNAs favor frameshifting, as do +1 frame codons with high levels of corresponding cognate tRNAs (12) (13) (14) (15) (16) .\nHigh levels of frameshifting are often achieved by the stimulatory action of a cis-acting element located downstream of the shift site. A wide variety of structures, most commonly H-type pseudoknots (17) , have been identified which stimulate À1 frameshifting in eukaryotes [for reviews see (18, 19) ]. Mutagenic and structural data for several of the frameshift stimulators have demonstrated that each pseudoknot has key structural features required for frameshift stimulation (20) (21) (22) (23) (24) (25) (26) (27) (28) . However, unifying structural feature essential for frameshifting has not yet been identified. This observation combined with recent reports that simple antisense oligonucleotides can functionally mimic cis-acting 3 0 stimulators of À1 frameshifting (29, 30) demonstrates that many different structures can stimulate frameshifting. Although it should be noted that not all structures of equal thermodynamic stability can stimulate frameshifting (Discussion). RNA pseudoknots have also been shown to stimulate programmed +1 frameshifting in many eukaryotic antizyme genes (31, 32) . Antizyme is a negative regulator of cellular polyamine levels through its ability to target ornithine decarboxylase (the rate-limiting enzyme in polyamine biosynthesis) for degradation (33) (34) (35) , inhibits polyamine import (36, 37) and stimulates export (38) . Antizyme expression is induced by high-intracellular polyamine levels, and decreased with lowered levels. The polyamine sensor is a programmed +1 frameshift event that is required for antizyme synthesis. *To whom correspondence should be addressed. Tel: +1 801 585 1927; Fax: +1 801 585 3910; Email: [email protected] Ó 2006 The Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.\nAt low polyamine levels, termination at the end of open reading frame 1 (ORF1) is efficient, whereas at high levels of polyamines, a substantial proportion of ribosomes shift to the +1 reading frame and then resume standard decoding to synthesize the full-length and active antizyme protein.\nFrameshifting at the mammalian antizyme mRNA shift site, UCC UGA, is stimulated by two cis-acting signals (39, 40) . One of these, the 5 0 element, encompasses $50 bases upstream of shift site and is important for the polyamine effect (39) (40) (41) . The other cis-acting element is a pseudoknot located 3 0 of the shift site. The mammalian antizyme pseudoknot and a structurally distinct counterpart in a subset of invertebrate antizyme mRNAs (31) are the only pseudoknots known to act as stimulators for +1 frameshifting in eukaryotes.\nAlthough it is unknown if pseudoknots stimulate À1 frameshifting and +1 frameshifting by different mechanisms, one notable difference is found in positioning of the downstream structure relative to the shift site. Naturally occurring pseudoknots or stem-loop stimulators of À1 frameshifting typically begin $6-9 nt downstream of the A-site codon of the shift site (18) , whereas +1 frameshift pseudoknots are located closer with only a 2-3 nt separation from the A-site codon (31) . Mutagenic studies have revealed that altering the size of the spacer affects frameshifting and, in general, reduces efficiency (27, 31, (42) (43) (44) .\nHere we have tested the ability of antisense oligonucleotides, annealed downstream of the shift-prone site, UCC UGA, to induce shifting of the ribosome to the +1 reading frame. The directionality of frameshifting (either into the +1 or À1 reading frame) is shown to be dependent upon the position of the duplex region relative to the shift site, and the efficiency of frameshifting is responsive to polyamine levels and enhanced by the inclusion of stimulatory sequences found upstream of the human antizyme +1 programmed frameshift site.\nComplementary oligonucleotides, to construct the sequences described in this paper, were synthesized at the University of Utah DNA/Peptide Core Facility such that when annealed they would have appropriate ends to ligate into the SalI/ BamHI sites of the dual luciferase vector, p2luc (45) . Dual luciferase constructs were prepared and their sequence was verified as described previously (46) .\nInsert sequences with shift site in boldface is given as follows:\nP2lucAZ1wt: TCGACGGTCTCCCTCCACTGCTGTAG-TAACCCGGGTCCGGGGCCTCGGTGGTGCTCCTGATG-CCCCTCACCCACCCCTGAAGATCCCAGGTGGGCGAG-GGAATAGTCAGAGGGATCACAACGGATC;\nP2lucAZ10sp: TCGACGGTCTCCCTCCACTGCTGTAG-TAACCCGGGTCCGGGGCCTCGGTGGTGCTCCTGAC-CCTCACCCACCCCTGAAGATCCCAGGTGGGCGAGG-GAATAGTCAGAGGGATCACAACGGATC;\nP2lucAZ1hp: TCGACGGTCTCCCTCCACTGCTGTAG-TAACCCGGGTCCGGGGCCTCGGTGGTGCTCCTGATG-\nP2lucAZ1PKdel: TCGACGGTCTCCCTCCACTGCTG-TAGTAACCCGGGTCCGGGGCCTCGGTGGTGCTCCT-GATGCCCCTGGATC;\nP2lucAZ1PKm1: TCGACGGTCTCCCTCCACTGCTGT-AGTAACCCGGGTCCGGGGCCTCGGTGGTGCTCCTG-ATGCCCCTCACCCACCGGGATCACAAGGATC;\nP2lucAZ1sl: TCGACGGTCTCCCTCCACTGCTGTAGT-AACCCGGGTCCGGGGCCTCGGTGGTGCTCCTGATG-CCCCTCACCCACCCGGATC;\nP2lucAZ1FS: TCGACGTGCTCCTGATGCCCCTG-GATC;\nP2lucAZ1FSUGG: TCGACGTGCTCCTGGTGCCCCTG-GATC. \nThe dual luciferase constructs (0.1 mg) described above were added directly to TNT coupled reticulocyte lysate reactions (Promega) with 35 S-labeled methionine in a volume of 10 ml. Reactions were incubated at 30 C for 1 h. Radiolabeled proteins were separated by SDS-PAGE and the gels were fixed with 7.5% acetic acid and methanol for 20 min. After drying under vacuum, the gels were visualized using a Storm 860 PhosphorImager (Molecular Dynamics) and radioactive bands quantified using ImageQuant software. Percent frameshifting was calculated as the percentage of full-length (frameshift) product relative to the termination product and the full-length product combined. The value of each product was corrected for the number of methionine codons present in the coding sequence. The reported values are the average and standard deviations obtained from at least three independent measurements. Tables showing percent frameshifting and standard deviations can be found in Supplementary Data.\nPlasmid p2lucAZ1PKdel was co-transfected into CV-1 cells with varying concentrations of AZ1B 2 0 -O-Methyl antisense oligonucleotides under the following conditions. CV-1 cells (1.5 · 10 4 ) in 50 ml of DMEM + 5% fetal bovine serum were added to wells (1/2 area 96-well tissue culture treated plates) containing 25 ng of DNA, varying amounts of AZ1B antisense oligonucleotides and 0.4 ml Lipofectamine 2000 (Invitrogen) in 25 ml of Optimem. Cells were incubated at 37 C (5% CO 2 ) for 20 h. Media were then removed from the cells and the transfected cells were lysed in 12.5 ml lysis buffer and luciferase activity determined by measuring light emission following injection of 25 ml of luminescence reagent (Promega). Percent frameshifting was calculated by comparing firefly/Renilla luciferase ratios of experimental constructs with those of control constructs: (firefly experimental RLUs/Renilla experimental RLUs)/(firefly control RLUs/Renilla control RLUs) · 100.\nThe ability of cis-acting RNA structures or trans-acting 2 0 -O-Methyl antisense oligonucleotides to induce ribosomal frameshifting was determined by in vitro transcription and translation of a dual luciferase reporter vector, p2Luc. p2Luc contains the Renilla and firefly luciferase genes on either side of a multiple cloning site, and can be transcribed using the T7 promoter located upstream of the Renilla luciferase gene (45) . Sequences containing shift-prone sites were cloned between the two reporter genes such that the downstream firefly luciferase gene is in the +1 reading frame. The resulting constructs were then transcribed and translated in vitro with or without complementary cis-Acting stimulators of frameshifting at the antizyme shift site Initially, three dual luciferase reporter vectors were generated containing the human antizyme 1 frameshift cassette (p2luc-AZ1wt) with the 5 0 and 3 0 stimulators of frameshifting, with the pseudoknot deleted (p2luc-AZ1PKdel), or replaced with a stem-loop (p2luc-AZ1hp) (Figure 1 ). Each constructs was then subjected to coupled transcription and translation reactions in the presence of increasing amounts of spermidine, and the 35 S-labeled products separated by SDS-PAGE. Table 2 ). Maximal levels of frameshifting were found to occur when 2-4 mM of antisense oligonucleotide was added to the transcription/translation reactions (Supplementary Table 3 ). In the presence of 0.4 mM exogenous spermidine, highly efficient shifting of ribosomes into the +1 reading frame (higher than that observed in the wild-type antizyme frameshift cassette) was observed with the addition of AZ1A (26.1%), AZ1B (51.8%) and AZ1C (31.8%) (Supplementary Table 2 ). The most efficient frameshifting is observed with the antisense oligonucleotide AZ1B which anneals such that spacing between the shift site and the beginning of the duplex region is the same as that observed between the shift site and the beginning of stem 1 of the natural antizyme 3 0 pseudoknot structure (i.e. each has a 3 nt spacer). To verify that the antisense oligonucleotide was activating ribosomal frameshifting and not transcription slippage, RNA was transcribed from p2luc-AZ1PKdel in the absence of oligonucleotide and added to reticulocyte lysate translations in the presence of increasing amounts of 2 0 -O-Methyl AZ1B oligonucleotide. Frameshifting levels were increased to the same level as that observed in coupled transcription and translation reactions demonstrating that the oligonucleotide acts to induce frameshifting during translation (Supplementary Figure) .\nSurprisingly, the addition of AZ1A (0 spacer) also induced high-level frameshifting into the À1 reading frame in a manner which was modestly inhibited by the addition of spermidine (19% in the absence and 10% in the presence of 0.4 mM exogenous spermidine) ( Figure 3A and Supplementary Table 2 ). No À1 frameshift product was observed when the wild-type antizyme cassette was examined in the absence of antisense oligonucleotide addition (Figure 2 ; AZwt). As the AZ1A antisense oligonucleotide was designed to anneal directly adjacent to the UGA codon of the shift site, it was of interest to determine whether the wild-type antizyme pseudoknot could induce À1 frameshifting when located in the equivalent position. To address this, a new construct p2luc-AZ1-0sp ( Figure 1A ) was made by deleting the 3 nt spacer between the pseudoknot and the shift site of p2luc-AZ1wt. In this case, the wild-type pseudoknot is directly 3 0 adjacent to the shift site. The products of in vitro transcription and translation were separated by SDS-PAGE. No À1 frameshift product was observed and levels of the +1 frameshift product were significantly reduced to $3% ( Figure 3D and Supplementary Table 2) .\nAZ1A, AZ1B and AZ1C were designed to complement RNA sequences encoded by the originating vector. To determine if duplexes formed between the antisense oligonucleotide and 3 0 adjacent antizyme sequences would result in more efficient frameshift stimulation, reporter vectors were designed to contain a portion of the antizyme 3 0 stimulator. Construct p2luc-AZ1PKm1 contains sequences from the 5 0 half of the axis formed by the stacking of stem 1 and stem 2 of the pseudoknot ( Figure 1A) . Two complementary 2 0 -O-Methyl antisense oligonucleotides were designed. First, PKm1 has perfect complimentarity to the region starting 3 nt and ending 21 nt downstream of the UGA shift site codon. Second, PKm2 is the same except that a mispaired C and bulged A were located at positions 9 and 16, respectively. These two alterations were included to more closely mimic the natural pseudoknot which also contains a mispaired C and bulged A at equivalent positions along the extended stem formed by the stacking of pseudoknot stems 1 and 2 ( Figure 1 ; compare p2luc-AZ1wt with the duplex formed between p2luc-PKm1 and antisense oligonucleotide PKm2). PKm1 and PKm2 induced 30 and 22% frameshifting, respectively, when added to coupled transcription and translation reactions of p2luc-AZ1PKm1 in the presence spermidine ( Figure 4A and B, and Supplementary Table 4 ). Neither PKm1 nor Pkm2 induced frameshifting to the same levels seen with AZ1B, suggesting that the sequence content of the duplex region can affect the efficiency of frameshift stimulation and that native antizyme sequences are not required.\nA second construct, p2luc-AZ1sl, was designed to contain only the 5 0 half of stem 1 of the antizyme pseudoknot downstream from the UCC UGA shift site ( Figure 1A ). 2 0 -O-Methyl antisense oligonucleotides were designed to anneal between 3 and 15 nt (SL1) or 3 and 22 nt (SL2) downstream from the UGA codon of the shift site. Frameshift efficiency induced by these two antisense oligonucleotides, 8 and 22% respectively, was somewhat lower than that observed with PKm1 and PKm2 ( Figure 4C and D and Supplementary Table 4 ). In these cases frameshift efficiency was higher for the longer antisense oligonucleotide (SL2), suggesting that frameshift efficiency most probably correlates with stability of the duplex. As was seen with AZ1A, AZ1B and AZ1C, frameshifting efficiency stimulated by antisense oligonucleotides PKm1, PKm2, SL1 and SL2 was also strongly correlated with the concentration of exogenously added spermidine (Supplementary Table 5) .\nThe importance of the antizyme 5 0 sequence context to antisense oligonucleotide induced ribosome frameshifting was examined by testing the frameshift site, UCC UGA, without the 5 0 and 3 0 stimulatory antizyme sequences. To this end, the 5 0 antizyme stimulatory sequences were deleted from p2luc-AZ1PKdel to make p2luc-AZ1FS. Each of the antisense oligonucleotides AZ1A, AZ1B or AZ1C was added to coupled transcription and translation reactions with p2luc-AZ1FS in the presence or absence of spermidine. Frameshift efficiency was measured at 11, 8 and 4%, in the presence of spermidine and 3, 0.4 and 0.2% in its absence for AZ1A, AZ1B and AZ1C, respectively ( Figure 5A and B) .\nTo determine whether the stop codon of the shift site is essential for frameshifting, the UGA codon of p2luc-AZ1FS was altered to UGG such that the shift site was UCC UGG (p2luc-AZ1-UGG). Frameshift efficiency was significant, but reduced, compared to the shift site UCC UGA, and shows little stimulation by the addition of spermidine; AZ1A, AZ1B and AZ1C induced 3, 1 and 1.4% frameshifting in the presence of spermidine, and 1.9, 0.8 and 1.7% frameshifting in its absence, respectively ( Figure 5C and D) .\nThe ability of antisense oligonucleotides to induce frameshifting in cultured mammalian cells was examined by co-transfection of CV-1 cells with p2lucAZ1PKdel and increasing amounts of 2 0 -O-Methyl antisense oligonucleotides AZ1B as described in Materials and Methods. In the absence of antisense oligonucleotide frameshifting levels were determined to be 1.1%, whereas a graded increase in frameshift levels was observed upon the addition of AZ1B ( Figure 6 ). Maximal frameshifting levels were 13% in the presence of 2 mM AZ1B in the transfection media.\nSeveral models attempting to explain pseudoknot stimulation of programmed À1 frameshifting have been proposed [for reviews see (18, 19) ]. Most models invoke a pausing mechanism whereby the ribosome is paused over the shift site such that time is allowed for the tRNAs to reposition in the new reading frame. This explanation is clearly too simplistic as stem-loops and pseudoknots of similar thermodynamic stability that cause ribosome pausing are not necessarily effective frameshift stimulators (47) (48) (49) . In addition, variations of the IBV pseudoknot have demonstrated a lack of correlation between the extent of pausing and the efficiency of frameshifting (47) . A recent publication by Brierley and co-workers (50) presents structural data demonstrating that the IBV frameshift stimulating pseudoknot blocks the mRNA entrance tunnel and leads to a structural deformation of the P-site tRNA. The resulting movement of the tRNA displaces the anticodon loop towards the 3 0 end of the mRNA. A model is presented in which this movement results in disruption of the codon-anticodon interactions, thus allowing for tRNA slippage relative to the mRNA. Similar tRNA movements were not observed with non-frameshift stimulating stem-loop structures. This model provides a feasible mechanistic explanation for the ability of some downstream structures to induce frameshifting.\nThe ability of antisense oligonucleotides to induce highlevel À1 frameshifting (29, 30) demonstrates that elaborate tertiary structures are not required, and that a duplex formed by complementary antisense oligonucleotides (with a variety of chemistries, including RNA, 2 0 -O-Methyl, morpholino) is sufficient to induce high-level frameshifting. Here we demonstrate for the first time that trans-acting antisense oligonucleotides may stimulate ribosome shifting to the +1 reading frame at surprisingly high levels, levels which are greater than those achieved by natural 3 0 cis-acting mRNA pseudoknot structures in programmed +1 frameshifting.\nStructural studies indicating that the mRNA begins to enter the ribosome 7-9 nt downstream from the A-site codon is of direct relevance to this study (50, 51) . Our results indicate that maximal frameshifting is induced when the antisense-mRNA duplex begins 3 nt downstream of the UGA of the shift site, in agreement with the distance found between the UGA of the shift site and the beginning of stem 1 of the pseudoknot stimulator found in antizyme genes. Given this distance, the implication is that the stimulatory secondary structure would be encountered by the ribosome when the UCC codon enters the A-site of the ribosome. Perhaps as suggested by the structural studies of the IBV-1 frameshift inducing pseudoknot, the codon-anticodon interactions between the UCC codon and Ser-tRNA Ser are disrupted during translocation to the P-site. Given the importance of the UGA codon during frameshifting at the UCC UGA shift site, subsequent events following translocation of the UCC codon to the P-site and UGA to the A-site must influence frameshifting efficiency. This latter event most probably involves competition between termination and +1 frame decoding when the UGA codon is in the A-site. Various discussions have been presented for the importance of A-site and P-site events during ribosomal frameshifting (7, 52) and clearly, further investigations of this topic are warranted.\nThe observation presented here that the antisense oligonucleotide, AZ1A, which anneals directly adjacent to the UGA stop codon can induce ribosome frameshifts to either the +1 or À1 reading frame is surprising. In light of the above discussion of spacing for naturally occurring cis-acting frameshift stimulators, it is possible that frameshifting may occur at codons upstream of the known UCC UGA shift site. However, visual examination of upstream codons does not reveal an obvious À1 or +1 frameshift site.\nThe ability of spermidine to stimulate antisense oligonucleotide induced ribosome frameshifting to the +1 reading frame at the UCC UGA shift site in the absence of the natural 3 0 stimulator demonstrates that this cis-acting element is not required for polyamine responsiveness. Similarly, spermidine stimulation was observed in the absence of the 5 0 element but virtually eliminated by altering the UGA codon of the shift site to UGG. These observations are in agreement with previous studies examining the importance of cis-acting elements for polyamine induced frameshifting during expression of antizyme genes (39) (40) (41) .\nFinally, the ability to direct ribosomes to the +1 reading frame in living cells ( Figure 6 ) suggests a potential therapeutic application for antisense oligonucleotides. Directed frameshifting to the +1 reading frame near a disease causing À1 frameshift mutation would cause some ribosomes to resume decoding in the wild-type ORF, thus restoring partial production of full-length protein from mutant alleles. The importance of the stop codon for efficient frameshifting suggests that the stop codon following the frameshift mutation presents a promising target for antisense induced phenotypic suppression, and that modulation of intracellular polyamine levels, although not essential, may increase the effectiveness of this approach. Further experiments are required to determine the therapeutic potential of this approach in vivo including the generality and efficiency of frameshift induction at non-programmed frameshift sites." }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: Translation is optimally accurate and the correspondence between the nucleotide and the protein sequences are often considered as an immutable dogma. However, the genetic code is not quite universal: in certain organelles and in a small number of organisms the meaning of different codons has been reassigned and all the mRNAs are decoded accordingly. More surprisingly, the standard rules of genetic decoding are altered in specific genes by different events that are globally termed recoding (1) . In all cases, translational recoding occurs in competition with normal decoding, with a proportion of the ribosomes not obeying to the 'universal' rules. Translational recoding has been identified in both prokaryotes and eukaryotes. It has crucial roles in the regulation of gene expression and includes stop codon readthrough, ribosome hopping and ±1 programmed frameshifting [for reviews see (2) (3) (4) ].\nIn stop codon readthrough a stop codon is decoded by a tRNA carrying an unusual amino acid rather than a translational release factor. Specific stimulatory elements downstream to the stop codon regulate this process (5) . Hopping, in which the ribosome stops translation in a particular site of the mRNA and re-start few nucleotides downstream, is a rare event and it has been studied in detail only in the bacteriophage T4 (6) . In programmed frameshifting, ribosomes are induced to shift to an alternative, overlapping reading frame 1 nt 3 0 -wards (+1 frameshifting) or 5 0 -wards (À1 frameshifting) of the mRNA. This process is regulated and its frequency varies in different genes. The ±1 programmed frameshifting has been studied extensively in viruses, retrotransposons and insertion elements for which many cases are documented (7) (8) (9) . Instead, this phenomenon is by far less common in cellular genes. A single case of programmed +1 frameshifting is known in prokaryotes (10, 11) while in eukaryotes, including humans, several genes regulated by this recoding event have been described previously [(4) and references therein]. Compared to +1 frameshifting, À1 frameshifting is less widespread with only two examples in prokaryotes (12) (13) (14) and few others in eukaryotes (15) (16) (17) .\nThe programmed À1 frameshifting is triggered by several elements in the mRNA. The slippery sequence, showing the X-XXY-YYZ motif, in which X can be any base, Y is usually A or U, and Z is any base but G, has the function of favouring the tRNA misalignment and it is the site where the shift takes place (3, 18) . Frameshifting could be further stimulated by other elements flanking the slippery sequence: a codon for a low-abundance tRNA, a stop codon, a Shine-Dalgarno sequence and an mRNA secondary structure. It has been *To whom correspondence should be addressed. Tel: +39 081 6132271; Fax: +39 081 6132277; Email: [email protected] Ó 2006 The Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.\nreported that these elements, alone or in combination, enhance frameshifting by pausing the translating ribosome on the slippery sequence (4, 18) .\nNoticeably, known cases of recoding in Archaea [recently reviewed in (19) ] are limited to termination codon readthrough events that regulate the incorporation of the 21st and 22nd amino acids selenocysteine and pyrrolysine, respectively (20) (21) (22) (23) .\nNo archaeal genes regulated by translational programmed frameshifting and ribosome hopping have been identified experimentally so far; therefore, if compared with the others domains of life, the study of translational recoding in Archaea is still at its dawn.\nWe showed that the a-L-fucosidase gene from the crenarchaeon Sulfolobus solfataricus is putatively expressed by programmed À1 frameshifting (24) . This gene, named fucA1, is organized in the open reading frames (ORFs) SSO11867 and SSO3060 of 81 and 426 amino acids, respectively, which are separated by a À1 frameshifting in a 40 base overlap ( Figure 1A ). We have reported previously that the region of overlap between the two ORFs had the characteristic features of the genes expressed by programmed À1 frameshifting including a slippery heptanucleotide A-AAA-AAT (codons are shown in the zero frame) flanked by a putative stem-loop and the rare codons CAC ( Figure 1A ) resembling the prokaryotic stem-loops/hairpins and the Shine-Dalgarnolike sites (24) . We showed that the frameshifting, obtained by mutating by site-directed mutagenesis the fucA1 gene exactly in the position predicted from the slippery site, produced a full-length gene, named fucA1 A , encoding for a polypeptide of 495 amino acids ( Figure 1B ). This mutant gene expressed in Escherichia coli a fully functional a-L-fucosidase, named Ssa-fuc, which was thermophilic, thermostable and had an unusual nonameric structure (24, 25) . More recently, we determined the reaction mechanism and the function of the residues of the active site of the mutant enzyme (26, 27) .\nThe functionality of the product of the mutant gene fucA1 A does not provide direct experimental evidence that programmed À1 frameshifting occurs in vivo and in S.solfataricus. To address these issues, we report here the study of the expression of the wild-type split gene fucA1 and of its mutants in the slippery sequence. We demonstrate here that fucA1 is expressed by programmed À1 frameshifting in both E.coli and S.solfataricus. This is the first experimental demonstration that this kind of recoding is present in the Archaea domain of life. The relevance of programmed À1 frameshifting in Archaea is also discussed. Figure 1 . The a-fucosidase gene. (A) Region of overlap in the wild-type split fucA1 gene. The N-terminal SSO11867 ORF is in the zero frame, the C-terminal SSO3060 ORF, for which only a fragment is shown, is in the À1 frame. The slippery heptameric sequence is underlined; the rare codons are boxed and the arrows indicate the stems of the putative mRNA secondary structure. The amino acids involved in the programmed À1 frameshifting and the first codon translated after this event in the À1 frame are shadowed. (B) Fragment of the full-length mutant fucA1 A gene. The small arrows indicate the mutated nucleotides.\nAnalysis of the a-fucosidase expression S.solfataricus cells were grown, and cell extracts obtained, as described previously (24, 28) .\nThe expression in the E.coli strain BL21(RB791) of the wild-type gene fucA1 and of the mutant genes fucA1 A [previously named FrameFuc in (24) ], fucA1 B , fucA1 sm and fucA1 tm as fusions of glutathione S-transferase (GST) and the purification of the recombinant proteins were performed as reported previously (23) . The nomenclature used in this paper for the different a-fucosidase genes is listed in Table 1 .\nFor the western blot studies, equal amounts of E.coli cultures expressing the wild-type and mutant fucA1 genes, normalized for the OD 600 , were resuspended in SDS-PAGE loading buffer containing 0.03 M Tris-HCl buffer, pH 6.8, 3% SDS (w/v), 6.7% glycerol (w/v), 6.7% 2-mercaptoethanol (w/v) and 0.002% blue bromophenol (w/v). The samples were incubated at 100 C for 5 min (unless otherwise indicated) and were directly loaded on to the gel. Western blot analyses were performed by blotting SDS-PAGEs of the concentrations indicated on Hybond-P polyvinylidenfluorid filters (Amersham Biosciences, Uppsala, Sweden); polyclonal anti-Ssa-fuc antibodies from rabbit (PRIMM, Milan, Italy) and anti-GST antibodies (Amersham Biosciences) were diluted 1:5000 and 1:40 000, respectively. The filters were washed and incubated with the ImmunoPure anti-rabbit IgG antibody conjugated with the horseradish peroxidase (HRP) from Pierce Biotechnology (Rockford, IL, USA). Filters were developed with the ECL-plus Western Blotting Detection system (Amersham Biosciences) by following the manufacturer's indications. The molecular weight markers used in the western blot analyses were the ECL streptavidin-HRP conjugate (Amersham Biosciences).\nThe protein concentration of the samples was measured with the method of Bradford (29) and the amounts of sample loaded on to the SDS-PAGEs are those indicated. The quantification of the bands identified by western blot was performed by using the program Quantity One 4.4.0 in a ChemiDoc EQ System (Bio-Rad, Hercules, CA, USA) with the volume analysis tool. The frameshifting efficiency was calculated as the ratio of the intensity of the bands of the frameshifted product/frameshifted product + termination product.\nThe mutants in the slippery sequence of the wild-type gene fucA1 were prepared by site-directed mutagenesis from the vector pGEX-11867/3060, described previously (24, 27) . The synthetic oligonucleotides used (PRIMM) were the following: FucA1sm-rev, 5 0 -TTTAGGTGATATTGGTGTT-CTGGTCTATCT-3 0 ; FucA1sm-fwd, 5 0 -GAACACCAATAT-CACCTAAAGAATTCGGCCCA-3 0 ; FucA1tm-rev, 5 0 -AGG-TGATATTGGTGTTCTGGTCTATCTGGC-3 0 ; FucA1tm-fwd, 5 0 -CCAGAACACCAATATCACCTCAAGAACTCGGCCCA-GT-3 0 , where the mismatched nucleotides in the mutagenic primers are underlined. Direct sequencing identified the plasmids containing the desired mutations and the mutant genes, named fucA1 sm and fucA1 tm , were completely re-sequenced.\nThe mutant Ssa-fuc B was prepared by site-directed mutagenesis from the vector pGEX-11867/3060, by using the same site-directed mutagenesis kit described above. The synthetic oligonucleotides used were FucA1sm-rev (described above) and the following mutagenic oligonucleotide: Fuc-B, 5 0 -GAACACCAATATCACCTAAAGAAGTTCGGCCC-AGT-3 0 , where the mismatched nucleotides are underlined. Direct sequencing identified the plasmid containing the desired mutations and the mutant gene, named fucA1 B , was completely re-sequenced. The enzymatic characterization of Ssa-fuc B was performed as described previously (24, 27) .\nSamples of the proteins expressed in E.coli from the wildtype gene fucA1 and the mutants fucA1 A and fucA1 sm , purified as described, were fractionated on an SDS-PAGE. Protein bands were excised from the gel, washed in 50 mM ammonium bicarbonate, pH 8.0, in 50% acetonitrile, reduced with 10 mM DTT at 56 C for 45 min and alkylated with 55 mM iodoacetamide for 30 min at room temperature in the dark. The gel pieces were washed several times with the buffer, resuspended in 50 mM ammonium bicarbonate and incubated with 100 ng of trypsin for 2 h at 4 C and overnight at 37 C. The supernatant containing peptides was analysed by MALDIMS on an Applied Biosystem Voyager DE-PRO mass spectrometer using a-cyano-4-hydroxycynnamic acid as matrix. Mass calibration was performed by using the standard mixture provided by manufacturer.\nLiquid chromatography online tandem mass spectrometry (LCMSMS) analyses were performed on a Q-TOF hybrid mass spectrometer (Micromass, Waters, Milford, MA, USA) coupled with a CapLC capillary chromatographic system (Waters). Peptide ions were selected in the collision cell and fragmented. Analysis of the daughter ion spectra led to the reconstruction of peptide sequences.\nGenomic DNA from S.solfataricus P2 strain was prepared as described previously (24) . A DNA fragment of 1538 nt containing the complete fucA1 gene, was prepared by PCR, by using the following synthetic oligonucleotides (Genenco, Florence, Italy): FucA1-fwd, 5 0 -CTGGAGGCGCGCTAA-TACGACTCACTATAGGTCAGTTAAATGTCACAAAA-TTCT-3 0 ; FucA1-rev, 5 0 -GACTTGGCGCGCCTATCTAT-AATCTAGGATAACCCTTAT-3 0 , in which the sequence corresponding to the genome of S.solfataricus is underlined. In the FucA1-fwd primer, the sequence of the promoter of the T7 RNA polymerase is in boldface and the sequence of the BssHII site is shown in italics. The PCR amplification was performed as described previously (24) and the amplification products were cloned in the BssHII site of the plasmid pBluescript II KS+. The fucA1 gene was completely re-sequenced to check if undesired mutations were introduced by PCR and the recombinant vector obtained, named pBlu-FucA1, was used for translation in vitro experiments.\nThe plasmids expressing the mutant genes fucA1 A , fucA1 sm and fucA1 tm for experiments of translation in vitro were prepared by substituting the KpnI-NcoI wild-type fragment, containing the slippery site, with those isolated from the mutants. To check that the resulting plasmids had the correct sequence, the mutant genes were completely re-sequenced.\nThe mRNAs encoding wild-type fucA1 and its various mutants were obtained by in vitro run-off transcription. About 2 mg of each plasmid was linearized with BssHII and incubated with 50 U of T7 RNA polymerase for 1 h 30 min at 37 C. The transcription mixtures were then treated with 10 U of DNAseI (RNAse free) for 30 min. The transcribed RNAs were recovered by extracting the samples twice with phenol (pH 4.7) and once with phenol/chloroform 1:1 followed by precipitation with ethanol. The mRNAs were resuspended in DEPC-treated H 2 O at the approximate concentration of 0.6 pmol/ml.\nIn vitro translation assays were performed essentially as described by Condò et al. (28) . The samples (25 ml final volume) contained 5 ml of S.solfataricus cell extract, 10 mM KCl, 20 mM Tris-HCl, pH 7.0, 20 mM Mg acetate, 3 mM ATP, 1 mM GTP, 5 mg of bulk S.solfataricus tRNA, 2 ml of [ 35 S]methionine (1200 Ci/mmol at 10 mCi/ml) and $10 pmol of each mRNA. The mixtures were incubated at 70 C for 45 min. After this time, the synthesized proteins were resolved by electrophoresis 12.5% acrylamide-SDS gels and revealed by autoradiography of the dried gels on an Instant Imager apparatus.\nCells of S.solfataricus, strain P2, were grown in minimal salts culture media supplemented with yeast extract (0.1%), casamino acids (0.1%), plus glucose (0.1%) (YGM) or sucrose (0.1%) (YSM). The extraction of total RNA was performed as reported previously (24) . Total RNA was extensively digested with DNAse (Ambion, Austin, TX, USA) and the absence of DNA was assessed by the lack of PCR amplification with each sets of primers described below. The RT-PCR experiments were performed as reported previously (24) by using the primers described previously that allowed the amplification of a region of 833 nt (positions 1-833, in which the A of the first ATG codon is numbered as one) overlapping the ORFs SSO11867 and SSO3060 (24) .\nFor real-time PCR experiments total cDNA was obtained using the kit Quantitect RT (Qiagen GmbH, Hilden, Germany) from 500 ng of the same preparation of RNA described above. cDNA was then amplified in a Bio-Rad LightCycler using the DyNAmo HS Syber Green qPCR Kit (Finnzymes Oy, Espoo, Finland). Synthetic oligonucleotides (PRIMM) used for the amplification of a region at the 3 0 of the ORF SSO3060 were as follows: 5 0 -Real: 5 0 -TAAATGGC-GAAGCGATTTTC-3 0 ; 3 0 -Real: 5 0 -ATATGCCTTTGTCGC-GGATA-3 0 for the gene fucA1. 5 0 -GAATGGGGGTGATA-CTGTCG-3 0 and 5 0 -TTTACAGCCGGGACTACAGG-3 0 for the 16S rRNA gene.\nFor each amplification of the fucA1 gene was used $2500fold more cDNA than that used for the amplification of the 16S rRNA. Controls with no template cDNA were always included. PCR conditions were 15 min at 95 C for initial denaturation, followed by 40 cycles of 10 s at 95 C, 25 s at 56 C and 35 s at 72 C, and a final step of 10 min at 72 C. Product purity was controlled by melting point analysis of setpoints with 0.5 C temperature increase from 72 to 95 C. PCR products were analysed on 2% agarose gels and visualized by ethidium bromide staining.\nThe expression values of fucA1 gene were normalized to the values determined for the 16S rRNA gene. Absolute expression levels were calculated as fucA1/16S ratio in YSM and YGM cells, respectively. Relative mRNA expression levels (YSM/YGM ratio) were calculated as ( fucA1/ 16S ratio in YGM cells)/(fucA1/16S ratio in YSM). Each cDNA was used in triplicate for each amplification.\nThe wild-type fucA1 gene, expressed in E.coli as a GST-fused protein, produced trace amounts of a-fucosidase activity (2.3 · 10 À2 units mg À1 after removal of GST), suggesting that a programmed À1 frameshifting may occur in E.coli (24) . The enzyme was then purified by using the GST purification system and analysed by SDS-PAGE revealing a major protein band ( Figure 2A ). The sample and control bands were excised from the gel, digested in situ with trypsin and directly analysed by matrix-assisted laser desorption/ ionization mass spectrometry (MALDIMS). As shown in Figure 2B and C, both spectra revealed the occurrence of an identical mass signal at m/z 1244.6 corresponding to a peptide (Peptide A) encompassing the overlapping region of the two ORFs. This result was confirmed by liquid chromatography online tandem mass spectrometry (LCMSMS) analysis of the peptide mixtures. The fragmentation spectra of the two signals showed the common sequence Asn-Phe-Gly-Pro-Val-Thr-Asp-Phe-Gly-Tyr-Lys in which the amino acid from the ORF SSO11867 is underlined. These results unequivocally demonstrate that the protein containing the Peptide A is produced in E.coli by a frameshifting event that occurred exactly within the slippery heptamer predicted from the analysis of the DNA sequence in the region of overlap between the ORFs SSO11867 and SSO3060 ( Figure 1A) .\nRemarkably, the MALDIMS analysis of the products of the wild-type fucA1 gene revealed the presence of a second Peptide B at m/z 1258.6 that is absent in the spectra of the Ssa-fuc control protein ( Figure 2B and C). The sequence of Peptide B obtained by LCMSMS ( Figure 2D ) was Lys-Phe-Gly-Pro-Val-Thr-Asp-Phe-Gly-Tyr-Lys. This sequence differs only by one amino acid from Peptide A demonstrating that the interrupted gene fucA1 expresses in E.coli two fulllength proteins originated by different À1 frameshifting events. Polypeptide A results from a shift in a site A and it is identical to Ssa-fuc prepared by site-directed mutagenesis (24) , suggesting that the expression occurred with the simultaneous P-and A-site slippage. Instead, polypeptide B, named Ssa-fuc B , is generated by frameshifting in a second site B as the result of a single P-site slippage ( Figure 2E) .\nTo measure the global efficiency of frameshifting in the two sites of the wild-type gene fucA1 we analysed the total extracts of E.coli by western blot using anti-GST antibodies ( Figure 2F ). Two bands with marked different electrophoretic mobility were observed: the polypeptide of 78.7 ± 1.1 kDa migrated like GST-Ssa-fuc fusion and was identified as originated from frameshifting in either site A or B of fucA1. The protein of 38.1 ± 1.2 kDa, which is not expressed by the mutant gene fucA1 A (not shown), had an electrophoretic mobility compatible with GST fused to the polypeptide encoded by the ORF SSO11867 solely (27 and 9.6 kDa, respectively). This polypeptide originated from the translational termination of the ribosome at the OCH codon of the fucA1 N-terminal ORF ( Figure 1A) . The calculated ratio of frameshifting to the termination products was 5%.\nTo test if the full-length a-fucosidase produced by the À1 frameshifting event in site B (Ssa-fuc B ), resulting from the single P-site slippage has different properties from Ssa-fuc, whose sequence arises from the simultaneous P-and A-site slippage, we prepared the enzyme by site-directed mutagenesis. The slippery sequence in fucA1 A-AAA-AAT was mutated in A-AAG-AAG-T where mutations are underlined. The new mutant gene was named fucA1 B . The first G, producing the conservative mutation AAA!AAG, was made to disrupt the slippery sequence and hence reducing the shifting efficiency. The second G was inserted to produce the frameshifting that results in the amino acid sequence of Peptide B. Therefore, the sequence of the two full-length mutant genes fucA1 A and fucA1 B differs only in the region of the slippery sequence: A-AAG-AAT-TTC-GGC and A-AAG-AAG-TTC-GGC, respectively (the mutations are underlined, the nucleotides in boldface were originally in the À1 frame) ( Table 1) .\nThe recombinant Ssa-fuc B was purified up to $95% (Materials and Methods). Gel filtration chromatography demonstrated that in native conditions Ssa-fuc B had the same nonameric structure of Ssa-fuc with an identical molecular weight of 508 kDa (data not shown). In addition, Ssa-fuc B had the same high substrate selectivity of Ssa-fuc. The two enzymes have high affinity for 4-nitrophenyl-a-L-fucoside (4NP-Fuc) substrate at 65 C; the K M is identical within the experimental error (0.0287 ± 0.005 mM) while the k cat of Ssa-fuc B (137 ± 5.7 s À1 ) is $48% of that of Ssa-fuc (287 ± 11 s À1 ). In addition, 4-nitrophenyl-a-L-arabinoside, -rhamnoside, 4-nitrophenyl-a-D-glucoside, -xyloside, -galactoside and -mannoside were not substrates of Ssa-fuc B as shown previously for Ssa-fuc (24) . This suggests that the different amino acid sequence did not significantly affect the active site. Both enzymes showed an identical profile of specific activity versus temperature with an optimal temperature higher than 95 C (data not shown). The heat stability and the pH dependence of Ssa-fuc and Ssa-fuc B are reported in Figure 3 . At 80 C, the optimal growth temperature of S.solfataricus, the half-life of Ssa-fuc B is 45 min, almost 4-fold lower than that of Ssa-fuc ( Figure 3A ). The two enzymes showed different behaviour at pH <6.0 at which Ssa-fuc B is only barely active and stable ( Figure 3B) ; however, the two enzymes showed similar values of specific activity at pHs above 6.0, which is close to the intracellular pH of S.solfataricus (30) .\nCharacterization of the slippery sequence of fucA1 in E.coli\nThe experimental data reported above indicate that the predicted slippery heptanucleotide in the region of overlap between the ORFs SSO11867 and SSO3060 of the wildtype gene fucA1 could regulate in cis the frameshifting events observed in E.coli. To test this hypothesis, we mutated the sequence A-AAA-AAT into A-AAG-AAT and C-AAG-AAC (mutations are underlined) obtaining the fucA1 single mutant ( fucA1 sm ) and triple mutant ( fucA1 tm ) genes, respectively. It is worth noting that the mutations disrupt the slippery sequence, but they maintain the À1 frameshift between the two ORFs (Table 1) . Surprisingly, the expression of fucA1 sm in E.coli produced a full-length polypeptide that, after purification by affinity chromatography and removal of the GST protein, showed the same electrophoretic migration of Ssa-fuc and Ssa-fuc B ( Figure 4A ). This protein was then characterized by mass spectrometry analyses following in situ tryptic digestion. Interestingly, the MALDI spectra revealed the presence of a single peptide encompassing the overlapping region between the two ORFs with a mass value of 1259.7 Da (peptide C; Figure 4B ). The sequence of peptide C, determined from the fragmentation spectra obtained by LCMSMS analysis, was Glu-Phe-Gly-Pro-Val-Thr-Asp-Phe-Gly-Tyr-Lys ( Figure 4C ). Remarkably, apart from the Glu residue, this sequence is identical to that of peptide B produced from fucA1, indicating that in the mutant gene fucA1 sm only one of the two frameshifting events observed in the wild-type fucA1 gene had occurred. The presence of a Glu instead of Lys was not unexpected. The mutation A-AAA-AAT!A-AAG-AAT in fucA1 sm was conservative in the zero frame of the ORF SSO11867 (AAA!AAG, both encoding Lys), but it produced the mutation AAA!GAA (Lys!Glu) in the À1 frame of the ORF SSO3060.\nIt is worth noting that the frameshifting efficiency of the gene fucA1 sm , calculated by western blot as described above, was 2-folds higher (10%) if compared to fucA1 (5%) ( Figure 4D ). This indicates that the mutation cancelled the frameshifting site A and, in the same time, enhanced the frameshifting efficiency of site B.\nIn contrast, the triple mutant fucA1 tm produced in E.coli only the low molecular weight band resulting from translational termination ( Figure 4D ). No full-length protein could be detected in western blots probed with either anti-GST ( Figure 4D ) or anti-Ssa-fuc antibodies ( Figure 4E ). These data show that the disruption of the heptameric slippery sequence completely abolished the frameshifting in E.coli confirming that this sequence has a direct role in controlling the frameshifting in vivo.\nTo test whether fucA1 is expressed in S.solfataricus we analysed the extracts of cells grown on yeast extract, sucrose and casaminoacids medium (YSM). Accurate assays showed that S.solfataricus extracts contained 3.4 · 10 À4 units mg À1 of a-fucosidase activity. These very low amounts hampered the purification of the enzyme. The extracts of S.solfataricus cells grown on YSM revealed by western blot a band of a molecular mass >97 kDa and no signals were detected with the pre-immune serum confirming the specificity of the anti-Ssa-fuc antibodies ( Figure 5A) . The different molecular mass may result from post-translational modifications occurred in the archaeon or from the incomplete denaturation of a protein complex. In particular, the latter event is not unusual among enzymes from hyperthermophilic archaea (31, 32) . To test which hypotheses were appropriate, cellular extracts of S.solfataricus were analysed by western blot extending the incubation at 100 C to 2 h. Interestingly, this treatment shifted the high-molecular mass band to 67.6 ± 1.2 kDa ( Figure 5B and C) , which still differs from that of the recombinant Ssa-fuc, 58.9 ± 1.2 kDa, leaving the question on the origin of this difference unsolved. To try to shed some light we immunoprecipitated extracts of S.solfataricus with anti-Ssa-fuc antibodies and we analysed the major protein band by MALDIMS. Unfortunately, we could not observe any peptide compatible with the fucosidase because the heavy IgG chain co-migrated with the band of the expected molecular weight (data not shown).\nTo test if the scarce amounts of the a-fucosidase in S.solfataricus extracts was the result of reduced expression at transcriptional level, we performed a northern blot analysis of total RNA extracted from cells grown either on YSM or YGM media. We could not observe any signal by using probes matching the 3 0 of the ORF SSO3060 (data not shown). These results suggest that fucA1 produced a rare transcript; therefore, we analysed the level of mRNA by RT-PCR and by real-time PCR. A band corresponding to the region of overlap between the ORFs SSO11867 and SSO3060 was observed in the RNA extracted from cells grown on YSM 2 mg) , the product of the gene fucA1 sm (2 mg), and Ssa-fuc (4 mg). The bands with faster electrophoretic mobility result from the proteolytic cleavage of the full-length protein (25) . (B) Partial MALDIMS spectrum of the tryptic digest from mutant fucA1 sm expressed in E.coli. The mass signal corresponding to peptide C encompassing the overlapping region is indicated. (C) LCMSMS analysis of peptide C. The amino acid sequence inferred from fragmentation spectra is reported. (D) Western blot of E.coli cellular extracts expressing fucA1 A , the wild-type fucA1, fucA1 sm and fucA1 tm genes (Materials and Methods). The blot was probed with anti-GST antibodies. (E) Western blot of partially purified protein samples expressed in E.coli fused to GST from wild-type and mutant fucA1 genes. Cellular extracts were loaded on GST-Sepharose matrix. After washing, equal amounts of slurries (30 ml of 300 ml) were denaturated and loaded on a 8% SDS-PAGE. Extracts of E.coli cells expressing the parental plasmid pGEX-2TK were used as the negative control (pGEX). The blot was probed with anti-Ssa-fuc antibodies. and YGM media, demonstrating that under these conditions the two ORFs were co-transcribed ( Figure 6A) .\nThe experiments of real-time PCR shown in Figure 6B demonstrated that rRNA16S was amplified after $17 cycles while the amplification of fucA1 mRNA was observed after 38 cycles, despite the fact that we used $2500-fold more cDNA for the amplification of fucA1. This indicates that the gene fucA1 is transcribed at very low level. No significant differences in the fucA1 mRNA level were observed in cells grown in YSM or YGM media. This is further confirmed by the analysis by western blot of the extracts of the same cells of S.solfataricus used to prepare the total RNAs, which revealed equal amounts of a-fucosidase in the two extracts ( Figure 6C) . Therefore, the low a-fucosidase activity observed under the conditions tested is the result of the poor transcription of the fucA1 gene.\nTo determine whether, and with what efficiency, the À1 frameshifting could be performed by S.solfataricus ribosomes, mRNAs obtained by in vitro transcription of the cloned wild-type fucA1 gene and the mutants thereof were used to program an in vitro translation system prepared as described by Condò et al. (28) . To this aim, a promoter of T7 polymerase was inserted ahead of the gene of interest to obtain RNA transcripts endowed with the short 5 0untranslated region of 9 nt observed for the natural fucA1 mRNA (24) . Autoradiography of an SDS-PAGE of the translation products (Figure 7 ) revealed that the wild-type fucA1 transcript produced a tiny but clear band whose molecular weight corresponded to that of the full-length Ssa-fuc obtained by site-directed mutagenesis (24); the latter was translated quite efficiently in the cell-free system in spite of being encoded by a quasi-leaderless mRNA. Judging from the relative intensity of the signals given by the translation products of the wild-type fucA1 and the full-length mutant fucA1 A , the efficiency of the À1 frameshifting in the homologous system was $10%. No signals corresponding to the polypeptides expected from the separated ORFs SSO11867 and SSO3060 (9.6 and 46.5 kDa, respectively) were observed. However, it should be noted that the product of SSO11867, even if synthesized, is too small to be detected in the gel system employed for this experiment. The larger product of ORF SSO3060, on the other hand, is certainly absent. These data unequivocally demonstrate that the ribosomes of S.solfataricus can decode the split fucA1 gene by programmed À1 frameshifting with considerable efficiency producing a full-length polypeptide from the two ORFs SSO11867 and SSO3060.\nRemarkably, under the same conditions at which fucA1 drives the expression of the full-length protein, we could not observe any product from the fucA1 sm and fucA1 tm constructs. These data demonstrate that the integrity of the heptanucleotide is essential for the expression of the fucA1 gene in S.solfataricus, thus further confirming that the gene is decoded by programmed À1 frameshifting in this organism. In addition, the lack of expression of fucA1 sm by translation in vitro in S.solfataricus contrasts with the efficient expression of this mutant in E.coli, indicating that the two organisms recognize different sequences regulating the translational frameshifting.\nThe identification of genes whose expression is regulated by recoding events is often serendipitous. In the framework of our studies on glycosidases from hyperthermophiles, we identified in the genome of the archaeon S.solfataricus a split gene encoding a putative a-fucosidase, which could be expressed through programmed À1 frameshifting (24) . We tackled this issue by studying the expression of fucA1 in S.solfataricus and in E.coli to overcome the problems connected to the scarcity of expression of the a-fucosidase gene and to the manipulation of hyperthermophiles. As already reported by others, in fact, it is a common strategy to study recoding events from different organisms in E.coli (23, 33) .\nThe expression in E.coli of the wild-type split gene fucA1 led to the production by frameshifting of two full-length polypeptides with an efficiency of 5%. This is a value higher than that observed in other genes expressed by translational frameshifting in a heterologous system such as the proteins gpG and gpGT (0.3-3.5%) (33) .\nThe gene fucA1 is expressed in S.solfataricus at very low level under the conditions tested. In particular, the transcriptional analysis of the gene revealed that it is expressed at very low level in both YSM and YGM media. Similarly, no differences in the two media could be found by western blot probed with anti-Ssa-fuc antibodies, indicating that the low expression of the enzyme in S.solfataricus is the result of scarce transcription rather than suppressed translation.\nWestern blots allowed us to identify a specific band $8.7 kDa heavier than that of the recombinant Ssa-fuc and experiments of translation in vitro showed that the wild-type gene expresses a full-length polypeptide exhibiting the same molecular mass of the recombinant protein. This demonstrates that the translational machinery of S.solfataricus is fully competent to perform programmed frameshifting. It seems likely that the observed discrepancy in molecular mass might arise from post-translational modifications that cannot be produced by the translation in vitro. Further experiments are required to characterize the a-L-fucosidase identified in S.solfataricus.\nMALDIMS and LCMSMS analyses of the products in E.coli of the wild-type split gene fucA1 demonstrated that two independent frameshifting events occurred in vivo in the proposed slippery site. In particular, the sequences obtained by LCMSMS demonstrate that peptide A results from a simultaneous backward slippage of both the P-and the A-site tRNAs ( Figure 8A) . Instead, the sequence of peptide B is the result of the re-positioning on the À1 frame of only the P-site tRNA; in fact, the next incorporated amino acid is specified by the codon in the new frame ( Figure 8B) . Therefore, the expression by À1 frameshifting of the wild-type gene fucA1 in E.coli follows the models proposed for ribosomal frameshifting (34) . We confirmed the significance of the slippery heptanucleotide in promoting the programmed frameshifting in vivo by mutating the putative regulatory sequence. The triple mutant fucA1 tm gave no full-length products; presumably, the mutations in both the P-and in the A-site of the slippery sequence dramatically reduced the efficiency of the À1 frameshifting as observed previously in metazoans (35) . This result confirms that the intact slippery sequence in the wild-type gene fucA1 is absolutely necessary for its expression in E.coli. In contrast, surprisingly, the single mutant fucA1 sm showed an even increased frequency of frameshifting (10%) if compared to the wild-type and produced only one polypeptide by shifting specifically in site B. We explained this result observing that the mutation in the P-site of the slippery sequence A-AAA-AAT!A-AAG-AAT created a novel slippery sequence A-AAG identical to that controlling the expression by programmed À1 frameshifting of a transposase gene in E.coli (36) . Therefore, apparently, the single mutation inactivated the simultaneous P-and A-site tRNA re-positioning and, in the same time, fostered the shifting efficiency of the tRNA in the P-site. It is worth noting that, instead, in S.solfataricus, only the simultaneous slippage is effective ( Figure 8B ) and even the single mutation in the slippery sequence of fucA1 sm completely annulled the expression of the gene. This indicates that this sequence is essential in the archaeon and that programmed frameshifting in S.solfataricus and E.coli exploits different mechanisms. Furthermore, since the only difference between the enzymes produced by the frameshifting sites A and B, Ssa-fuc and Ssa-fuc B , respectively, is the stability at 80 C, which is the S.solfataricus physiological temperature, the functionality of Ssa-fuc B in the archaeon appears questionable.\nThe reason why fucA1 is regulated by programmed À1 frameshifting is not known. However, the physiological significance of programmed frameshifting has been assigned to a minority of the cellular genes while for most of them it is still uncertain [see (4) and reference therein; (16) ]. This mechanism of recoding is exploited to set the ratio of two polypeptides such as the t and g subunits of the DNA polymerase III holoenzyme in E.coli (12) . Alternatively, programmed frameshifting balances the expression of a protein, as the bacterial translational release factor 2 and the eukaryotic ornithine decarboxylase antizyme [see (4) and (18) and references therein]. In the case of fucA1, the polypeptide encoded by the smaller ORF SSO11867 could never be detected by western blots analyses. In addition, the modelling of Ssa-fuc on the high-resolution crystal structure of the a-L-fucosidase from Thermotoga maritima (25, 37) showed that the fucA1 N-terminal polypeptide is not an independent domain. Moreover, we have shown recently that SSO11867 includes essential catalytic residues (27) , excluding the possibility that a functional a-fucosidase can be obtained from the ORF SSO3060 alone. Therefore, several lines of evidence allow us to exclude that programmed À1 frameshifting is used to set the ratio of two polypeptides of the a-fucosidase from S.solfataricus. More probably, this translational mechanism might be required to control the expression level of fucA1.\nNoticeably, this is the only fucosidase gene expressed by programmed À1 frameshifting. Among carbohydrate active enzymes, the only example of expression through this recoding mechanism is that reported for a gene encoding for a a(1,2)-fucosyltransferase from Helicobacter pylori that is interrupted by a À1 frameshifting (38) . In this case, the expression by programmed frameshifting would lead to a functional enzyme synthesizing components of the surface lipopolysaccharides to evade the human immune defensive system. It is hard to parallel this model to fucA1. Nevertheless, the monosaccharide fucose is involved in a variety of biological functions (39) . Therefore, the a-L-fucosidase might play a role in the metabolism of fucosylated oligosaccharides; experiments are currently in progress to knockout the wild-type fucA1 gene and to insert constitutive functional mutants of this gene in S.solfataricus.\nFucA1 is the only archaeal a-L-fucosidase gene identified so far; hence, it is probably the result of a horizontal gene transfer event in S.solfataricus. However, since there are no a-fucosidases genes regulated by programmed frameshifting in Bacteria and Eukarya, it is tempting to speculate that this sophisticated mechanism of translational regulation preexisted in S.solfataricus and it was applied to the fucosidase gene for physiological reasons. The identification of other genes interrupted by À1 frameshifts in S.solfataricus would open the possibility that they are regulated by programmed À1 frameshifting. Recently, the computational analysis of prokaryotic genomes revealed that seven Archaea harbour interrupted coding sequences, but S.solfataricus is not included in this study (40) . A computational analysis on several archaeal genomes revealed that 34 interrupted genes are present in the genome of S.solfataricus, 11 of these genes are composed by two ORFs separated by À1 frameshifting and could be expressed by recoding (B. Cobucci-Ponzano, M. Rossi and M. Moracci, manuscript in preparation).\nWe have experimentally shown here, for the first time, that programmed À1 frameshifting is present in the Archaea domain. This finding is the missing piece in the puzzle of the phylogenetic distribution of programmed frameshifting demonstrating that this mechanism is universally conserved." }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: Helicases are enzymes that separate in an energy-dependent manner stretches of duplexed DNA and/or RNA into singlestranded components. Currently, based on characteristic motifs and the sequence comparisons, three superfamilies (SF1 through 3) and two smaller families (F4, F5) of helicases have been identified (1) . Superfamily 1 and 2 contain helicases which share seven or more recognized signature amino acid motifs while SF3 and F4 and F5 helicases are characterized generally by three conserved motifs (2) ; the F4 and F5 proteins are largely bacterial and bacteriophage proteins. Currently, it should be cautioned that many 'helicases' are not bona fide helicases, but may only function as RNA translocases, perhaps to fulfill functions in the remodeling of ribonucleoprotein complexes (RNP).\nDEAD-box and the related DEAH, DExH and DExD (3) helicases are the most numerous members of SF2 and are ubiquitously present in eukaryotic genomes. These helicases share eight conserved motifs and are commonly refered to as the DExH/D family of helicases. Humans, Arabidopsis and Saccharomyces have 38, 55 and 25 such entities, respectively (4) . Differing from DNA helicases and DExH proteins, DEAD helicases are poor in unwinding long nucleic acid duplexes and are best suited for separating short RNA hybrids. DEAD-box proteins bind with high-affinity RNAprotein complexes while exhibiting little RNA sequence preference. This suggests that the specificity determinants for DEAD helicases may be through the recognition of protein factors. In this regard, a better understanding of the roles for DEAD proteins depends on the clear characterization of their respective interacting proteins.\nAlthough the precise substrate for most helicases awaits definition, DEAD helicases are generally thought to participate pleiotropically in many aspects of RNA metabolism including transcription, mRNA splicing, mRNA export, translation, RNA stability and mitochondrial gene expression (5) (6) (7) (8) . Some examples of helicases and their attributed functions include the following. UAP56, Brr2, Prp16, Prp22 and Prp43 play roles in RNA-splicing (4, 9) , while Dbp5 (10, 11) and DDX3 (12) chaperone RNAs from the nucleus into the cytoplasm. eIF4a and Ded1 serve for translation of mRNAs while Rh1B, Ski2, Dob1, Dhh1 helicases contribute to mRNA stability (4) . Other DEAD helicases act in ribosome biogenesis through regulation of small nucleolar RNAs and ribosomal RNAs (rRNAs) interactions (13, 14) . Finally, Neurospora and Trypanosoma DEAD proteins contribute to mitochondrial gene expression (15, 16) ; a Cryptococcus DEAD helicase is required for cryptococcosis pathogenesis (17) , and the dipteran Chironomus tentans uses a hrp84 DEAD helicase to regulate mRNA transport from the nucleus into the cytoplasm onto polyribosomes (18) .\nGiven that helicases significantly contribute to normal cellular metabolism, are they similarly essential to viruses?\nThe operational answer appears to be a qualified 'yes'. Indeed, when DEAD/DEAH-box helicase motif (InterPro IPR001410) was used to search the EMBL-EBI database, 1561 matches to individual viral sequence entries were found (http://www.ebi.ac.uk/interpro/DisplayIproEntry?ac¼ IPR001410), suggesting that many viruses have evolved to encode directly helicase or helicase-like proteins. The strongest biological evidence which supports the importance of a helicase in the virus life cycle comes from those viruses with an RNA genome. Hence, all positive-strand RNA viruses encode one or more helicase/helicase-like open reading frame (ORF) which, aside from the RNA-dependant RNA polymerase, is the most highly conserved viral sequence.\nAlthough less ubiquitous, helicases are also found in other types of viruses (see some examples listed in Table 1 ). Direct mutagenesis studies have confirmed that a helicase function is biologically required for the replication of many viruses including vaccinia virus (19) , poliovirus (20) , alphaviruses (21) , brome mosaic virus (22) , nidoviruses (23, 24) and flaviviruses (25) (26) (27) .\nIn 1981, the first cases of acquired immunodeficiency syndrome (AIDS) were described in American homosexual men. Thereafter, within three short years, French and American scientists confirmed that the human immunodeficiency virus (HIV) is the causative agent for AIDS. In the ensuing 20 years, >20 million individuals have died from AIDS; and currently, in 2006, 50 million people worldwide are infected by HIV-1 with 3 million incremental AIDS deaths and 4-5 million new infections occurring annually. The magnitude of this burden casts urgency to medical research on HIV/AIDS.\nHIV-1 is a retrovirus of the lentivirus genus with an RNA genome of 9 kilobases which encodes nine polypeptides. The major HIV-1 structural proteins are encoded by three genes, gag (group-specific antigen), pol (polymerase) and env (envelope), while the accessory proteins, Vif, Vpu, Vpr and Nef, and the regulatory proteins, Tat and Rev, are the primary translation products of multiply-spliced mRNA. HIV-1 infects CD4+ human T-cells and macrophages and integrates as a provirus into the host cell's DNA. Gene expression of HIV-1 is governed transcriptionally by a viral protein, Tat (28, 29) , via its binding to a nascent viral TAR RNA (30) , and post-transcriptionally by a second viral protein Rev (31,32) through its association with the viral RRE RNA. Both Tat and Rev interact with several host cell proteins in their transcriptional and post-transcriptional functions (33) . HIV-1 does not encode for any RNA helicase; however, findings suggest that host cell RNA helicases may be involved in the reverse transcription of HIV-1 RNA, in HIV-1 mRNA transcription and in the nucleus-to-cytoplasm transport of viral mRNA.\nA recent unexpected finding revealed the possibility that an RNA helicase may potentially contribute roles in HIV-1 particle assembly and reverse transcription (34) . Using proteomic analyses, Roy et al. (34) reported that the DEAH protein RNA helicase A (RHA) was found associated with HIV-1 Gag and packaged into HIV-1 virions in an RNAdependent manner. When RHA was knocked down in cells, HIV-1 particles which were produced from these cells were significantly less infectious. This appears to be compatible with two possible explanations. First, it is conceivable that RHA participates in the formation of infectious virus particles either by shaping Gag-RNA interaction during viral particle assembly or by budding. Failure of RHA to properly restructure viral RNP could explain the observed reduced infectivity. Second, Roy et al. (34) reported evidence that HIV-1 particles that do not contain RHA showed reduced virionendogenous reverse transcriptase activity. In this respect, it may be that RHA assists HIV-1 reverse transcriptase to more efficiently copy RNA by unwinding RNA secondary structure or by promoting the interaction of viral RNA with the nucleocapsid protein in order to assemble a better reverse transcription complex.\nSeparate from reverse transcription, the unwinding of highly structured RNAs might also be reasoned to be important for transcription (35) . However, direct evidence for an RNA helicase role has been somewhat elusive. There are several examples which seemingly support an activity for RNA helicase in transcription. First, in vaccinia virus, it has been postulated that the NPH-II helicase assists transcription by strand-separating duplexed RNA structures to prevent R-loop formation behind the elongating RNA polymerase (36) . Second, RHA has been invoked to provide a factorrecruitment role, bridging at the promoter the CREB-binding protein (37) and RNA polymerase II (37) . Third, the p68 DEAD-box helicase was shown recently to be a novel transcriptional co-activator for p53's transcriptional function (38) . Interestingly, in the latter two instances, neither the ATPase nor the helicase activity of RHA and p68 is apparently required for their attributed transcriptional roles.\nFor HIV-1, two recent studies provide clues that RNA helicases may also serve co-factor function for transcription from the viral long terminal repeat (LTR). Fujii et al. (39) observed that RHA conserves in its N-terminus two double-stranded RNA-binding (dsRBD) domains characterized previously for the TAR RNA-binding protein, TRBP (40, 41) . These investigators found in both reporter and virus replication assays that RHA activated, in a TAR RNA-binding dependant manner, HIV-1 LTR-directed transcription (39 roles in transcription or indirectly influence the milieu of polymerase II initiation/elongation at the LTR. Downstream from transcription, the fate of HIV-1 encoded RNA is regulated at the step of export of unspliced/partially spliced moieties from the nucleus into the cytoplasm. Unspliced and partially spliced viral RNAs code for genomic RNAs that are packaged into progeny virions and structural proteins. Hence, the egress of these RNAs from the nucleus into the cytoplasm is critical to the life cycle of the virus. Exit of HIV RNAs from the nucleus is a significant issue because unspliced/partially spliced cellular mRNAs are routinely retained in and not permitted export from the nucleus (43) (44) (45) (46) . A large body of work has suggested an elegant solution to this conundrum. Thus, it was established that the HIV-1 encoded Rev protein binds a highly secondary structured element (Rev responsive element; RRE) present in all unspliced and partially spliced HIV transcripts (47) (48) (49) (50) (51) (52) (53) (54) (55) (56) (57) ; and this binding specifically distinguishes, for purposes of nuclear export, viral transcripts from cellular RNAs.\nNew evidence now suggests that RNA helicases are also co-factors for Rev-directed export of HIV-1 mRNAs (58) . In its role of transporting unspliced and incompletely spliced viral RNAs from the nucleus, Rev directly interacts with nuclear export receptor CRM1 (59, 60) , and CRM1 is required for Rev-mediated export of HIV RNAs (59, 61, 62) . A recent report provides data that an RNA helicase, DDX3, is an additional player in the Rev-CRM1-RRE complex (12) . Thus, it was shown that DDX3 over-expression enhanced Revdependent, but not other export, pathway; and that DDX3 is a nucleo-cytoplasmic shuttling protein which binds CRM1 and Rev. Moreover, DDX3's necessity for Rev/RRE/CRM1 function was demonstrated by knock-down of cell endogenous DDX3. Finally, because DDX3 locates to nuclear pore complexes (NPC), Yedavalli et al. (12) further proposed that this human helicase, like the analogous yeast Dbp5p (11), may function with Rev/CRM1 to remodel and 'thread' large unspliced HIV-1 RNAs through the nuclear pore, facilitating their final release to the cytoplasmic side of the NPC.\nThe above DDX3 results are consistent with two additional papers which described similar findings for a related RNA helicase, DDX1. Thus, Pomerantz and co-workers (63) showed that DDX1 binds directly to the N-terminus of Rev and to the RRE-RNA motif and participates in the export of unspliced HIV-1 RNA from the nucleus to the cytoplasm. Additionally, they illustrated that reduced expression of DDX1 in astrocytes explains the previously observed tissue restricted function of HIV-1 Rev (64). Fully spliced viral mRNAs encoding for viral Tat, Rev and Nef, proteins have been shown previously to exit the nucleus using the cellular mRNA export pathway. Export of these mRNA may require the RNA helicase Dbp5 (65, 66) . As yet, the involvement of Dbp5 in export of spliced HIV-1 viral RNA has not been fully clarified.\nThe story of HIV-1 and RNA helicases is, however, likely to be more complex than and unlikely to conclude simply with RHA, RH116, DDX1 and DDX3 (Figure 1 ). HIV-1 RNAs are extensively regulated through splicing. Splicing is a multiple-step process requiring the recognition of splice sites by spliceosomes. It is generally believed that remodeling of RNA-RNA and RNA-protein interactions within the spliceosome is catalyzed by a family of DEAD/DExH box RNA helicases. To date, seven mammalian proteins that are RNA helicases have been implicated in mRNA splicing (67, 68) . Whether there is specific preference by subclasses of RNA helicases for viral mRNA splicing remains to be clarified. Moreover, how cellular RNA helicases might contribute to the translation of viral mRNAs also require further investigation.\nRecently both Van't Wout et al. (69) and Krishnan and Zeichner (70) have provided evidence that the expression of several cellular RNA helicases including DDX24, DDX21, DDX18, DDX11 and DDX9 is modulated during HIV-1 infection; however, the precise cellular role and significance of these helicases for HIV-1 pathogenesis have not be characterized. Interestingly, Krishnan and Zeichner reported microarray data which examined the transition of HIV-1 infection from latency to productive replication, and found that several cellular RNA helicases were upregulated (71) . For future understanding of functions, it will be important to design experiments which can segregate helicases which serve direct, although perhaps overlapping and redundant, roles on HIV-1 from those that might participate indirectly in the viral life cycle. Nevertheless, the convergence of evidence would support that several discrete cellular RNA helicases contribute importantly to the efficient execution of several steps in the HIV-1 replicative cycle.\nGiven that the HIV-1/AIDS disease burden has reached pandemic global proportions, new antiviral strategies that target molecularly delineated mechanisms used by this virus are urgently needed (72) . Is there a possibility that host cell helicases can be therapeutic targets for anti-HIV-1 chemotherapy? Implicit within this question is the concept that one could attack a host cell protein in order to treat an infecting pathogen. Although targeting a cellular protein involved in a viral pathway risks obvious cytotoxicity, this approach avoids the inherent problem posed by rapid HIV-1 mutation to all currently utilized chemotherapeutics targeted to virus-encoded proteins. We note that inhibition of cell-encoded enzymes in medical therapy is not an unprecedented strategy. Suppression of angiotensin-converting enzyme (ACE) is widely used to treat hypertension, congestive heart failure, myocardial infarction, endothelial dysfunction and renal disease (73) . Elsewhere, aromatase inhibitors have been used to treat hormone-dependent breast cancer (74) , and inhibitors of cellular secretory proteases are contemplated for Alzheimer's disease (75) . We recently inhibited the cellular polyprotein convertase, furin, at minimal toxicity to the cell in order to block HIV-1 replication (76). Thus, a priori exclusion of cellular helicase as an antiviral target is not warranted.\nGuarded optimism that small molecule helicase inhibitors can be developed against viruses arises from encouraging progress in non-retroviral systems. Unlike HIV-1, human herpesviruses physically encode helicases. The herpes simplex virus UL5 and UL9 genes are helicases in superfamily 1 and 2, respectively (77) . HSV UL5 together with UL8 and UL52 form a heterotrimeric helicase-primase complex responsible for unwinding duplex viral DNA at replication forks. Two recent studies provide proof-of-concept that the HSV helicase-primase can be targeted at low host cell toxicity by two new classes of drugs, amino-thiazolyphenylmolecules (78) and thiazole amide derivatives (79) . In addition, other studies suggest that the NS3 protein, a RNA helicase encoded by Hepatitis C virus and related West Nile virus and Japanese Encephalitis virus can be targeted to inhibit viral replication (80) (81) (82) . This conceptual break through in drug development is important because it indicates that target discrimination between different helicases by small molecule inhibitors is possible. Of relevance to HIV-1, a synthetic immunomodulator Murabutide was shown recently to suppress HIV-1 replication in macrophages and T cells. Murabutide was shown to inhibit the activity of RNA helicase RH116, blocking its positive transcriptional activity for HIV-1 gene expression (42) .\nIf one looks beyond the signature motifs conserved amongst helicases, then it becomes clear that the different proteins are widely divergent in their coding sequences. In principle, this suggests that individual helicases can be abrogated with specificity in a knowledge-directed manner.\nIn theory, a helicase can be attacked by (i) inhibition of NTPase activity through direct competition for NTP binding, (ii) inhibition of substrate binding through direct competition at active site, (iii) allosteric mechanisms to affect NTPbinding/NTP hydrolysis and/or polynucleotide binding, and (iv) inhibition of unwinding activity by steric hinderance of helicase translocation along the polynucleotide substrate (83, 84) . Because the NTP-binding and substrate-binding pockets may be sufficiently similar between various helicases, specificity of inhibition through these sites will likely be extremely difficult, although perhaps not impossible. On the other hand, the tremendous variations in sequence and sizes of helicases, in their oligomerization states, in their discrete domains responsible for protein-protein interactions and/or for targeting to specific nucleic acids (85) , and in their differential localizations within cells (86) offer interventional possibilities outside of the NTP-or polynucleotidebinding sites.\nWe are in the preliminary stages of screening ringexpanded nucleoside analogs found previously to be successful NTPase/helicase inhibitors of West Nile virus, Hepatitis C virus and Japanese encephalitis virus (81, 82) . We have observed that a few of these candidate inhibitors have substantial anti-HIV-1 activity at doses that do not incur cytotoxicity to cells treated in tissue culture for 1 week. Further studies are needed before concluding that these compounds exert specific inhibition of DDX3, one of the other cellular helicases, or some other target altogether.\nThere is another area where a cellular helicase activity and HIV-1 are likely to intersect. An emerging research focus is the role of small interfering RNAs (87) and microRNAs (miRNA) as innate cell defenses against viruses including HIV-1 (87) (88) (89) (90) . In human cells, the precursor for miRNA (pre-miRNA) is processed by DICER (Figure 2A) which is a ribonuclease with a bona fide RNA helicase domain (91, 92) . A surprising recent finding revealed that the human TRBP, which has been shown to be a potent binder of the HIV-1 TAR RNA RNA (40, 41) , is an indispensable dsRNA-binding partner of DICER which allows the latter to associate with pre-miRNA (91, 92) . Without TRBP, DICER's miRNA processing activity is lost. Thus an intriguing scenario can potentially unfold. Accordingly, whereas the ribonuclease-helicase protein DICER requires TRBP to process duplex-structured miRNAs in order that the cell can use such matured miRNAs for antiviral defense, it could be speculated that HIV-1 has evolved to restrict this defense by the ability to transcribe viral TAR RNA to squelch TRBP away from DICER (Y. Bennasser, M. L. Yeung and K. T. Jeang, manuscript submitted) ( Figure 2B ). If this thinking is correct, then HIV-1 has developed mechanisms not only to co-opt the active functions of a virus-propitious cellular helicase (i.e. DDX3) but also to inactivate the role of a second virus-pernicious helicase (i.e. DICER) for purposes of selfish gain.\nIn a separate perspective, virus infection can trigger through double-stranded viral RNAs an innate antiviral immune response. Thus viral dsRNAs can be recognized by cellular proteins [pattern-recognition receptors (PRRs)] which initiate antiviral responses by inducing the production of a variety of cytokines including type I interferons (IFN-a and IFN-b) and initiating additional inflammatory and adaptive immune responses. Recently, DExD/H RNA helicases such as RIG-1 (retinoic acid inducible gene-1) (93) and Mda5 (melanoma differentiation-associated gene 5) (94) have been identified as suppressors of viral replication by binding to virus associated dsRNA and activating type I interferon-dependent antiviral immunity. Over-expression of RIG-1 and Mda5 was found to enhance dsRNA induced type I interferon antiviral response. Currently, it remains speculative whether helicases like RIG-1 and Mda5 may recognize HIV-1 dsRNA and trigger an innate immune response. Intriguingly, several reports exist in the literature that HIV-1 infection does induce activation of type 1 interferons (95, 96) .\nIn conclusion, by studying helicase proteins one can gain insights into normal cellular metabolic processes, abnormal inherited human diseases (e.g. Bloom syndrome, Werner syndrome, Cockayne's syndrome and xeroderma pigmentosum; all diseases with mutations in cellular helicases), and remarkably also the biology of viruses." }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: Since the pioneering work of Smith and co-workers [1] [2] [3] , phage display technology has been widely used in both basic research such as the exploration of protein-protein interaction sites and networks [2] [3] [4] [5] , and applied research such as the development of new drugs, diagnostics, and vaccines [6] [7] [8] . Phage display has also become a promising epitope mapping method, which has been applied in many fields such as allergology [9] and oncology [10] . The phage display based epitope mapping is usually accomplished through comparing the sequence of mimotopes (antibody-selected phage displayed peptides) to the anti-gen. In some cases, the mimotope sequence is identical or very similar to a sequence in the antigen [2] , there by indicating the location of the native epitope. These cases are rare however, and usually the mimotope sequence has little, if any, similarity with the antigen sequence. Compared with traditional epitope mapping methods such as solving the crystal structure of the antigen-antibody complex or scanning overlapping peptides of the antigen, phage display based epitope mapping is generally much cheaper and less arduous.\nThough epitope mapping based on phage display can be done manually [11] , it is quite tedious and time-consuming to compare a set of mimotopes to the antigen without computational support. The low sequence similarity between the mimotope and the antigen often makes the mapping even harder. To solve these problems, several groups have researched algorithms and programs that assist and automate phage display based epitope mapping [12] [13] [14] [15] [16] [17] . According to their dependency on antigen structure, the existing programs for phage display based epitope mapping can be classified into three categories. Program in the first category such as FINDMAP, only work with sequence data from the mimotopes and antigen [13] . The second category needs both the sequence data and the antigen structure. SiteLight [12] , 3DEX [14] , and Mapitope [16, 17] belong to this category. A very recently published work: MIMOP [15] makes the third category, which integrates the two different approaches and can work with or without the antigen structure. Though implemented differently, all the existing programs have succeeded in given cases. However, most of the existing tools have not been implemented as a freely available online service until now, making it less convenient for the community to access, utilize, and evaluate them.\nIn the present study, we describe a web-based tool for phage display based epitope mapping named MIMOX. It was coded with Perl as a CGI program and can be used to align a set of mimotopes and derive a consensus sequence. The consensus sequence, or a single mimotope sequence, can then be mapped on to the antigen structure, and potential epitopes determined by spatial clustering of the mapped residues. The results mapped on to the antigen's 3D structure can then be viewed interactively. To validate this web-based tool, we compared the results from MIMOX with the results from other computational tools and experimentally identified native epitopes in several case studies.\nOverall architecture of MIMOX MIMOX was coded with Perl using modules from the Bioperl [18] project. The whole online service provided by MIMOX is accomplished through a set of CGI scripts. The MIMOX service can be divided into two main sections. In the first section, MIMOX provides a simple interface for ClustalW [19] to align a set of mimotope sequences; this is implemented as the script mimosa.pl. The alignment can then be used to derive the consensus sequence through a simple statistical method; this is implemented as the script mimocs.pl. The alignment can also be viewed and managed through an embedded Java applet version of JalView [20] ; this is implemented as the script jalviews.pl. In the second section, MIMOX tries to map the user supplied sequence on to the given antigen structure. This is implemented as the script mimox.pl. The program NAC-CESS [21] is also wrapped into mimox.pl and used to calculate the surface accessibility of the mapping results. All mapping results are ranked based on their solvent accessible surface. Each mapping result has detailed information of the accessibility of each candidate residue, which is parsed through the script parsa.pl and displayed as a table in a new window. Each mapping result can also be viewed interactively on the antigen structure. This is implemented as the script jmol.pl, which wraps a Java applet version of Jmol [22] . The overall architecture of MIMOX is shown schematically in Figure 1 .\nAs described above, MIMOX wraps ClustalW to align a set of mimotope sequences and then allows the alignment to be viewed, edited, and analyzed through an embedded version of JalView. Based on the review by Smith et al [3] , we also implemented a simple statistical method in the script mimocs.pl to derive a consensus sequence from the alignment. Firstly, the script counts the appearance of each kind of amino acid at each position in the alignment and calculates the percentage frequency of each one. The frequency of a given amino acid X at the position i of the alignment (f xi ) is defined as where Xi means the times that the given amino acid X appears at the position i of the alignment and N is the number of sequences in the alignment. All frequencies are compared to a threshold value, which is 25% by default. If a frequency is more than the threshold, the corresponding residue is considered as a motif residue at that position. If the sum frequency of similar residue at the same position is above the threshold, the similar residues are also regarded as motif residues. In MIMOX, there are five similar residue groups (L, I, V; T, S; E, D; Q, N; K, R; F, W); other residues are considered unique. This classification scheme is the same as that used by Mapitope [16, 17] . If no motif residue is found at a given position, then X is used to stand for any amino acid residue. Motif residues at all positions of the alignment are then displayed in a table.\nOverall architecture of MIMOX Figure 1 Overall architecture of MIMOX. MIMOX has two sections. The first section has 3 perl scripts. The script mimosa.pl aligns a set of mimotope sequences powered by ClustalW. The script jalviews.pl wraps JalView to view and manage the alignment. The script mimocs.pl derives a consensus sequence from the alignment. The second section also has 3 perl scripts. The script mimox.pl maps the user supplied sequence on to the given antigen structure and utilizes NACCESS to calculate the accessibility. The script parsa.pl displays the detailed accessibility information of each mapping result. The script jmol.pl wraps Jmol to view the mapping result interactively on the antigen structure. Thus a consensus sequence is suggested by the program. The script also creates a 3D bar figure based on the statistical analysis above, where the X axis represents the 20 amino acid types and gap type, Y axis is the frequency and Z axis stands for the position of the aligned sequences (Shown in Figure 2 ).\nSince the mimotopes and the native epitope on the antigen bind to the same antibody, it is assumed that the mimotopes and the native epitope have similar physicochemical properties and similar spatial organization. This assumption is the basis of the MIMOX algorithm. The mapping process of MIMOX is based on the input sequence (such as the consensus sequence) and the uploaded antigen structure. A fragment of the sequence can also be used as input.\nFirstly, for each position in the input sequence, MIMOX searches the uploaded PDB structure for matching residues and places them into an array of candidate residues for that position. Two matching modes are available at present. One is strict mode, which means the type of mimotope residue must match the antigen residue exactly. The other is called conservative mode, which means similar residues are also included in the candidate residue array. There are 5 groups of similar residues (L, I, V; T, S; E, D; Q, N; K, R; F, W) in MIMOX, which has been described in previous section.\nWeb interface of MIMOX section 1 Figure 2 Web interface of MIMOX section 1. Mimotopes selected out with trastuzumab [10] are input and aligned with wrapped Clus-talW. The frequency of a given amino acid at each position of the alignment is calculated and displayed in a table. A 3D bar figure is also created, where the X axis represents the 20 amino acid types and gap, Y axis is the frequency and Z axis stands for the position of the aligned sequences. A consensus sequence is then suggested, which can be used in further mapping with MIMOX. The alignment can also be managed with the embedded JalView [20] .\nThe array of candidate residues for each position is then added to an array of arrays. MIMOX finds all the residue neighbour pairs between consecutive candidate residue arrays in the array of arrays. Whether two residues are neighbours is determined by the distance between the two residues and the distance threshold value. If the distance between two residues is below the threshold, the two residues are taken as a neighbour pair. MIMOX provides three methods to calculate neighbour residue pairs. One method is to take the distance between the Cα atoms of the two amino acids as the distance between the two residues. Using Cα atoms may better reflect the backbone positions. The second method is to use the distance between the Cβ atoms, which may better reflect the side chain position (Cα atom is still used when it is a glycine because it does not have a Cβ atom). The third method described below, is based on the distances between all the heavy atoms of the two amino acids. All of the distances mentioned above are Euclidean distances, calculated as:\nwhere D 21 means the distance between atom 2 and atom 1 and x 2 , y 2 , z 2 , x 1 , y 1 , z 1 are coordinates of atom 2 and atom 1. When the methods based on Cα or Cβ atom position are used, the default distance threshold is 7.0 angstroms, as it approximates the upper limit for noncovalent interactions in macromolecular structures [23] . When the third method is used, the distance threshold is calculated as\nwhere DT is the distance threshold, DF is the Distance Factor (given by user), and vdwAtom is the Van der Waals radius of the atom. The default DF value is 1.11. If two residues have a pair of heavy atoms which are nearer than the distance threshold calculated from their Van der Waals radius, the two residues are taken as a neighbour pair.\nMIMOX then recursively links the neighbour pairs until all possible ways of forming the input sequence are made. Each result is then ranked according to the sum of the absolute residue accessibility of each residue calculated from the NACCESS result file. In the end, the results are displayed in a table with hyperlinks to call the script parsa.pl which can parse and display the accessibility data in detail, and the script jmol.pl to view the result interactively mapped onto the antigen structure.\nWeb interface of MIMOX MIMOX has successfully been implemented as an online service, which has a simple web interface both for input and output. As described previously, MIMOX can be divided into two sections; we show here the input and output of the two sections in Figure 2 and Figure 3 respectively.\nTo test MIMOX, we have applied it to several cases taken from other similar research and literature. We compared the results from MIMOX with the results from other computational tools and the native epitope itself if the epitope is known in the CED database [24] . It should be pointed out that cases using monoclonal antibodies are most appropriate for testing [15] . However, in order to compare with previously published tools, some less appropriate cases (using polyclonal antibodies) taken from the corresponding literature are also used. More case studies [see Additional file 1] can also be found on the test dataset page of MIMOX[25].\nThe first case is taken from FINDMAP [13] . In 1999, Jesaitis and co-workers used an anti-actin polyclonal antibody to select a phage displayed random peptide library; VPHPTWMR was one of the consensus sequences they derived from the selected mimotopes. They manually mapped VPHPTWMR to the known structure of actin [PDB: 1ATN] and suggested that it might correspond to residues: V129, P130, H101, P102, T358, W356, M355, R372 [11] . In 2003, Mumey et al used FINDMAP to align VPHPTWMR to the actin sequence without utilizing information on the antigen structure. The result from FIND-MAP shows VPHPTWMR can be mapped to residues as V129, P130, H101, P102, T103, W356, M355, R372 [13] . FINDMAP mapped the input sequence to a slightly different set of residues (using T103 instead of T358). When running MIMOX with all parameters as defaults, we got no result. However, after the distance threshold is changed to 12 Å (the maximum distance allowed in MIMOX), we find that the two mappings above are returned as candidate cluster 5 and candidate cluster 17. As the side chain of some amino acids (such as arginine) can span a distance as great as 12 Å, MIMOX takes this value as the maximum allowable distance. This distance restriction is also used by Mapitope [16, 17] . In this case, the need for the higher distance threshold is due to R372 which lies some distance from the other mapped residues. MIMOX also suggested other possibilities such as cluster 1(V96, P102, H101, P130, T358, W356, M355, R372) which has a bigger solvent accessible surface, and cluster 26 (V96, P98, H101, P102, T103, W356, M355, R372), which clearly has 3 sequential segments, i.e. VPHPT, WM, and R.\nThe second case is taken from work by Enshell-Seijffers [16] . They used monoclonal antibody 17b, which is against HIV gp120 envelope glycoprotein, to select a phage displayed random peptide library and got a set of Figure 4 . As the latter two mapping results suggested by MIMOX are more exposed, they might be able to bind to the antibody more easily.\nThe last case is taken from MIMOP [15] . BO2C11 is a human monoclonal antibody against human coagulation factor VIII. Villard et al selected two phage displayed random peptide libraries with BO2C11 and got a set of 27 mimotopes [26] . Very recently, Moreau et al have applied their newly developed tool MIMOP to analyze these mimotopes. Combining the two methods MimAlign and Mim-Cons in MIMOP, the BO2C11 epitope is predicted be composed of a segment YFTNMF (2195-2200) and residues T2202, K2207, R2215, R2220, Q2222. The structure of human coagulation factor VIII in complex with\nComparison of three mapping results. MIMOX was used to map LLTTNKD to HIV gp120 with three different methods. Taking together, our initial case studies show that MIMOX can fully or partially repeat results from manual mapping, other existing tools, and also provide novel suggestions. MIMOX is designed to be a tool which is more interactive than automatic. We acknowledge that tuning the probe sequences and parameters are often required to get good results. This interactive process gives hints to users step by step and greatly decreases the load of the server and prevents the loss of some reasonable results. MIMOX lists all the matched results with no prediction threshold. This allows users to find the reasonable results by themselves based on their background knowledge on a given antibody, a given antigen and a given phage display experiment. Nevertheless, according to the test dataset page of MIMOX, the true epitope (or its segments) often falls in the top 5 (if the there are only a few result entries) or top 10% (if the there are many result entries) of the results.\nWhere the real epitope is unknown, we would suggest running MIMOX with a range of parameters and consensus sequence derived fragments to find overlapping or otherwise promising (high surface accessibility) candidate.\nAs we have mentioned previously, several groups have researched algorithms and programs that may assist and automate phage display based epitope mapping. Based on the dependency on antigen structure, the existing programs can be classified into three categories. FINDMAP belongs to the first category, which is independent of any structural information. FINDMAP has been implemented as a C++ program. It aligns a probe (e.g. a consensus sequence derived from a set of mimotopes) to the sequence of native antigen, allowing any permutation of the probe sequence. It uses a two-part scoring system to evaluate the quality of alignments and a branch-andbound algorithm to find an alignment with maximum score [13] .\nThe programs in the second category include SiteLight, 3DEX, Mapitope, and MIMOX. SiteLight was implemented in C++ and it has been tested on Red Hat Linux. First, the program divides native protein surface into overlapping patches based on geodesic distances between residues; then aligns each mimotope in the library with each patch and scores and sorts them; finally, high scoring matches are selected iteratively until 25% of the native protein is covered [12] . Another program 3DEX was implemented in Visual Basic and could only run on Windows. It divides a sequence into a set of overlapping subsequences with a user-defined length (3-maximum length of mimotope). Then, it searches for matching residues at each position of the above subsequences against the sequence or PDB structure of native protein and links the neighbours iteratively until the first subsequence is complete. This is repeated for the following subsequences to complete the mimotope and return the result [14] . Mapitope was also implemented in C++ and its algorithm was first described by Enshell-Seijffers in 2003. Briefly, Mapitope deconvolutes a set of mimotope sequences into a set of overlapping amino acids pairs (AAP). Then a set of major statistically significant pairs (SSP) are identified based on the AAP. Later, the SSP are mapped and clustered in the antigen structure. Finally, the most elaborate and diverse clusters on the antigen surface are identified and regarded as the predicted epitope candidates [16, 17] . [15] . It seems that MIMOP can work with or without the antigen structure from the published case studies. However, the sequence of the only case that is independent of antigen structure is just a continuous subsequence of the antigen sequence. Thus, more studies are still needed to prove that MimAlign can work without antigen structure information.\nAll the existing programs described above have succeeded in given cases. However, a systematic evaluation on these tools is absent. Moreover, as shown in the Table 1 , most of the existing tools have not been implemented as a publicly accessible online service until now, making it less convenient for the community to access, utilize, and evaluate them.\nLike all software, bugs will have crept into MIMOX during the programming. We expect users will send their feedback to help us maintain and improve MIMOX in the future. A new version of MIMOX with more user definable options and supporting multiple-chain antigens will be implemented in the future, allowing epitopes formed by residues from different polypeptide chains to also be predicted. A systematic evaluation and comparison study with all available tools including MIMOX that assist phage display based epitope mapping is also under our consideration.\nMIMOX, a web application for phage display based epitope mapping has been coded with Perl. It is helpful for molecular biologists to identify the native epitope of an antibody based on the antigen structure and a set of mimotope sequences they get through phage display technology. As a publicly accessible web tool in this area, MIMOX is very convenient for the community to access, utilize, and evaluate, complementing other existing programs. " }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: In central nervous system (CNS) demyelinating diseases such as multiple sclerosis (MS), myelin damage impairs impulse conduction along denuded axons. Limited remyelination occurs in MS lesions (1Y3) but is typically insufficient to prevent long-term neurologic disability. After a demyelinating event, improved remyelination could maximize functional recovery of viable axons and prevent associated axonal damage and degeneration.\nIn rodent models, extensive spontaneous remyelination and functional recovery is possible after an episode of acute transient demyelination (4, 5) . Proliferation of immature cells and differentiation into myelinating oligodendrocytes is required for this extensive remyelination (6, 7) . Similarly, immature oligodendrocyte lineage cells persist in the adult human CNS and may proliferate to increase in number within and near MS lesions (8, 9) . However, these immature cells often fail to differentiate sufficiently to remyelinate throughout the extent of MS lesions. Therefore, the repair capacity of endogenous cells may be limited by nonpermissive signals in chronic MS lesions.\nGrowth factors, cytokines, and cell adhesion molecules may regulate oligodendrocyte progenitor (OP) differentiation into remyelinating oligodendrocytes in the environment of a demyelinated lesion. Among these potential signaling molecules, we have examined the in vivo role of endogenous fibroblast growth factor 2 (FGF2), which is upregulated during postnatal development and in acute demyelination (10Y 12). During remyelination after acute demyelination, FGF2 null mice exhibit enhanced oligodendrocyte repopulation of demyelinated lesions (12) . This improved oligodendrocyte regeneration was further studied using in vivo retroviral lineage analysis in wild-type versus FGF2 null mice, which demonstrated that the predominant effect of FGF2 in vivo during remyelination is inhibition of OP differentiation (11) .\nThe current study examines whether this FGF2 effect on OP differentiation and oligodendrocyte regeneration has a significant impact on remyelination. Acute demyelination models in rodents typically exhibit spontaneous remyelination that may be so robust as to prevent appropriate evaluation of strategies for improving remyelination. Therefore, the current study challenges the capacity of endogenous cells to regenerate oligodendrocytes and remyelinate chronically demyelinated lesions.\nWe induced active demyelination in mice by adding 0.2% cuprizone to the diet so that return to normal chow would allow analysis of spontaneous remyelination without ongoing disease pathogenesis. Cuprizone reproducibly demyelinates the corpus callosum of mice (13) . Spontaneous remyelination is greatly reduced after chronic cuprizone demyelination in contrast to acute demyelination (14 Y 16) . Importantly, after chronic cuprizone demyelination, axons remain viable and can be remyelinated by transplanted OP cells (16) . In this chronic demyelination model, the current study demonstrates dramatically improved remyelination by endogenous cells in mice with genetic deletion of FGF2 as compared with wild-type mice. Thus, FGF2 expression in chronic lesions may limit remyelination and attenuation of FGF2 may generate a sufficiently permissive environment for spontaneous remyelination by endogenous cells.\nMice were bred and maintained in the Uniformed Services University of the Health Sciences (USUHS) animal housing facility and all procedures were performed in accordance with guidelines of the National Institutes of Health, the Society for Neuroscience, and the USUHS Institutional Animal Care and Use Committee. FGF2 knockout mice on the 129 Sv-Ev:Black Swiss genetic background were obtained from breeding heterozygous pairs (provided by Dr. Doetschman, University of Cincinnati). This FGF2 knockout was generated by a targeted deletion replacing a 0.5-kb portion of the FGF2 gene, including 121 bp of the promoter and the entire first exon with an Hprt minigene (17) .\nCuprizone ingestion results in a reproducible pattern of extensive corpus callosum demyelination (12, 13) . Cuprizone treatment was started at 8 weeks of age and only male mice were used. Cuprizone (0.2% (w/w), finely powdered oxalic bis(cyclohexylidenehydrazide) (Sigma-Aldrich, St. Louis, MO), was thoroughly mixed into chow (diet TD.01453; Harlan Teklad, Madison, WI), which was available ad libitum. Mice were maintained on the cuprizone diet until perfused for analysis or returned to normal chow after 6 weeks or 12 weeks of cuprizone ingestion.\nMice were perfused with 4% paraformaldehyde and then brains were dissected before overnight postfixation in 4% paraformaldehyde (18) . Brain tissue was cryoprotected overnight at 4-C in 30% sucrose and frozen in OCT compound for immunostaining and in situ hybridization.\nIn situ hybridization and preparation of digoxigeninlabeled riboprobes were performed as previously detailed (18, 19) . Antisense riboprobes were used to detect mRNA transcripts for proteolipid protein (PLP; gift from Dr. Lynn Hudson; National Institutes of Health [20] ) and PDGF>R (gift from Dr. Bill Richardson; University College London [21] ). The digoxigenin-labeled riboprobes were hybridized to 15-Km-thick coronal brain sections. Digoxigenin was detected with an alkaline phosphatase-conjugated sheep antidigoxigenin antibody (Boehringer Mannheim, Indianapolis, IN) followed by reaction with NBT/BCIP substrate (DAKO, Carpinteria, CA).\nTo identify OPs in situ, 15-Km coronal brain sections were immunostained for NG2 and PDGF>R (12, 19) . Primary antibodies used were rabbit polyclonal antiNG2 antibody (gift from Dr. William Stallcup, La Jolla, CA) and rat monoclonal antiPDGF>R antibody (APA5; Pharmingen, San Diego, CA). Donkey antirabbit IgG F(ab') 2 conjugated with Cy3 (Jackson Immunoresearch, West Grove, PA) was used to detect NG2, whereas the PDGF>R was detected with biotinylated donkey antirat IgG F(ab') 2 (Jackson Immunoresearch) followed by coumarin tyramide amplification (New England Nuclear, Boston, MA).\nMyelin was immunostained with monoclonal antibody 8-18C5, which recognizes myelin oligodendrocyte glycoprotein (MOG). Hybridoma cells were provided by Dr. Minetta Gardinier, University of Iowa (22) . MOG immunolabeling was detected with donkey antimouse IgG F(ab') 2 conjugated with Cy3 (Jackson Immunoresearch).\nCell proliferation was estimated with immunostaining for Ki-67 antigen, which is expressed in the nuclei of actively dividing cells but absent at G 0 (23). Ki-67 was recognized with a rat monoclonal antibody to mouse Ki-67 antigen (DAKO) followed by detection with the ABC elite kit using 3,3-diaminobenzidine (DAB) as a substrate (Vector Labs, Burlingame, CA).\nImages of in situ hybridization and immunostaining results were captured with a Spot 2 CCD digital camera using Spot Advanced image acquisition software (Diagnostic Instruments, Sterling Heights, MI) on an Olympus IX-70 microscope. Images were prepared as panels using Adobe Photoshop (San Jose, CA).\nFor comparing cell densities, all quantification was performed by an investigator blinded to the treatment condition. Cells expressing PLP mRNA were quantified using unbiased stereologic morphometric analysis (12) (Stereologer System from Systems Planning and Analysis, Inc., Alexandria, VA). Analysis was restricted to the corpus callosum region from the midline and extending laterally to below the cingulum in 15-Km-thick coronal sections. Using the Stereologer System, the specimen thickness contributes to the sampled volume so that measurements reflect cells/mm 3 . The unbiased stereologic method could not be used appropriately for conditions with relatively few cells of interest in any chosen category. Therefore, quantification of cells in the corpus callosum expressing PDGF>R or Ki-67 required counting all labeled cells and using the Spot 2 CCD camera and software to measure the area sampled, resulting in density units of cells/mm 2 (12) .\nQuantification of corpus callosum myelination was estimated from MOG immunofluorescence detected with a Spot 2 CCD camera. Using Metamorph software, pixel intensity values were normalized between sections by thresholding to exclude values below the level of immunoreactivity in the dorsal fornix, which was selected as an adjacent white matter tract that is not demyelinated by cuprizone. The percent area of the corpus callosum (midline bilaterally to a point under the cingulum apex) with MOG immunoreactivity above the threshold level was then used as an estimate of the myelinated area.\nEach category analyzed included three or more tissue sections per mouse and three or more mice per condition, except where larger sample sizes are noted in text and/or figure legends. One-way analysis of variance (ANOVA) with post hoc Tukey's multiple comparison test was used to determine significant differences among stages of disease progression or treatment. Unpaired Student t-test was used to compare between FGF2 genotypes in nonlesioned mice. Significance of an FGF2 genotype effect across multiple treat-ment conditions was calculated using a two-way ANOVA. No statistical comparisons were made between mice with different genetic backgrounds (i.e. C57Bl/6 mice and FGF2 mice).\nChronic Cuprizone Demyelination Provided a Relevant Model of Insufficient Remyelination to Characterize the Repair Capacity of Endogenous Cells C57Bl/6 mice were used to establish parameters for analyzing, and later manipulating, the capacity of endogenous FIGURE 1. Spontaneous remyelination of the corpus callosum was compromised after chronic demyelination of C57Bl/6 mice. (A) Corpus callosum myelination was estimated by immunofluorescence for myelin oligodendrocyte glycoprotein (MOG). Pixel intensity values were normalized between tissue sections by thresholding to exclude values below the immunoreactivity in the dorsal fornix, which was not demyelinated by cuprizone (see DF in [C]). The percent area of the corpus callosum with MOG immunoreactivity above the threshold level was then used as an estimate of myelinated area. Bar colors indicate treatment conditions: no cuprizone (white), acute cuprizone (up to 6 weeks, gray), or chronic cuprizone (9 weeks and above, black). At least three sections were quantified per mouse and at least four mice per condition. After acute cuprizone, myelination returned to near nonlesioned values (p > 0.05; no cuprizone, n = 4; 6 weeks cuprizone 6 weeks off, n = 5). Recovery to nonlesioned levels did not occur after chronic cuprizone (p G 0.001; 12 weeks cuprizone 6 weeks off; n = 6). Values for 12-week cuprizone three off were not significantly different than either 12-week cuprizone or 12-week cuprizone 6 off. Significant differences, p G 0.05 or less, are noted by an asterisk (*) for comparisons with no cuprizone. oligodendrocyte lineage cells to repopulate and remyelinate chronically demyelinated lesions. We used the cuprizone model to take advantage of the ability to stop active demyelination simply by returning the mice to a normal diet. As characterized in C57Bl/6 mice, continuous cuprizone ingestion results in persistent demyelination with a variable degree of partial remyelination, indicating a regenerative potential of endogenous cells even while mice are still fed cuprizone (24) . However, after 12 weeks of continuous 0.2% cuprizone ingestion in C57Bl/6 mice, spontaneous remyelination remained limited through the 6-week recovery period (Fig. 1 ). This poor remyelination after chronic demyelination contrasted with the almost complete remyelination seen within the same recovery period after acute demyelination (i.e. 6 weeks on cuprizone followed by 6 weeks off cuprizone; Fig. 1 ).\nTo better evaluate the underlying causes of limited remyelination after chronic demyelination, we characterized the oligodendrocyte lineage cell responses relative to disease progression with a focus on the repair potential. Oligodendrocyte and OP populations were characterized during multiple stages of disease progression: nonlesioned (no cuprizone), acute demyelination (3Y5 weeks of continuous cuprizone), chronic demyelination (12 weeks of continuous cuprizone), and recovery after chronic demyelination (12 weeks of continuous cuprizone followed by normal chow for 3 or 6 weeks). In situ hybridization was used to identify oligodendrocytes expressing PLP mRNA transcripts (Fig. 2) and OP cells expressing FIGURE 2 . Oligodendroglial repopulation of the corpus callosum was compromised after chronic demyelination of C57Bl/6 mice. (A) Quantification of the density of oligodendrocytes in the corpus callosum of C57Bl/6 mice. In situ hybridization for PLP mRNA, which identifies premyelinating and myelinating oligodendrocytes, was quantified using unbiased stereology with at least three sections per mouse and at least four mice per condition. Bar colors indicate no cuprizone (white), acute cuprizone (gray), or chronic cuprizone (black). The oligodendrocyte density after 12 weeks of cuprizone followed by 6 weeks for recovery was still significantly below nonlesioned values (p > 0.05; n = 5 for both conditions). Significant differences, p G 0.05 or less, are noted by an asterisk (*) for comparisons with no cuprizone and with a carrot (^) for comparisons of recovery stages with 12 week cuprizone. platelet-derived growth factor > receptor (PDGF>R) mRNA transcripts (Fig. 3) .\nQuantitative analysis of the oligodendrocyte population revealed ongoing partial regeneration during and after chronic demyelination (Fig. 2) . The oligodendrocyte density was lowest after the initial 3 weeks of cuprizone in the acute phase. After 12 weeks of continuous cuprizone, the oligodendrocyte density continued to be severely reduced relative to values from nontreated mice. Ongoing partial regeneration during chronic demyelination was indicated by the fact that the oligodendrocyte density after 12 weeks was higher than after the initial 3 weeks. After ending the chronic cuprizone treatment, this oligodendrocyte regeneration improved during the recovery period. Interestingly, as shown in Figure 1 , remyelination did not progress similarly during this 6-week recovery period. The PLP mRNA in situ hybridization used to identify oligodendrocytes should detect both premyelinating and myelinating oligodendrocytes because PLP transcription precedes myelin formation (25) . These results indicate that after chronic demyelination, a proportion of premyelinating oligodendrocytes that are generated during the recovery period may fail to differentiate into myelinating oligodendrocytes.\nThe OP population dynamics were dramatically different in response to acute versus chronic demyelination (Fig. 3) . In response to the initial episode of acute demyelination, the OP population was amplified almost fourfold (Fig. 3) . However, by the end of the chronic demyelination period, the OP density was greatly reduced and remained low during the recovery period. In the chronic lesions, similar results were observed with OP identification using PDGF>R (Fig. 3A ) and using NG2 (12 weeks cuprizone = 155 cells/mm 2 , n = 5; 12 weeks cuprizone plus 3 weeks recovery Figure 1 ) of corpus callosum myelination estimated by immunostaining for myelin oligodendrocyte glycoprotein (MOG) in cuprizone-treated FGF2 j/j mice (A) and FGF2 +/+ mice (B). The corpus callosum showed persistent demyelination with 0.2% cuprizone ingestion throughout 9 weeks and 12 weeks. After 12 weeks of cuprizone treatment, on return to normal chow, remyelination in FGF2 j/j mice significantly improved (p G 0.001; 12 weeks cuprizone, n = 4; 12 weeks cuprizone 6 weeks off, n = 4) and recovered to nonlesioned levels (p > 0.05; no cuprizone, n = 5; 12 weeks cuprizone 6 weeks off). In contrast, values in FGF2 +/+ mice did not increase after chronic demyelination (p > 0.05; 12 weeks cuprizone, n = 4 vs. 12 weeks cuprizone 6 weeks off, n = 3) and remained significantly below nonlesioned levels (p G 0.001; no cuprizone, n = 5 vs. 12 weeks cup). = 169 cells/mm 2 , n = 6; 12 weeks cuprizone plus 6 weeks recovery = 115 cells/mm 2 , n = 5). At 12 weeks of cuprizone treatment, very few cells in the corpus callosum were identified as actively undergoing mitosis using DAPI counterstaining to reveal mitotic chromatin figures, but each actively dividing cell was also immunostained for NG2 (inset, Fig. 3A ). Immunostaining for Ki-67 nuclear antigen was used to more broadly identify actively cycling cells within the corpus callosum (Fig. 3B) . Ki-67-labeled cycling cells were present at less than half the density of OP cells. This cycling population, although still relatively small, was significantly increased within the corpus callosum of mice fed cuprizone for 12 weeks as compared with age-matched controls or with mice that had recovered for 6 weeks after chronic demyelination. Together, these findings demonstrated compromised but continued OP cycling and regeneration of oligodendrocytes during chronic demyelination and the subsequent recovery period (Figs. 2, 3 ).\nFGF2 expression is increased during acute cuprizone demyelination and can inhibit OP differentiation and oligodendrocyte regeneration during subsequent remyelination (11, 12) . Using in situ hybridization for FGF2 mRNA transcripts, we have shown that elevated FGF2 expression persists within chronic cuprizone lesions, as compared with nonlesioned corpus callosum (Fig. 4) . FGF2 mRNA transcript abundance related to changes in FGF2 protein detection in developing white matter and after acute demyelination (10, 19) . We predicted that in chronic lesions, FGF2 inhibition of differentiation could contribute to the limited capacity to generate remyelinating oligodendrocytes (Figs. 1, 2) , especially in the context of a reduce OP pool (Fig. 3A) .\nAlthough the endogenous populations did not efficiently remyelinate after chronic cuprizone demyelination in C57Bl/6 mice (Fig. 1) , transplantation of OP cells has shown that the axons remain viable for remyelination (16) . Therefore, we used this chronic demyelination model in FGF2 FIGURE 6 . Oligodendroglial repopulation of the corpus callosum after chronic demyelination was dramatically improved in FGF2 j/j mice as compared with FGF2 +/+ mice. (A) Quantification of the density of oligodendrocytes in the corpus callosum of FGF2 mice. In situ hybridization for PLP mRNA, which identified premyelinating and myelinating oligodendrocytes, was quantified using unbiased stereology with at least three sections per mouse and at least four mice per condition. The oligodendrocyte density after 12 weeks of continuous cuprizone was significantly below nonlesioned values (noted by asterisks [*]; p G 0.01 for FGF2 +/+ ; p G 0.05 for FGF2 j/j mice; n = 4 for each condition). After 12 weeks of cuprizone and a subsequent 3-and 6-week period for recovery on normal chow, oligodendrocyte densities in FGF2 j/j mice were no longer significantly different as compared with nonlesion values (p > 0.05 at 3 weeks and at 6 weeks; n = 4 for each condition). Comparison of the FGF2 +/+ values with the matching conditions in FGF2 j/j mice demonstrated a significant effect of genotype (p = 0.0196). knockout mice to test whether repair from the endogenous OP population might be improved by removing endogenous FGF2 from the lesion environment. The extent of demyelination after 12 weeks of cuprizone ingestion was similar in C57Bl/6 mice and mice of both FGF2 wild-type (FGF2 +/+ ) and null (FGF2 j/j ) genotypes (Figs. 1, 5) . The percentage of the corpus callosum area with myelin immunolabeling was 45.8 T 14.1% in C57Bl/6 mice, 50.05 T 7.8% in FGF2 +/+ mice, and 46.9 T 4.3% in FGF2 j/j mice. After the chronic demyelination, only in FGF2 j/j mice did myelin immunostaining in the corpus callosum significantly increase during the recovery period (Fig. 5) . Surprisingly, the myelin immunostaining recovered to approximately nonlesion levels by 6 weeks after removal of cuprizone from the diet of FGF2 j/j mice. In contrast, in C57Bl/6 mice (Fig. 1) or FGF2 +/+ mice (Fig. 5) , the myelinated proportion of corpus callosum area did not significantly increase during the recovery period.\nThis striking improvement in remyelination corresponded with a dramatic increase in oligodendrocyte repopulation of chronic lesions in FGF2 j/j mice, which was not found in FGF2 +/+ mice (Fig. 6) . Before cuprizone treatment, nonlesioned FGF2 +/+ and FGF2 j/j mice had a similar density of oligodendrocytes in the corpus callosum at 26 weeks of age, which corresponded with the longest cuprizone treatment and recovery protocol. In addition, the cuprizone-induced loss of oligodendrocytes in FGF2 +/+ and FGF2 j/j mice was similar at the end of the chronic cuprizone treatment. In conjunction with the quantitative myelin immunostaining analysis, these findings indicate that FGF2 +/+ and FGF2 j/j mice experienced a similar chronic disease severity. Importantly, during the 6-week recovery period after chronic demyelination, the oligodendrocyte density returned to nonlesioned values in the FGF2 j/j mice, but not in FGF2 +/+ mice. Analysis of an additional intermediate recovery period in FGF2 j/j mice confirmed this increased oligodendrocyte regeneration and demonstrated that this repopulation could occur within 3 weeks after removal of cuprizone from the diet.\nWe previously showed that the absence of FGF2 did not significantly alter OP amplification in response to acute cuprizone demyelination (12) . A robust OP proliferative response occurred after 5 weeks of cuprizone in FGF2 null and wild-type mice that was similar to the previously mentioned data for C57Bl/6 mice (Fig. 3) . However, the OP population was depleted during chronic demyelination in contrast to the amplified OP response to acute demyelination in C57Bl/6 ( Fig. 3) . Therefore, we examined the effect of FGF2 genotype on the density of OP cells and cycling cells in the corpus callosum after chronic demyelination. FGF2 genotype did not appear to alter the OP population dynamics of chronically demyelinated mice or age-matched nonlesioned mice (Fig. 7) . The density of OP cells, identified by in situ hybridization for PDGF>R mRNA, was similar in FGF2 +/+ mice and FGF2 j/j mice (Fig. 7A) . Somewhat unexpectedly, mice of both FGF2 null and wild-type genotypes, which are on a 129 Sv-Ev:Black Swiss genetic background, exhibited an increase in OP cell density after 12 weeks of cuprizone that was not observed in C57Bl/6 mice (Fig. 3A ). This strain difference in OP accumulation after chronic demyelination corresponded with fewer oligodendrocytes observed during the recovery period in FGF2 +/+ mice (Fig. 6 ) as compared with C57Bl/6 mice (Fig. 2) . Therefore, the FGF2 background appears to be even less favorable than the C57Bl/6 background for OP differentiation and oligodendrocyte regeneration, yet the detrimental effects of the chronic lesion environment can still be overcome in the FGF2 j/j mice.\nKi-67 immunostaining (Fig. 7C ) indicated that the density of cycling cells in the corpus callosum was less than 20% of the density of OP cells, indicating a relatively low level of ongoing proliferation. Cells with nuclear Ki-67 immunoreactivity were often observed as doublets, as is appropriate for confirming detection of a cycling population. The density of cells immunolabeled for Ki-67 nuclear antigen increased in chronically lesioned corpus callosum with a return to nonlesioned levels during the recovery phase. The density of endogenous cells that were proliferating in the corpus callosum of FGF2 j/j mice was similar to the values for FGF2 +/+ mice (Fig. 7B ) and for C57Bl/6 mice (Fig. 3) . The similarity of FGF2 j/j mice and FGF2 +/+ mice in our analysis of OP cells and cycling cells indicates that the dramatic differences observed in oligodendrocytes and remyelination may result from a permissive effect of FGF2 removal on OP differentiation into remyelinating oligodendrocytes.\nIn MS lesions, an initial episode of transient demyelination may be followed by spontaneous remyelination. However, remyelination typically fails with recurring or chronic myelin damage. The current studies show the advantages of the cuprizone model of chronic demyelination for focusing on this compromised repair response. Importantly, our analysis in FGF2 j/j mice demonstrates that it is possible to overcome chronic lesion effects on endogenous oligodendrocyte lineage cells to increase remyelination. More specifically, taken together with our previous work (11, 12) , our findings in FGF2 j/j mice indicate that removing FGF2 inhibition of OP differentiation promotes spontaneous remyelination from a depleted pool of endogenous progenitors that persists after chronic demyelination.\nThe current findings in C57Bl/6 mice are consistent with previous reports of cuprizone treatment causing extensive demyelination of the corpus callosum in this mouse strain (13, 16, 24, 26) . To take further advantage of this model for examining mechanisms to promote remyelination from endogenous cells, we have further characterized the endogenous oligodendrocyte lineage population responses during the recovery period. We used C57Bl/6 mice for this part of our study to facilitate comparison with other studies, because differences in mouse strain can influence the response to cuprizone as well as overall cellular responses to CNS injury (13, 27) . After a transient episode of demyelination from 6 weeks of cuprizone ingestion, spontaneous remyelination is effective throughout the corpus callosum. In contrast, after a prolonged period of demyelination, from 12 weeks of continuous cuprizone, the remyelination remains limited and does not progress during a recovery interval that was examined out to 6 weeks. This limited remyelination corresponds with depletion of the OP pool ( [16] current study). However, the number of oligodendrocytes increases somewhat during the recovery period without a corresponding increase in remyelination of the corpus callosum. Taken together, these findings indicate that the depleted OP pool has a limited capacity to generate oligodendrocytes, and available OP cells may fail to fully differentiate into remyelinating oligodendrocytes in the environment of a chronic lesion. In lesions of some MS cases, immature oligodendrocyte progenitors and premyelinating oligodendrocytes may be present in relatively normal densities without efficiently remyelinating (9, 28) . Therefore, further studies will be important for better understanding regulation of the OP pool size and the transition of OP cells into premyelinating and then myelinating oligodendrocytes.\nRobust OP proliferation in response to acute demyelination appears to be required for spontaneous remyelination (29) . During acute demyelination, the OP pool is dramatically amplified so that efficient differentiation into mature oligodendrocytes may not be required for extensive remyelination. PDGF-AA ligand, acting through PDGF>R activation, is an important mitogen for this OP amplification in response to acute cuprizone demyelination (11, 30) . In PDGF>R heterozygous mice, amplification of the OP pool in response to acute cuprizone demyelination is compromised and the corresponding generation of oligodendrocytes is reduced (11) . Crossing these PDGF>R heterozygous mice to FGF2 j/j mice actually increased oligodendroglial repopulation of lesions after acute cuprizone demyelination (11) . Therefore, in both acute and chronic lesion environments, FGF2 removal may promote the generation of oligodendrocytes from OP cells during remyelination.\nEndogenous FGF2 has been predicted to contribute to proliferation of neonatal and adult OPs, especially when present in combination with PDGF-AA, based on in vitro studies (31Y33). In our current analysis, the densities of OP cells and Ki-67-immunolabeled cells were similar between FGF2 wild-type and null genotypes. These results are consistent with our previous BrdU incorporation studies indicating that endogenous FGF2 is not a predominant mitogen for OPs in the acute cuprizone model (11) . However, subtle changes in the proliferation rate among an asymmetrically dividing OP pool could be relatively difficult to detect in a small population of cells over a prolonged disease and recovery course. FGF2 is also predicted to stimulate neural stem cells in the subventricular zone (SVZ) to contribute to repopulation of corpus callosum lesions (34) . During the prolonged demyelination period of the current study, the contribution of cells derived from the SVZ should have been evident in the dynamics of the OP pool in the corpus callosum. The lack of a detrimental effect of FGF2 absence on the OP pool may indicate that FGF2 effects on the SVZ cells may not be a significant contribution to the remyelination of the overlying corpus callosum. However, more direct analysis of the response of cells in the SVZ is required to make this determination. In addition, endogenous elevation of FGF2 in lesions may elicit specific effects that are not replicated with in vivo methods of elevating exogenous FGF2 levels (34Y37).\nFGF2 is among the most effective neuroprotective growth factors in diverse models of CNS injury, including stroke, trauma, excitotoxicity, and axotomy (38, 39) . In contrast to this expected protective effect of FGF2 on neurons, removal of endogenous FGF2 actually improved regenerative parameters in lesions that involved remyelination. Improved regeneration of oligodendrocytes was observed in FGF2 j/j mice after acute cuprizone demyelination of the corpus callosum and after murine hepatitis virus strain A59 demyelination of spinal cord ( [11, 12] current study). In an example from the peripheral nervous system, FGF2 knockout mice were used to prevent the normal upregulation of FGF2 and FGF receptor (FGFR) activation associated with Schwann cells and macrophages at a site of sciatic nerve crush injury (40) . Compared with wild-type mice, the FGF2 knockout mice exhibited improved myelination and increased axon diameter during regeneration from sciatic nerve crush (40) . These results indicate that a detrimental effect of endogenous FGF2 on remyelination may outweigh FGF2 protection from acute axon damage in these lesion models. Indeed, the improved remyelination in nerves of the FGF2 knockout mice may provide significant protection of axons from damage subsequent to the initial crush injury. Correlative support for a similar role of FGF2 in a chronic autoimmune model of MS can be found in a study of neural stem cell transplantation improved into mice with experimental allergic encephalomye-litis (EAE) (41) . The transplanted mice exhibited improved remyelination, with a major contribution from endogenous cells, and had less axonal loss in lesion areas. Neural stem cell transplantation was also associated with a significant decrease in FGF2 expression and reduced astroglial scar formation in the EAE lesions. FGF2 levels may modulate scar formation in demyelinated lesions because expression of FGF2 and FGFR1 corresponds with scarring, but not nonscarring, astroglial responses after other forms of CNS injury (42) .\nIt is not yet clear which FGFR type, or types, mediates the effect of endogenous FGF2 in the context of demyelination and remyelination. Differential effects of FGF2 at different stages of the oligodendrocyte lineage may occur through differential expression and activation of FGFR isoforms (43) . Oligodendrocyte lineage cell expression of high-affinity FGFRs and coreceptors varies with developmental stage and FGF2 exposure (44) . Furthermore, expression of multiple FGFR types is significantly increased in response to demyelination (19) . FGF2 may also have differential effects based on changing interactions with other signaling pathways that regulate oligodendrocyte lineage differentiation and myelination such as Notch1 (45, 46) and neuregulin (47, 48) . Interpreting a direct effect of FGF2 is complicated because neurons and glial cells express multiple FGFR types within and near demyelinated lesions (19) . Therefore, future studies will be required to identify the specific signaling components mediating endogenous FGF2 signaling in the in vivo context of demyelination and remyelination.\nIn MS lesions, the pathology is heterogeneous and the effect on the oligodendrocyte lineage population varies dramatically (49) . Oligodendrocyte density is severely compromised in demyelinated lesions in some patients with MS. Further studies are imperative to optimize regenerative responses from immature oligodendrocyte lineage cell populations that have been observed in some MS lesions (9, 28) . Targeting inhibitory signals that are upregulated in lesions should be a viable strategy for therapeutics to promote differentiation as needed near lesions. FGF2 expression has been reported in reactive astrocytes of acute and chronic MS plaques (50) . In addition, in cultures from adult human brain white matter, FGF2 inhibited the differentiation of preoligodendrocytes into oligodendrocytes (51) . Strategies to promote remyelination by attenuating FGF2 inhibition of OP differentiation may take advantage of reagents to modulate FGF2 signaling, which are currently being developed for angiogenesis and cancer treatments. In our in vivo analysis, absence of FGF2 was not detrimental in the normal adult or throughout the disease process. Therefore, inhibition of FGF2 signaling may be acceptable across demyelinating disease stages, which would improve treatment feasibility in diseases such as MS that have an unstable disease course. Treatments to promote remyelination should be a valuable complement to strategies that abrogate ongoing causes of demyelination such as immunomodulatory therapies." }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: For both surveillance and diagnostic applications, fine-scale pathogen identification and near-neighbor discrimination is important; therefore, an assay that monitors at this very specific level is desirable for many types of samples such as clinical and environmental (1) (2) (3) . To successfully use any method based on DNA or RNA detection, these assays must be coupled with large databases of nucleic acid sequence information for assay design to ensure that the desired information is provided and for the interpretation of raw data. Several well-established techniques use PCR to amplify individual target pieces of sequenced genomes to provide detection of organisms (4) . These methods can roughly be divided into approaches that target individual short sequence lengths or probes (<40 bp) and methods that examine longer probes. The advantage of using short probes is that when the uniqueness of the probe has been assured and unique primers are also selected, this method gives good specificity. This approach is capable of providing fine-scale identification of several genetically close organisms by selecting a sufficient number of probes. However, this can rapidly lead to a very large number of total probes being required to detect all organisms of interest. In addition these selected probes, which in the initial selection process were determined to be unique, are often later found to be less specific as more organisms are sequenced or are less specific under conditions that differ from the original conditions. This is particularly a problem for organisms belonging to a family with a high mutation rate and also for pathogens that have relatively few neighboring pathogens sequenced. In addition, PCR approaches focused on short unique probes are not capable of detecting the presence of new significant mutations nor can they easily resolve base sequence details. Approaches that use longer individual probes avoid many of these issues at the cost of being less specific. This issue means most of these approaches are not suitable for providing the information desired, providing impetus to this work.\nHigh-density resequencing microarrays produce variable length segments, 10 2 -10 5 bp, of direct sequence information. This target sequence falls in the longer target regime of PCR approaches but rather than being hybridized to a longer lessspecific probe on the microarray, many shorter specific probes are placed on the microarray to allow more detailed determinations from the entire PCR amplicon. This also means that the specificity of the primers used can be relaxed. They have been successfully used to detect single nucleotide polymorphism (SNP) and genetic variants from viral, bacterial and eukaryotic genomes (5) (6) (7) (8) (9) (10) (11) (12) . Their use for SNP detection has clearly established their ability to provide reliable quality sequence information. In most cases, the microarrays were designed to study a limited number of genetically similar target pathogens and for many cases, the detection methods relied only on recognizing hybridization patterns for identification (6, 9, 10, 13, 14) . Taking advantage of the sequential base resolution capability of resequencing microarrays that is required for SNP detection, resequencing has recently been successfully adapted recently using a different approach for organism identification of multiple bacterial and viral pathogens while allowing for fine detailed discrimination of closely related organisms and tracking mutations within the targeted pathogen (15) (16) . The new methodology differed from earlier work by using the resolved bases as the query of a similarity search of DNA databases to identify the most likely species and variants that match the base calls from the hybridization observed. The system was capable of testing for 26 pathogens simultaneously and could detect the presence of multiple pathogens. A software program, resequencing pathogen identifier (REPI), was used to simplify data analysis by performing similarity searches of a genetic database using basic local alignment search tool (BLAST) (17) . The REPI program used BLAST default settings and would only return sequences that might represent the hybridization if the expect value, a quantity calculated by the BLAST program that indicates the likelihood that the sequence match found would have occurred by random chance in the database, was <10 À9 . This screened out all cases that had insufficient signal; however, the final determination of what pathogen(s) was detected and to what degree discrimination was possible required manual examination of the returned results. This method successfully allowed fine discrimination of various adenoviruses and strain identifications of Flu A and B samples in agreement with conventional sampling results (15, 16) . Two important advantages of this approach were that the information was always recovered at the most detailed level possible and that it was capable of still recognizing organisms with recent mutations. This approach also maintained specificity well, as it was not dependent on the uniqueness of a few individual short probes.\nAlthough this analysis method has utility, there are several shortcomings: it is time consuming, not optimized to maximize sensitivity, has complicated results, is suitable only for an expert, and contains redundant or duplicate information. The process was time consuming because only the initial screening was handled automatically while the remaining steps required manual interpretation before the detection analysis was complete. Because a simple criterion (expect value cutoff of 10 À9 ) and non-optimized BLAST parameters were used to consider a pathogen detected, the REPI algorithm provided a list of candidate organisms but did not make a final simple conclusion or relate the results of one prototype sequence to another. Instead a manual process was used to make the final determination, but because the REPI program provided all similar results and the use of public nucleic acid databases containing redundant entries, a large amount of data was presented to a user that was not useful. In addition, with a manual process it was not possible to establish that the algorithm developed was generally applicable for any organism where nucleic acid base resolved sequence information has been provided.\nIn this paper, we describe a new software expert system, Computer-Implemented Biological Sequence Identifier system 2.0 (CIBSI 2.0), that successfully uses resolved base sequence information from custom designed Affymetrix resequencing microarrays to provide a simple list of organisms that are detected. This algorithm addresses the most important shortcoming of previous methods by incorporating new features to completely automate pathogen identification. We have demonstrated the effectiveness of this algorithm for identification via several examples. The single program is capable of making correct decisions for all 26 pathogens contained on the Respiratory Pathogen Microarray v.1 (RPM v.1), whether detected alone or in combinations, with improved sensitivity. Although the program is currently applied to resequencing microarrays, the methodologies developed remain generally applicable. Only the first portion of the algorithm handles issues specific to microarrays while the remainder deals with sequences that are suitable for use as a query by the BLAST algorithm. In developing the general identification algorithm, we have identified and resolved issues specific to resequencing microarrays that complicate their use. Because the entire decision process for what is detected has been automated, it is straightforward to test whether the rules used to make identifications are rigorous and applicable to any pathogen. With this efficient program, resequencing based assays can provide a competitive method to test simultaneously for many possible pathogens, providing output that can be interpreted by a nonexpert.\nThe details of the RPM v.1 design and the experimental methods have been discussed in previous work (15, 16, 18) (Lin et al., submitted for publication). Briefly, the RPM v.1 chip design includes 57 tiled regions allowing resequencing of 29.7 kb of sequences from 27 respiratory pathogens and biothreat agents. These were selected based upon clinical relevance for the population of immediate interest (United States military recruit in training) (19) (20) (21) . Partial sequences from the genes containing diagnostic regions were tiled for the detection of these pathogens. The experimental microarray data used in the present analysis were obtained using a variety of purified nucleic acid templates and clinical samples culture (throat swabs and nasal washes) using random and multiplexed RT-PCR amplification schemes (for more detail description of amplification methods see Supplementary Data). Resequencing microarrays provide base call resolution by comparing the intensities between a set of four 25mer probes that differ from each other at the same position (13th base). An amplicon or target sequence is represented by numerous overlapping probe sets. GCOSÔ software v1.3 (Affymetrix Inc., Santa Clara, CA) was used to align and scan hybridized microarrays to determine the intensity of each probe in every probe set. Base calls were made based on the intensity data of each probe set using GDAS v3.0.2.8 software (Affymetrix Inc.) which used an implementation of the ABACUS algorithm (5) . The sequences were represented in FASTA format for later analysis steps.\nIn this paper, target pathogens are the organisms the assay was specifically designed to detect. The sets of probes that represent reference sequence selected from target pathogen genomes are referred to as a Prototype Sequence or 'ProSeq' for brevity. The set of resolved bases that result from hybridization of genomic material to a ProSeq is referred to as the hybridized sequence or 'HybSeq'. The HybSeq is split into possible subsequences or 'SubSeqs'.\nThe CIBSI 2.0 program implemented in Perl described in this study handled a hierarchy of three tasks ( Figure 1 ): (I) Pro-Seq identification; (II) ProSeq grouping; and (III) pathogen determination. The most developed and important portion of the algorithm deals with ProSeq identification Task(I) and is handled in three important subtasks: initial filtering of individual HybSeqs into SubSeqs suitable for sequence similarity comparisons ( Figure 2 ), database querying of individual SubSeqs ( Figure 3 ) and taxonomic comparison of BLAST returns for each SubSeq (Figure 4 ). The NCBI BLAST and taxonomy databases were used for the queries and images were obtained on February 7, 2006. For the ProSeq grouping Task(II), ProSeqs were compared to determine if they supported the same identified organism. In the pathogen determination Task(III), detected organisms were compared to the list of target pathogens the assay was designed for in order to determine if any were positively detected or were possibly related close genetic near neighbors. The level of discrimination that a particular sample supported was automatically determined.\nAn initial filtering algorithm, REPI, was developed previously (16) and the general concepts with revisions were incorporated into the current (automated detection) algorithm used in the CIBSI 2.0 program. Filtering and subsequence selection were used to remove potential biasing caused by reference sequence choice and by other sources (i.e. primers). When PCR amplification was used, microarrays were hybridized in the presence of only primers to determine locations where they resulted in hybridization. Any portions of the ProSeqs that hybridized with the primers were masked as N calls so that the HybSeq did not contain biased information. Normally the primers are designed to be outside the Pro-Seq region to minimize the interference caused by primers, and so minimize the bases to be masked. There is still the chance that some bases require masking because with the large number of primers used in the multiplex, short stretches of a ProSeq not corresponding to primer locations may still hybridize with the primers. Such regions could be removed from the reference sequences and so not appear on the microarray. However, determining such locations are a difficult and time-consuming task that for most cases is not worth the effort. The first subtask of ProSeq identification Task(I) is noted in Figure 1 and shown schematically in detail in Figure 2 . This subtask uses a procedure to examine a Hyb-Seq to find the longest possible subsequence of base calls (SubSeq) that can be submitted as a query to BLAST.\nIt produces a group of SubSeq that contain all portions of a HybSeq that have a chance of producing a limited list of returns from a BLAST query. When a HybSeq has two regions separated by a long stretch of continuous N calls, the relational positioning of the two regions cannot be trusted and so must be sent as separate queries. In addition for shorter subsequences, the number of base calls that must be made is dependent on the length. It was also recognized that for very long sequences a longer WORD size in BLAST may be used. A detailed description of the criteria and process used for each step is contained in Supplementary Data. Upon completion, the algorithm returned to the Task(I) loop and performed the BLAST subtask.\nThe database query subtask performed a batch similarity search of a database using SubSeq as the queries. The BLAST program used was the NCBI Blastall -p blastn version 2.12 with a defined set of parameters. The masking of low complex regions was performed for the seeding phase to speed up the query; however, low complexity repeats were included in the actual scoring. The entire nucleotide database from NCBI acquired on February 7, 2006 was used as the reference database. (Note that earlier images of the database were used during development but all experiments were rerun with the algorithm as described with the Figure 3 . Detailed schematic representation of the second subtask of ProSeq identification Task(I), organism identification for an individual SubSeq. Each SubSeq sent to BLAST returned a list of possible matches contained in a Return array that was sorted through to find best bit score/expect value pair (MaxScore). If the MaxScore was greater than MIN (10 À6 ), all returns that had this best Score were sorted into a new array Rank1. Detailed SubSeqs requirement is described in Supplementary Data.\nimage of the database obtained on this date.) The default gap penalty and nucleotide match score were used. The nucleotide mismatch penalty, -q, parameter was set to À1 rather than the default. The results of any BLAST query with an expect value <0.0001 were returned in tabular format from the blastall program. The information about each return (bit score, expect value, mismatches, length of match) was placed in the Return{hash key}{info} hash using the SubSeq identity as the hash key for further analysis.\nThe next subtask of ProSeq identification Task(I) carried out was the determination of SubSeq() states and is shown in Figure 3 . The BLAST algorithm gives a ranking score which can be reported as accounting for the size of the database (expect value) or not (bit score). The full taxonomic classifications of every return for a SubSeq were retrieved from the NCBI taxonomy database obtained on February 7, 2006. Using the scores and taxonomy relationships it was possible to find a reduced number of returns that had the best match with the HybSeq. These results were summarized by identifying the taxonomic class to which all the returns belonged to, 'identified organism', and a parameter that indicated how they are related to each other, 'organism uniqueness'. A detailed description of the steps is contained in Supplementary Data. After each SubSeq was examined, the algorithm moved to the next subtask, which was to determine the identified organism of the ProSeq from the SubSeq (Figure 4 ). The subsequences from the same ProSeq were only allowed to support a single 'identified organism' determination. The procedure shown in Figure 4 demonstrates the decision method used to arrive at this determination (detailed description in Supplementary Data). After the subtask covered in Figure 4 was completed, the ProSeq identification Task(I) loop continued until all ProSeqs were examined. A list of ProSeqs that had detected organisms was built up in the Result1 array.\nAfter the ProSeq identification Task(I) was completed, ProSeq grouping Task(II) (Figure 1 ) was used to examine the identified organism values listed in Result1 and grouped them together if they identified the same taxonomic class. Each entry in Result1 was examined and a new entry was created in Result2 if the identified organism did not appear in this list. The entries of Result2 represented the distinct individual organisms identified, but might still contain redundant information. When the ProSeqs were designed to detect the same organism and they all hybridized well, this grouping led to a reduction in redundant information being reported. But, when one ProSeq did not hybridize as well for a variety of possible reasons, multiple entries would appear in Result2 that actually represent hybridization from the same pathogen. This is because there is an alternative cause for the ProSeq hybridizing in this manner. This hybridization could be caused by two different but closely related organisms both being present in a sample and hybridizing to the microarray. Because we have not yet developed methods to distinguish these cases, no further reduction of the list of organisms is made for in ProSeq grouping Task(II) in cases where the level of identification varied on different ProSeq targeted for the same organism.\nAlthough it was difficult to relate results from separate Pro-Seqs to each other, it was desired to have a simple final detection decision be made in pathogen determination Task(III). The first task was specifically implemented so that information about what a ProSeq was intended to detect was not considered and the second task only minimal consideration of this was taken into account. This allowed these initial tasks to be capable of recognizing not only just positive and negative identifications of target pathogens but also cases that were indeterminate. In the final task, the algorithm considered whether the identified organisms belonged to the list of organisms the ProSeqs were designed to detect. The task would group organisms from ProSeq grouping Task(II) together that belonged to or were child classes of the taxonomic class of a target organism. The taxonomic class reported was the common taxonomic group of all the organisms. When all the ProSeqs for a pathogen hybridized well, a fine level discrimination was reported. But if one or more ProSeqs hybridized less well, the reported positive target pathogen was only identified at the level of the less detailed level. This is conservative because methods have not yet been developed to clearly discriminate mixtures of very closely related organisms causing different ProSeqs to hybridize from variable hybridization of a single organism on several ProSeqs. The results of all When there was a single SubSeq or one scored much better than the other, the ProSeq inherited the properties of that SubSeq. Detailed SubSeqs requirement is described in Supplementary Data. three tasks were reported and a more experienced user can view ProSeq grouping Task(II) results to clarify some cases. Note that organisms identified in ProSeq grouping Task(II) that only belonged to target pathogens were reported as positives. Clear negative ProSeqs were not mentioned in the output. ProSeqs that were indeterminate or that detected close genetic organisms were never reported as positives. These organisms were instead reported as being detected.\nA resequencing microarray (RPM v.1) was designed previously for detection and sequence typing of 20 common respiratory and 6 CDC category A biothreat pathogens known to cause febrile respiratory illness based on ProSeqs without relying on predetermined hybridization patterns (15, 16, 18 \nPurified Chlamydia pneumoniae nucleic acid samples with 10-1000 genome copies (via method in Lin et al., submitted for publication) were chosen to illustrate how pathogen detection and identification were done when multiple ProSeqs were targeted for the same pathogen. RPM v.1 has three highly conserved ProSeqs selected from the genes encoding for the major outer membrane proteins VD2 and VD4, and the DNA-directed RNA polymerase (rpoB) gene. The HybSeqs from the different samples differed only in the number of unique base calls as shown in Table 1 . The percentage of the ProSeq called varied from 80 to 100% except for one case at a concentration of 10 that had only 11% of the rpoB ProSeq producing unique base calls. Because the samples at this concentration are not reproducibly generating the same percentage of base calls, this is probably the detection limit of this ProSeq of the assay. Table 1 listed the determinations made for the SubSeq and at the end of each task for the various samples. The ProSeq from the different cases produced the same number of SubSeqs. These SubSeqs from different samples reported different bit scores for the same top ranked returns from BLAST. In fact VD2 and VD4 produced exactly the same results. The NCBI taxonomy database classified the returns into four distinct groups, which represented the C.pneumoniae taxonomic group and three child strain groups. AE001652, AE002167, AE017159 and BA000008 appeared in the returns of all the ProSeqs for each sample, since they represented database entries of completely sequenced genomes. One rpoB Sub-Seq produced for its organism uniqueness, SeqUniqu. All other SubSeqs were TaxAmbig as multiple returns from different taxonomic classes were returned. Since the VD2 and VD4 ProSeq each have a single SubSeq, Task(I) assigned the Pro-Seq the state of the SubSeq. For the rpoB ProSeq, the bit score of one SubSeq was large enough that the algorithm assigned that SubSeq's identification to the ProSeq. Task II of the algorithm grouped all three ProSeqs together since they all had the same identified organism and TaxAmbig was assigned. The result of Task(III) was positive for target pathogen C.pneumoniae and this decision was straightforward as all the ProSeqs agreed with each other and belonged to the same target pathogen taxonomic class. Although the rpoB ProSeq was SeqUniqu, this was not the final conclusion for Task(II) as the ProSeq that was SeqUniqu was not the child taxonomic group and other ProSeq were TaxAmbig. The three recognized strains scored the same, which indicated that the sequence selected for the ProSeqs was very conserved and would not allow discrimination between the strains. Influenza and Human Adenovirus (HAdV) were the only pathogens that had ProSeq selected that would permit detailed strain level discrimination as discussed in previous work (15, 16) . This previous work using manual analysis found that the microarray results were in excellent agreement with the conventional sequencing results for clinical samples. A few of the results of running the CIBSI 2.0 program using the updated NCBI database on the raw microarray results are presented in Table 2 (the results for all samples used in the previous work are presented in Supplementary Table A) . The identified organisms were not identical to the original findings due to the difference in database used and because all ProSeqs were considered rather than only the Flu A and B hemagglutinin. In fact, the conventional sequencing results that were submitted to NCBI from that work were found for every sample to be among the returns with the best score for the hemagglutinin ProSeq (Supplementary Table B ). It should be noted that the previous work based its analysis upon only the results of the hemagglutinin ProSeq. For 8 of 13 Influenza A and 3 of 12 Influenza B cases, the results of ProSeq identification Task(I) and ProSeq grouping Task(II) found that the conventional sequencing was the single best return for the hemagglutinin ProSeq. Owing to the large number of isolate sequences in the database for the hemagglutinin gene it was not surprising that in some cases a single unique entry was not found. In each of the remaining five Influenza A samples, the other sequences returned differed by <0.2% from the conventional sequence. The fewer samples with unique isolate identifications for Influenza B were due to an older reference sequence used for the ProSeq, which allowed less Note: Within a row the first listing of a specific strain was followed by a two-letter abbreviation used in the remaining columns of that row. hybridization to occur (18) . This also meant that when multiple sequences were returned for a sample they represented greater genetic variation, up to 2%. As a result of the current method of making pathogen determination Task(III) level identification, the final organism reported was less specific (H3N2 or Flu B) for every sample than what was reported as possible in ProSeq grouping Task(II). For HAdV samples, the algorithm also reproduced the finer scale discriminations that had been made previously by manual methods (data not shown).\nThe next example of detection for the Mycoplasma pneumoniae pathogen demonstrated a case where there was only a single ProSeq for the target pathogen. A total of 48 test samples were performed using multiplex PCR (via method in Lin et al., submitted for publication) where for 46 of the samples M.pneumoniae organism was spiked into nasal wash with several other pathogens from 100 to 100 000 colony forming units per ml, the remaining 2 samples were purified with nucleic acid from culture stock at a concentration of 1000 genome copies per reaction volume. This ProSeq was also not optimal for fine discrimination because it was selected from a highly conserved region (345 bp) of the cytadhesin P1 gene. In every case taxonomic database entries for M.pneumoniae or its one recognized distinct strain tied for MaxScore (Supplementary Table 3 ). To better understand these returns, the database sequences were examined and subdivided into three groups of sequences, A, B and C, based on how well they matched the reference sequence used to make the ProSeq. The placement of the database entries into the three groups was determined from a CLUSTAL alignment of the sequences of this gene. This alignment confirmed that the database entries differed significantly more from each other in regions not represented by the ProSeq and contained sufficient variability that would have allowed finer discrimination. Members of Group A exactly matched the ProSeq and could not be distinguished between on the microarray. Similarly, members of group B matched the Pro-Seq except at the 199th position where the base called was C rather than T. Group C sequences contained a few database entries that were more variable and might be distinguished from other entries within the ProSeq. For the 48 experimental tests of M.pneumoniae, as much as 80% of the ProSeq hybridized for 19 samples, yet only 5 of these samples had an unambiguous base call at the 199th position. When it was unambiguous, it always matched group B sequences. In the cases where an N base call was made at the 199th location, both groups A and B sequences were returned with the same score. Regardless of this, the target pathogen positively identified was M.pneumoniae for every sample tested.\nThese examples showed how decisions were made independent of whether single or multiple ProSeqs were dedicated to a target pathogen. They also illustrated that the level of discrimination possible was strongly determined by the quality of the selected ProSeq. It is possible that for some pathogens fine level discrimination is not required and the currently tested selections on RPM v.1 would provide satisfactory information. The CIBSI 2.0 algorithm demonstrated its capability to automatically report the maximum level of discrimination that could be supported by the HybSeq information.\nTo demonstrate how the algorithm handled closely related genetic species, a sample of a non-targeted pathogen was considered using multiplex PCR (via method in Lin et al., submitted for publication). For Variola major virus, one of the biothreat pathogens on the RPMv.1, the validation runs demonstrated that Variola major virus purified DNA templates of plasmids were always positively identified when detected (Table 3) . Table 4 shows the results when purified Vaccinia genomic DNA was spiked into nasal washes and processed at various concentrations using multiplex PCR. The array has two ProSeqs from hemagglutinin (VMVHA, $500 bp) and cytokine response modifier B (VMVcrmB, $300 bp) genes for Variola major virus detection. The percentage of the ProSeq that hybridizes is sufficient that if hybridization patterns were only considered one might assume that this tile is identifying the presence of its target. This would indicate that reference sequence selected was not the best choice. However, when our algorithm was applied none of the samples is in fact identified as Variola major or minor virus. Vaccinia was always one of the Orthopoxvirus species listed with the highest scores for VMVcrmB ProSeq, but in only seven cases was it uniquely identified as the probable species detected. In only one sample at the lowest concentration and fraction of VMVcrmB hybridizing, did this ProSeq even identify Variola major and minor virus as one among the Orthopoxvirus species that could be the cause of the hybridization. The lower limit of detection for the amplification method used was between this concentration and the one above it for Variola major itself. Table 2 showed that HybSeqs split into multiple SubSeqs were capable of very specific identification.\nTo illustrate the other filtering performed, when multiplex strategies rather than generic were used for amplification Figure 5 also contains the raw and mask filtered results of this region for the A/Puerto Rico/8/34 sample. It was necessary to perform additional filtering to remove potential biasing caused by the specific primers as described in the methods. In the case shown in Figure 5 , a sequence of 18 bases present in the raw result are made N after filtering since they are in a location that interacts with the primers. If these base calls were included in the subsequences constructed, even though the HybSeq would still be split into the same number of SubSeq, the query for the ProSeq would favor an incorrect strain.\nThe algorithm we have developed successfully provided pathogen identification to the maximum level of detail possible (species or strain) depending on the quality of each Pro-Seq. This identification capability requires minimal input on the identity of the pathogens, making non-expert use feasible. The crucial feature incorporated that allowed complete automation was the use of taxonomic databases, which classify organisms into ordered groups and provide relationships between organism entries, allowing removal of redundancies, comparison of different related prototype sequences and simplification of data presentation. This allows databases, i.e. NCBI, that are redundant and subject to minimal curation but which constantly receive updated and new sequence information to be used with great success. Although we have demonstrated this using only the NCBI databases, other databases or custom made ones could have easily been used, which might improve performance. The algorithm is capable of providing accurate identifications at all analysis levels for pathogens that are less variable or are represented by highly conserved ProSeqs. For more variable or rapidly mutating pathogens, e.g. Influenza A virus, ProSeq identification Task(I) and ProSeq grouping Task(II) still provided accurate detailed identifications, but the pathogen determination Task(III) was unable to report fine scale discrimination. The comparison of the conventionally sequenced Influenza virus gene sequences illustrated that the algorithm is capable of automatically adjusting for updates in databases. The algorithm demonstrated its capability to properly distinguish hybridizations on a ProSeq caused by the specified pathogen from those caused by genetically close (near neighbor) strains and did not make incorrect identifications, eliminating one potential cause of false positives. Filtering the raw hybridization results served to reduce the computation time, accounted for potential primer interference and more importantly reduced potential biasing. This simple integrated algorithm provided sufficient and accurate identification, so that immediate use of the RPM v.1 or similar resequencing arrays and assay is possible. Although not discussed in this paper, the algorithm has successfully detected the presence of simulated multi-infections (Lin et al., submitted for publication). The algorithm as currently developed will detect mixtures when the organisms have sufficient variation; however, detection of a mixture of an organism and its mutation strain in a sample is uncertain in its present phase. In principle it may be possible to detect such mixtures as the resequencing microarray can detect and sequence diploid organisms.\nBesides demonstrating the success of the CIBSI 2.0 program, the work involved in developing the algorithm allowed insight into the importance of proper ProSeq selection. The RPM v.1 was the first resequencing array designed specifically for multiple pathogen detection using database similarity searching and served as a prototype for this application. We have demonstrated that a single ProSeq with as few as 100 bp, when designed correctly, can be sufficient to unambiguously identify an organism. However, it is clearly indicated that several longer ProSeqs provide better confirmation and more detailed information of a pathogen. Although the algorithm provides accuracy equivalent to manual analysis for determinations of individual ProSeqs, the current algorithm is only partially successful in integrating information from multiple ProSeqs. The emphasis of the design to this point has been on capabilities that are generally applicable to any pathogen. We are incorporating these insights in our newer more comprehensive resequencing array designs. Improving on level of detail reported in pathogen determination Task(III) will require more information about an individual pathogen and may have to be developed for each specific pathogen or class of pathogens. This information is also required for the algorithm to identify which differences between a sample and database entries represent significant mutations. Future work will involve improving the use of the current taxonomic database or potentially developing a new relational database that is specific to our needs and then incorporating more specific information of target pathogens. The hierarchal design of the data analysis makes it easy to incorporate analysis that build upon the analysis already performed.\nWe have met with some success in the current version but want to have increased automated discrimination. We have a well-defined path to completing this aim. The use of properly designed resequencing microarrays and this automated detection algorithm provides a way forward to developing assays that can test for multiple organisms simultaneously while providing fine strain level discrimination giving access to information about detailed strain recognition, antibiotic resistance markers and pathogenicity. This is a capability that other approaches cannot currently provide. In addition, since the design of the original 30 kb RPM microarray, the possible sequence content of the current array has increased 10-fold to 300 kb and further increases in array density are still attainable. This, coupled with our identification algorithms, will allow the analysis of partial sequence information from even more organisms for applications such as differential diagnostics for illnesses with multiple potential causes (i.e. febrile respiratory illness), tracking of emergent pathogens, distinction of biological threats from harmless near genetic neighbors in surveillance applications and for tracking the impact of coinfections or super infections. The concept of categorizing and reporting different degrees of identification depending on the quality of samples and set of target sequences is not limited to resequencing microarrays but is more generally applicable to any platform that is capable of returning sequence level calls that can be used to query a reference DNA database. As the trend for assays that test for multiple pathogens increases, automated analysis tools, such as this one, become more crucial for rapid identification in simple formats useful to the non-expert on a day to day basis. The remaining hurdle to using resequencing microarrays as a routine assay method is now clearly the sample processing methods. Further automating these steps is an important area of future research and development.\nThe program can be obtained free of charge for research purposes by contacting the authors." }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: Ditchfield, R., Hehre " }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: Nucleic acid assays utilizing cleavage enzymes to generate a fluorescent signal from dual-dye-labeled oligonucleotide probes have been extensively used in diagnostics (1) (2) (3) . In these assays, the ability of fluorescent dyes to transfer energy absorbed from light to nearby molecules forms the basis of homogeneous nucleic acid based assays (1, 4, 5) . In the unhybridized state the fluorophore and quencher are within close proximity, which allows the quencher to absorb energy from the fluorophore to affect quenching. The use of fluorogenic probes in a 5 0 -nuclease polymerase assay, where the probe is enzymatically cleaved to release the fluorophore (2) , is well known. The signal amplification reaction utilizes invasive cleavage with structure-specific 5 0 -nucleases (6) . The method requires annealing of two oligonucleotides, called the upstream oligonucleotide and the probe, to a target sequence, which results in the formation of a unique substrate for the 5 0 -nuclease. In the DNAzyme-PCR strategy the primer contains a target-specific sequence and harbors the antisense sequence of a 10-23 DNAzyme. During amplification, amplicons are produced containing active sense copies of DNAzymes that cleave a reporter substrate included in the reaction mixture (7) . A target nucleic acid sequence can be amplified exponentially in vitro under isothermal conditions by using three enzymatic activities essential to retroviral replication: reverse transcriptase, RNase H (RNA cleavage) and a DNA-dependent RNA polymerase (8) . Cycling probe technology represents a simple method for the detection of DNA target sequences, utilizing a chimeric DNA-RNA-DNA probe which is cleaved by RNase H when hybridized with its complementary target (9) .\nApurinic/apyrimidinic (AP) sites in DNA arise via spontaneous or mutagen-induced hydrolysis of the N-glycosylic bond, or through the repair activity of DNA glycosylases (10) . If left unrepaired, AP sites are potentially lethal or mutagenic (11). To cope with the deleterious consequences of AP sites, organisms possess AP endonucleases that initiate the repair of these DNA lesions (10) . Class II AP endonucleases initiate repair by catalyzing the hydrolysis of the 5 0 -phosphodiester of an abasic site to generate a 3 0 -OH group and a 5 0 -abasic phosphate residue (10) . The substrate specificity of human apurinic endonuclease on synthetic substrate analogs has been reported previously (12) .\nHere we describe a novel endonuclease IV (Endo IV) assay substrate ( Figure 1C ) that mimics the abasic lesions that normally occur in double-stranded DNA ( Figure 1A and B). The first lesion ( Figure 1A ) is a typical abasic (apurinic or apyrimidinic site generated by spontaneous or enzymatic loss of a nucleic acid base. The second lesion ( Figure 1B) is an atypical abasic site appearing as a result of inherent instability of the 3 0 -phosphodiester bond in abasic deoxyribose in lesion 1 or its cleavage by a Class I AP endonuclease. As shown by the arrow, AP endonucleases cleave the phosphodiester linkage at the abasic sites. In the novel Endo IV assay a short probe with a fluorophore linked through a phosphate at the 3 0 end and a short enhancer oligonucleotide generate an artificial, lesion 2 type Endo IV substrate. This arrangement allows the specific and efficient cleavage of the phosphodiester bond of the probe by Endo IV and release of fluorescent dye ( Figure 1C ). The addition of a quencher at the 5 0 end of the probe allows quenching of the fluorescence in the uncleaved probe. The specific cleavage of the phosphate bond and the generation of fluorescence is the basis of the new assay.\nIn this study we used endonuclease IV from Escherichia coli (Endo IV). The enzyme functions as a Class II AP endonuclease and as a 3 0 terminal phosphodiesterase (13, 14) . Endo IV is enzymatically similar to exonuclease III, the major AP endonuclease of E.coli, but it differs from the latter enzyme mainly in lacking the associated exonuclease activity (14) . It was also found that E.coli Endo IV is fairly heat stable to $70 C (15, 16) .\nFull characterization for all the compounds described below can be found in the Supplementary Data.\nA total of 102 unrelated Centre Etude Polymorphism Humaine DNA samples were obtained from the Coriell Institute of Medical Research (http://locus.umdnj.edu/) after specifying that the DNA samples should be used for research purpose only. The list of templates used is available at http:// snp500cancer.nci.nih.gov.\nFor the synthesis of the fluorophore-modified controlled pore glass(CPG) supports, please see the Supplementary Data.\nFluorogenic probes were synthesized on fluorophore-linkerbased CPG supports (Scheme 1) using standard 3 0 -DNAphosphoramidites. An Eclipse Dark Quencher was introduced at the 5 0 end using the corresponding phosphoramidite (www. glenresearch.com). Probes were purified by reverse-phase high-performance liquid chromatography (HPLC), dried and re-dissolved in 1· TE buffer. A nearest-neighbor model was applied to calculate extinction coefficient (e 260 ) of oligonucleotides (17) . A 260 measurements were made in PBS (pH 7.2) at ambient temperature and assumed to be a random coil DNA structure in solution. For each Eclipse Quencher, fluorophore 1 (FL1), fluorophore 2 (FL2) or fluorophore 3 (FL3) substitution an e 260 correction of +6600, +18 500, or +25 100 or 28 600 M À1 cm À1 was used, respectively.\nPreparation of labeled probes with aminohexanoylextended hydroxyprolinol linker (FI 4, Table 1) 3 0 -Hydroxyprolinol modified probe precursor was synthesized using a modified CPG support (18) , and 3 0 -DNAphosphoramidites and Eclipse Quencher phosphoramidite on a 0.2 mmol scale. The probe was purified by reversephase HPLC using a gradient of acetonitrile in 0.1 M triethylammonium bicarbonate buffer (pH 8-9) followed by drying in a SpeedVac evaporator. The probe precursor was redissolved in 50 ml of dry DMSO and treated with 10 mmol of pentafluorophenyl N-FMOC-6-aminohexanoate (19) for 10 h. The FMOC-aminohexanoyl modified probe was isolated by reverse-phase HPLC and deprotected by treatment with concentrated ammonium hydroxide for 1 h at room temperature. The deprotected oligonucleotide was further purified by reverse-phase HPLC using the triethylammonium bicarbonate buffer as described above, dried and re-dissolved in 50 ml of dry DMSO. To this solution, 10 mmol compound 8c (Scheme 2) and 1 ml triethylamine were added. After being kept at room temperature for 10 h the reaction was combined with 1 ml of 2% solution of NaClO 4 in acetone. The precipitated crude conjugate was further purified by reverse-phase HPLC, dried and reconstituted in 1· TE buffer. An additional molar absorbance (e 260 ) of 28 500 M À1 cm À1 was used in the probe concentration calculation to correct for the presence of the dye.\nThe synthesis of special CPG supports for the preparation of required oligonucleotide probes are shown in Schemes 1-3. Methyl 3-(3-chloro-2,4-dihydroxyphenyl) propanoate (4a) (Scheme 1) was synthesized starting from 3-chloro-2,4dimethoxybenzaldehyde (1) (20) , which was converted to substituted cinnamic acid 2 by Knoevenagel condensation. Intermediate 2 was hydrogenated in the presence of Pd/C to yield substituted phenyl propionic acid 3, which was first demethylated with HBr/HOAc and then converted to the methyl ester 4a.\nSynthesis of the pentafluorophenyl ester dye intermediates 11 required to synthesize the dye-modified CPG supports 12 is shown in Scheme 2. Compounds 4a and b were reacted with substituted phthalic anhydrides in the presence of AlCl 3 to give the benzophenones 5, which were reacted with the resorcinol analogs 6 (a-c) in trifluoroacetic acid/ methanesulfonic acid to yield the carboxyethyl substituted dyes 7. Treatment of compounds 7 with trifluoroacetic anhydride generated lactones 8, which were first reacted with the DMT-protected hydroxyprolinol 9 (21) and then with trimethylacetic anhydride to afford the protected dyes 10. Reaction of intermediates 10 first with succinic anhydride and then with pentafluorophenyl trifluoroacetate, yielded the PFP esters 11.\nThe reaction of the PFP ester 11 with the long chain aminoalkyl CPG (LCAA-CPG) yielded the desired CPG supports 12 as shown in Scheme 1.\nEndo IV cleavage activity was measured in real-time as shown in Figure 2 . The model system in Figure 3A was used to determine optimum E.coli Endo IV (Trevigen, Gaithersburg, MD) concentration, magnesium concentration and pH; the latter two were determined to be at 20 mM Tris buffer, pH 8. 6 \nThe endo IV reaction was prepared by adding 5 ml Endo IV Master Mixture to 5 ml PCR mixture. Reaction was incubated at 50 C for 50-60 min. Fluorescent detection was performed on ABI 7700 or 7900 in the two channels typically used to detect FAM (for FL1) and VIC (for FL2, FL3 and FL4) utilizing the ABI software for Allelic Discrimination. Alternatively, the fluorescence could also be detected in a Figure 3B and D, respectively. The reaction mixture contained Endo IV at 0.04 U/ml concentration, probe and enhancer at 150 nM, and the target at 5 nM. The experiment was performed on LightCycler. LightCycler Ò LC24 Real-Time PCR System. All assays unless otherwise indicated were performed at least in triplicate.\nThermodynamic parameters of duplex formation were derived by the van't Hoff analysis methods. The shape of each melting curve was fitted to the two-state model with linear base lines (22) using a nonlinear least-square program (23) . Unless otherwise stated, T m of DNA duplexes were measured at a concentration of 5 · 10 À7 M in 1· PCR buffer containing 10 mM Tris-HCl (pH 8.5), 5 mM MgCl 2 and 40 mM NaCl, as described earlier (23) .\nThe MGB Eclipse Ò Design Software 3.0 (http://www. nanogen.com/products/customassays/mgb_ecliplse/) was used to design the primers for PCR. It was also used to calculate the T m of the enhancer and probe.\nAn example of the new Endo IV-based assay is shown in Figure 2 . A probe and an enhancer are hybridized to a complementary synthetic target with a one base gap between them.\nThe signal is substantially diminished in the absence of the enhancer. A rigid hydroxyprolinol linker ( Figure 3B ) between the oligonucleotide and fluorophore FL1, critical for sensitivity and specificity, was used in this experiment. The results in Figure 2 illustrate the importance of all components used in the assay. The cleavage of the fluorophore is highly target dependent and its rate is amplified in the presence of the enhancer oligonucleotide. Very low non-target-dependent cleavage is an important feature of the Endo IV assay.\nEffects of pH and cations on E.coli Endo IV activity was determined using the probe, enhancer and target ( Figure 3A) . It was shown that Endo IV cleavage of the probe has a relatively flat pH optimum in the range between pH 8.5 and 9.5. For compatibility with post-PCR Endo IV amplification the assay buffer with pH 8.6 was chosen, similar to that of the PCR master mixture. Inclusion of the divalent magnesium cation in the buffer ranging from 0 to 20 mM, resulted in more than 2-fold increase in cleavage rate, the optimum range was $5-8 mM. A concentration of 5 mM was chosen for the Endo IV assay. It was determined that the monovalent cations, such as Li, Na, K and Rb, inhibit Endo IV cleavage activity for the concentrations studied in the range from 0 to 100 mM. An inhibition of $30% was observed at 20 mM monovalent cation concentration, which increased to $60-70% at 100 mM concentration. The optimum probe concentration was determined to be $600 nM, please see Supplementary Figures 1-3 . Figure 3A shows a model Endo IV assay that requires a target complementary to an enhancer and a probe labeled at the 5 0 end with an Eclipse Dark Quencher and a fluorescent dye at the 3 0 end. As shown, the probe (calculated T m ¼ 46.7 C) and enhancer (calculated T m ¼ 50.2 C) are separated by one base to mimic a natural abasic site. Four probes with different fluorescent dyes and linkers ( Figure 3B ) were evaluated for their performance in the new assay. Fluorophores FL1, FL2 and FL3 of the probes I, II and III (with similar quantum yields) contained a hydroxyprolinol linker between the 3 0 -phosphate of the probe sequence and the core of the dyes. Fluorophore FL4 of probe IV was analogous to FL3 of probe III, with an additional aminocaproic spacer introduced between the hydroxylprolinol moiety and the dye; the purpose of this spacer was to distance the fluorophore from the cleavage site.\nThe probe specificity was evaluated by comparing the cleavage rates in the presence and absence of target. Targetdependent (specific) and target-independent (non-specific) cleavage rates of the probe were measured as the percent target cleaved per minute. The results are summarized in Table 1 . Probes I-III demonstrated similar specific cleavage. A 2-to 3-fold decrease in the rate of non-specific cleavage for probes II and III versus probe I was observed. These low, non-specific cleavage rates translate into high-specific to non-specific cleavage ratios. It appears that chloro-or chloro/ methyl substitutions in the fluorophores of probes II and III, respectively, are beneficial for the reduction of non-specific cleavage. One possible explanation for this reduction may be an effect of increased hydrophobicity of FL2 and FL3 on enzyme activity. In the case of probe IV, with the extended hydroxyprolinol linker, the rate of specific cleavage almost doubled. However, the corresponding increase in the rate of non-specific cleavage was $20 times higher than that observed in probe III, substantially compromising the ratio of specific to non-specific cleavage rates. Once again, this result implies the existence of inhibiting interactions between Endo IV enzymatic activity and fluorophore which can be adjusted by moving the fluorophore away from the active center.\nThe rigid hydroxyprolinol linker with low non-specific cleavage rates utilized in probes I-III was identified as the linker of choice in the Endo IV assays.\nThe use of a flexible, straight chain linker (C6) instead of hydroxyprolinol-based linker increased the rates of nonspecific cleavage to even higher degree than the extended hydroxyprolinol linker and reduced the specific to nonspecific ratios to an unacceptable level (data not shown).\nIt appears that both the structure of the sterically constrained hydroxyprolinol linker and proximity of the bulky dye moiety to the cleavage site moderate cleavability of the 3 0 -terminal phosphodiester bond and practically eliminate non-template-dependent cleavage. Figure 4A shows the target, probe and the enhancer aligned with the target to indicate different gaps. The T m of the enhancer is typically chosen to be at least 5 C higher than that of the probe. The cleavage rates in the presence of enhancer are shown with 1-5 base gaps ( Figure 4B ). As expected, based on the natural abasic substrate requirement shown in Figure 1B , a one base gap gave the highest cleavage rate. The probe in the absence of enhancer showed a cleavage Table 1 . Effects of dyes and linkers on the rates and ratios of specific and non-specific cleavage Probe nos Dye Substitution on Figure 3B Cleavage rate Specific/non-specific rate ratios Target-specific %/min Target-non-specific %/min rate nearly equivalent to that of a 4 base gap in the presence of enhancer. As shown in Figure 4C , the maximum cleavage rate is achieved at a concentration of enhancer greater than $80 nM. In practice, the concentration of the enhancer is typically at least equivalent or larger than that of the probe. Some cleavage is observed with no enhancer present.\nI FL1 R 1 -R 7 ¼ H; n ¼ 0 0.97 0.0031 313 II FL2 R 1 ¼ R 3 ¼ R 4 ¼ R 5 ¼ R 6 ¼ R 7 ¼ Cl; R 2 ¼ H; n ¼ 0 0.93 0.0012 775 III FL3 R 1 ¼ R 3 ¼ R 5 ¼ R 6 ¼ Cl; R 4 ¼ R 7 ¼ H; R 2 ¼ CH 3 ; n ¼ 0 1.04 0.0010 1040 IV FL4 R 1 ¼ R 3 ¼ R 5 ¼ R 6 ¼ Cl; R 4 ¼ R 7 ¼ H; R 2 ¼ CH 3 ; n ¼ 1 1.90 0.0260 73 - - - - - - - - - - - - - - - - -4 - - - - - - - - - - - - - - - -2 - - - - - - -3 - -5 3'-GCTGAGCCGGGAACGGGCGGTGTAACCGTGGCTGACACTGA -5' |||||||||| 5'-Q-CTCGGCCCTT Dye\nProbe T m optimization Figure 5 shows how the cleavage rate of probes with different T m s changes with temperature. Probe lengths ranging from a 6mer to a 14mer were investigated and are shown in Figure 5 with calculated T m s of between 14 and 60 C. All the probes showed a bell-shaped relationship for cleavage rate with temperature. As expected, the cleavage rate for all probes increased with increased temperature. A relatively sharp cleavage rate optimum was observed for the probes a, b and c with calculated T m s of 60, 48 and 42, respectively. It appears that the best probe cleavage occurs close to the calculated T m or slightly above it. Performing the assay at a temperature slightly higher than the T m ( Figure 5 ) showed not only optimum activity but also appears to allow probe cleavage cycling, an important feature of this assay.\nThe ability of the Endo IV assay to detect different target concentrations is shown in Figure 6 . It was observed that in the case of 125 nM target concentration, the probe-based fluorescence plateaued in $1 h, while in the presence of 25 nM target a fluorescence plateau was reached in $3 h. The limit of detection of this assay is 0.04 nM, clearly distinguishable from the stable background ( Figure 6 ). This stable background, a characteristic feature of the assay, is maintained over a period of 15 h. This property is also observed in the assay with other nucleic acid targets.\nThe ability of the Endo IV assay to discriminate mismatches at different positions of a 14mer probe is illustrated in Figure 7 . Mismatches were introduced in the probe one at a time, from positions 1 to 8. The Endo IV assay shows excellent specificity for the different mismatches from base 1 to base 6 with large match/mismatch signal ratios. As shown in Figure 7 , the mismatch signal is often about the same as that of the NTC or slightly higher. The exceptions are the difficult 4-G/T-and 8-T/G-mismatches (24) where match/mismatch ratios of $16 and 7 were observed, respectively. These ratios are quite adequate to differentiate matched and mismatched sequences. It appears (data not shown) that the discrimination in positions 1 and 2 is largely determined by substrate requirements while discrimination in other positions is determined by thermodynamic considerations. Experiments with a large number of assays have also indicated that, with 10mer to 12mer probes, satisfactory discrimination is observed with the mismatch position in all except the last three bases at the 5 0 end.\nThe exquisite specificity of the Endo IV assay appears to be the result of at least two factors. The first is the strict \nRelative Flourescence @ 60°C intrinsic substrate specificity requirement of the Endo IV enzyme which appears to require the rigid prolinol linker ( Figure 3B , n ¼ 0) used in the probes I-III. Second, the use of short probes (10mer to 12mer) gives the Endo IV a distinct advantage over most other assays where probes are typically much longer (1,2,6,25).\nThe application of the Endo IV cleavage as a post-PCR detection system is shown in Figures 8 and 9 . Following an asymmetric PCR using primers to produce predominantly single-stranded amplicon, two allele-specific probes, an enhancer and Endo IV enzyme are added to the amplification mixture and incubated isothermally for about an hour. The cleaved fluorescence from the two allele-specific probes, each labeled with a different fluorescent dye, is then plotted in a scatter diagram as shown in Figures 8 and 9 for the genotyping analysis of two single nucleotide polymorphisms (SNPs) which are of immediate importance to in cancer (http:// snp500cancer.nci.nih.gov). The scatter diagrams of two polymorphisms, namely agouti signal molecular epidemiology studies protein (ASIP-01) and adenomatous polyposis coli (APC-03) (http://snp500cancer.nci.nih.gov) are shown in Figures 8 and 9 , respectively. In both cases, a probe specific to the wild-type allele is labeled with FL1 and a probe specific for the mutant allele labeled with FL3 is used. The probe sets, designed to analyze ASIP-01 and APC-03 polymorphisms, were used to assay 102 unrelated human DNA samples obtained from the Coriell Institute. In the case of the ASIP-01 target the mismatch was situated two bases from the 3 0 end of the probe, while in the case of APC-03, the mismatch was six bases from the 3 0 end of the probe.\nFrom our experience, the spacing of the allele clusters in Figures 8 and 9 are comparable or better than those scatter plots observed in other methods (2, 26, 27) . The exquisite specificity achieved with the Endo IV enzyme, combined with the shorter probes used and the high signal accumulation, gives this assay an advantage over other SNP assays. In comparison with other signal accumulating assays (2), the Endo IV assay has similar levels of high signal accumulation, but uses shorter probes with higher specificity than those assays. In the case of hybridization-based assays (5, 25) , the Endo IV assay has not only a signal accumulation advantage, but also uses shorter probes with a specificity advantage. In addition, the shorter probes allow much more flexibility in assay design compared to other methods with longer probes.\nWe disclosed a novel nucleic acid signal detection system based on the cleavage of a phosphodiester linkage in a dualdye-labeled oligonucleotide probe by the Endo IV enzyme from E.coli. The parameters influencing the activity of the Endo IV enzyme were optimized. This optimized Endo IV detection assay requires, in addition to an oligonucleotide probe substrate, a fluorophore attached through a relatively rigid linker to the oligonucleotide and a helper oligonucleotide. The rigid linker conveys the exquisite specificity of Endo IV cleavage of the phosphodiester linkage in the probe and shows high-specific/non-specific rate ratios. The synthesis of fluorophore analogs coupled to a rigid linker and solid support, used in the synthesis of labeled oligonucleotides, was also disclosed. Excellent mismatch discrimination is observed from positions 1 to 6 in a 12mer probe. The ability to put the mismatch in any one of positions 1-6, allows for flexibility in probe design for genotyping assays where sequence and secondary structural constraints are considered. This method allows the use of probes shorter than those typically used in other methods (28) contributing to the improved mismatch discrimination.\nThe combination of the Endo IV assay with a PCR as a post-amplification detection system has been demonstrated. Post-PCR Endo IV genotyping shows excellent spacing of scatter plot endpoint signals of no template controls, wildtype, mutant and heterozygous samples allowing clear allele differentiation. In contrast, other endpoint genotyping methods may suffer from overlapping endpoint signals due to PCR efficiency issues (25, 29) . This Endo IV post-PCR genotyping has been successfully used in an industry setting for >5000 genotype assays." }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: In this paper, we present an ethical framework for pandemic influenza planning. The ethical framework was developed with expertise from clinical, organisational and public health ethics and validated through a stakeholder engagement process. The ethical framework includes both substantive and procedural elements for ethical pandemic influenza planning. The incorporation of ethics into pandemic planning can be helped by senior hospital administrators sponsoring its use, by having stakeholders vet the framework, and by designing or identifying decision review processes. We discuss the merits and limits of an applied ethical framework for hospital decision-making, as well as the robustness of the framework.\nThe need for reflection on the ethical issues raised by the spectre of a pandemic influenza outbreak is great. Our efforts to address the normative aspects of pandemic planning in hospitals have generated interest from other hospitals and from the governmental sector. The framework will require re-evaluation and refinement and we hope that this paper will generate feedback on how to make it even more robust.\nAs the world prepares for the emergence of a pandemic strain of influenza, trans-national, national and local organisations and agencies are designing plans to manage community outbreaks. In addition, the medical commu-nity is identifying scientific research priorities and needs related to the anticipated pandemic [1] [2] [3] [4] [5] . There is also a need to examine the ethical issues that arise from planning for a public health crisis of this magnitude. Who should get the limited supply of antivirals? Are health care workers duty-bound to care for the ill in a pandemic when they may have competing familial obligations? Who will be prioritized for scarce ventilated hospital beds? When should hospitals cancel elective surgeries or restrict hospital visitation? To date, the bioethics community has been slow to respond to public health issues in general [6, 7] , and pandemic influenza planning in particular [8, 9] . In this paper we discuss the need for ethics in pandemic influenza planning and discuss the ethical framework we developed to guide pandemic planning in hospitals.\nIn the only article we could find that has an in-depth analysis of the ethics of pandemic planning, Kotalik offers an ethical analysis of the pandemic plans of three countries. His arguments are primarily about the ethics of pandemic planning efforts, as opposed to the ethics in pandemic planning. For example, he argues persuasively that it is problematic that all three countries' plans accept particular conditions of resource scarcity as planning assumptions [10] . While Kotalik has raised important issues about the ethics of pandemic planning in his article, our ethical framework focuses specifically on providing guidance to decision-makers about ethical issues in pandemic planning. This includes providing guidance on how to design an ethical process for decision-making, and providing guiding ethical values for the consideration of substantive issues.\nThe framework here proposed is an example of practical ethics that attempts to provide decision-makers with an introduction to and articulation of generally accepted ethical principles or values. The significance of this ethical framework is a) in the unique collaborative approach taken to its development that involved ethicists with different areas of expertise and a variety of health care stakeholders, and b) that it fills an important need in pandemic planning for an ethical framework to guide decision-making that has been unmet in most pandemic planning processes world wide.\nOne of the characteristics of a public health crisis is that health needs overwhelm available human and material resources. Difficult decisions must be made about how, where and to whom resources should be allocated. Medical science provides valuable information to help make these decisions. However, science alone is insufficient. Now consider that resource allocation decisions are just one kind of decision decision-makers face in preparing for, and getting through an influenza pandemic [9] . As a few scholars have begun to point out, pandemic planning needs to take ethical considerations seriously, and not allow the urgency of logistical and scientific needs to sideline a discussion of ethical considerations [10, 11] .\nKotalik argues that as \"every discourse about health care has not only a scientific but also a moral dimension, [pandemic influenza] plans also presuppose certain ethical values, principles, norms, interests and preferences\" [10] . It is important to make these presuppositions explicit, because, as the SARS experience in Toronto taught health care organisations, the costs of not addressing the ethical concerns are severe: loss of public trust, low hospital staff morale, confusion about roles and responsibilities, stigmatization of vulnerable communities, and misinformation [12] [13] [14] . Another key insight from SARS that we overlook at our peril was that in times of crisis, \"where guidance is incomplete, consequences uncertain, and information constantly changing, where hour-by-hour decisions involve life and death, fairness is more important, rather than less [emphasis added]\" [14] . As we shall argue, fairness considerations are both procedurally and substantively important: there is a need for fair decision-making processes, as well as equitable distributions of scarce human and material resources.\nTake the example of triaging ventilated beds in an ICU. In theory, decision-makers rely on scientific evidence to determine how best to maximise benefit in the allocation of ventilated beds, but science cannot tell us whether or not the initial decision to maximise benefit is just. Because the notion of maximising benefit is derived from a reflection on values, ethical analysis is required to determine why a utilitarian approach to triage though maximisation of benefit is preferable to the assignment of ventilated beds on a different basis, for example that of greatest need. Even if the utilitarian maximisation of benefit is thought to be ethically sound, how to implement a system based on this criterion is not ethically straightforward, and requires ethical reflection about what counts as good stewardship, and about the moral obligation to demonstrate transparency, accountability, fairness and trustworthiness in the allocation of scarce resources.\nThe importance of ethics to pandemic planning is in the \"the application of value judgements to science\" [15] , especially as they are embedded in planning assumptions, and within the practice of medicine itself. For example, while ethics might have little to contribute to understanding the mechanism of influenza virus transmission, it can make a significant contribution to debates such as what levels of harm the public are prepared to accept, how the burdens of negative outcomes should be distributed across the population and whether or not more resources should be invested in stockpiling antiviral medications.\nThe use of ethical frameworks to guide decision-making may help to mitigate some of the unintended and unavoidable collateral damage from an influenza pandemic. As Kotalik argues, the incorporation of ethics into pan-demic plans can help to make them \"instruments for building mutual trust and solidarity at such time that will likely present a major challenge to our societies\" [10] . Using ethical frameworks to help guide decisions can offer greater assurance that the values instantiated within them, such as accountability, transparency and trust, will be carefully thought about in decision-making and when reviewing decisions with stakeholders.\nOne of the key lessons from the Toronto SARS experience was that health care institutions and their staff could benefit from the development of ethical frameworks for decision-making [12] . The intention of this section is not to systematically derivate from normative theory the values and principles in the framework. This paper has a more narrow focus -it is an example of applied/practical ethics that attempts to introduce and articulate values that are already commonly accepted. It is not our intention to comprehensively defend the values in the framework, but rather to show from which areas of scholarship they were drawn, articulate their relevance to pandemic planning, and to demonstrate their discursive legitimacy through a process of stakeholder engagement and vetting. To our knowledge, no other pandemic planning process has attempted to a) develop an ethical framework to guide pandemic influenza planning and b) assess an ethical framework's robustness and resonance in the community of its intended users. Thus, the significance of the procedural elements of the development of the framework is not to be minimized, nor are the insights we have gleaned from implementing the framework in health care organisations and in a governmental setting.\nBuilding on key lessons from SARS [12] [13] [14] and the \"emergency ethics\" literature and drawing on our expertise in clinical, organisational, and public health ethics, we identified key ethical processes and values that are relevant for health care organisations. These values were presented to and vetted by a variety of health care stakeholders. Thus, this framework is the product of an iterative and inclusive process.\nIn Ontario the need for guidance on the ethical issues pertaining to an influenza pandemic has been widely acknowledged. As word of our work on an ethical framework for Sunnybrook and Women's College Health Science Centre (S & W) became known, we were invited to join other hospitals' pandemic planning efforts. There was also broader sectoral interest in ethics, and we were invited to join the Ontario Ministry of Health and Long Term Care's (MOHLTC) efforts to design a pandemic plan.\nOur working group was formed in response to the pandemic planning initiative that took place at S & W in early 2005. The hospital's Clinical Ethics Centre was invited to provide ethics support in this planning initiative. It soon became apparent that the scope of the issues went beyond the purview of clinical ethics to include organisational and public health ethics. Expertise in organisational and public health ethics was quickly procured through the University of Toronto Joint Centre for Bioethics which is a partnership between the University and sixteen affiliated healthcare organizations that includes S & W among its partners. S&W was subsequently de-amalgamated into Sunnybrook Health Sciences Centre and Women's College Hospital, thus the ethical framework is currently being implemented at Sunnybrook HSC.\nAs the framework took shape, we were invited to join the MOHLTC planning efforts. We began to work with the Vaccine and Antiviral working group at the MOHLTC, and we adapted our work to meet the related but distinct challenges facing government. While our work with the MOHLTC began with the Vaccine and Antiviral working group, the ethical framework we developed for the MOHLTC was eventually included in the Ontario Health Pandemic Influenza Plan [16] not as an annex to the section on vaccines and antivirals as we had originally anticipated, but as an ethical framework for the plan as a whole.\nExpertise in clinical ethics was important to the development of this framework because of the knowledge, skills and experience clinical ethicists need to address dilemmas or challenges found in the daily clinical arena. An obvious challenge was how to integrate expertise in public health ethics into a framework designed to guide decision-making in clinical health care settings. A related challenge was to thoughtfully integrate generally accepted principles and values from clinical ethics with those in public health ethics. In order to meet this challenge, the authors turned not only to the respective ethics literature, but also to the SARS experiences of Toronto hospitals and health care providers. A review of the SARS literature, and that of public health ethics more generally, guided the integration of the public health and the clinical ethics perspectives [6, 9, 10, [12] [13] [14] [17] [18] [19] . The Toronto experience with SARS demonstrated that organisations faced unique ethical challenges when dealing with a public health crisis, and much of the ethics literature identified a need for greater forethought in how organisations can foster ethical decision-making in times of crisis [12] [13] [14] . We reasoned that the legitimacy of this framework would be enhanced by including insights from the analysis of a recent public health crisis like SARS.\nNot surprisingly, the literature on clinical ethics has little to say about disaster preparedness and how to make decisions about such things as triage under extraordinary circumstances. The ethics literature on bioterrorism and battle-field triage informed our thinking and called our attention to important issues such as the duty to care, reciprocity, equity and good stewardship [20] [21] [22] [23] [24] [25] . The importance of having ethically robust criteria and policies developed in advance of a pandemic influenza outbreak is underscored in this literature, for \"critical decisions like these should not be made on an individual case-by-case basis\" and \"physicians should never be placed in a position of individually deciding to deny treatment to patients without the guidance of policy or protocol\" [22] . Robust disaster preparedness requires practising preventive ethics.\nThe ethical framework was vetted through S & W's Pandemic Planning Committee, the Joint Centre for Bioethics' Clinical Ethics Group (comprised of the affiliated health care organizations' clinical ethicists), the MOHLTC Vaccine and Antiviral Working Group, and the MOHLTC pandemic planning committee. Through this process, we refined the framework and we are grateful to these groups for their valuable insights.\nThe ethical framework is intended to inform decisionmaking, not replace it. It is intended to encourage reflection on important values, discussion and review of ethical concerns arising from a public health crisis. It is intended also as a means to improve accountability for decisionmaking and may require revision as feedback and circumstances require.\nThe framework is divided into two distinct parts, and begins with the premise that planning decisions for a pandemic influenza outbreak ought to be 1) guided by ethical decision-making processes and 2) informed by ethical values. Ethical processes can help to improve accountability and it is hoped that, to the extent that it is possible for ethical processes to produce ethical outcomes, the substantive ethical quality of decisions will be enhanced. Recognising, however, that ethical processes do not guarantee ethical outcomes, we have identified ten key ethical values to guide decision-making that address the substantive ethical dimensions of decision-making in this context.\nIn planning for and throughout a pandemic influenza crisis, difficult decisions will be made that are fraught with ethical challenges. Our framework around ethical proc-esses is based upon the \"accountability for reasonableness\" model developed by Daniels & Sabin [26] and adapted by Gibson, Martin & Singer [27] . This model provides a useful means of identifying the key elements of ethical decision-making processes. An extensive literature has developed around Daniels' and Sabin's accountability for reasonableness framework. The Daniels and Sabin framework has broad applicability across institutional settings and priority setting situations [28] [29] [30] [31] [32] [33] [34] [35] . Because the Daniels and Sabin framework applies deliberative theories of democratic justice to the specific problem of health care priority setting, and because it is unique in this regard, we felt it promoted the kind of deliberative approach to pandemic planning that this ethical framework is intended to support. Table 1 outlines the characteristics of an ethical decision-making process. Stakeholders will be more able to accept difficult decisions during a pandemic influenza crisis if the decisionmaking process has, and is perceived to have, ethical legitimacy.\nThe second part of the framework identifies ten key ethical values that should inform the pandemic influenza planning process and decision-making during an outbreak. These values are intended to provide guidance, and it is important to consider that more than one value may be relevant to a situation. Indeed, the hallmark of a challenging ethical decision is that one or more value(s) are in tension and that there is no clear answer about which one to privilege in making the decision. When values are in tension with one another, the importance of having ethical decision-making processes is reinforced (see above.)\nThe values identified in our ethical framework were based initially on previous research findings on ethics and SARS at the University of Toronto Joint Centre for Bioethics (JCB). This work was funded by a Canadian Institutes of Health Research grant in 2004 through 2006 and has led to several key publications on the ethical dimensions of SARS [14, [36] [37] [38] [39] . In particular, Singer et. al., in their seminal British Medical Journal article begin to identify key ethical values that were of relevance during the SARS epidemic in Toronto. These values were then further articulated by our working group and adapted for the pandemic influenza planning context. Through a discursive process of stakeholder consultation with public health specialists, ministry officials, S & W's pandemic influenza committee, and the Clinical Ethics Group at the JCB, we augmented the values to include two new values (stewardship and trust [40, 41] ) and refined the definitions of each value in light of the anticipated demands of a pandemic influenza crisis compared to a hospital-based epidemic such as SARS. The substantive values identified and articulated in the framework are not intended to be an exhaustive set, and they may underdetermine how best to achieve the overall goals of pandemic planning, which generally include the minimization of morbidity, mortality, and societal disruption. Nevertheless, this is not to say that that a procedural engagement about the overall goals of a pandemic response would not benefit from using the ethical framework to guide and shape debate. A description of the values that should guide decision-making can be found in Table 2 .\nIncluded in the framework are \"hot button\" ethical issues that we identified through our work with Toronto hospitals and the MOHLTC. These issues were as follows: These \"hot button\" issues are not intended to be exhaustive, but rather they serve to illustrate how the values in the ethical framework can be used to identify key ethical aspects of decision-making.\nLet us take the issue of targeting and prioritizing populations for vaccine and antivirals to illustrate how the values in the ethical framework can help guide decision-making. The values of solidarity and protecting the public from harm would require that priorities be set to maximize the capacity to help society ensure that the ill are cared for during a pandemic. Furthermore proportionality would require that decision-makers consider who within the community are most vulnerable to the contagion as well as who are most likely to benefit from immunization. A well-informed public conversant with the values in the ethical framework and aware of the expertise that informed the ranking of priorities for immunisation would be consistent with value of trust and the principle of transparency.\nLastly, while knowing how to use the framework to inform decision-making is vital, there is more to ensuring that the framework will be used or useful.\nWe have identified three necessary, if not exhaustive elements to the successful integration of ethics into hospital pandemic planning processes. These elements are 1) sponsorship of the ethical framework by senior hospital administration; 2) vetting of the framework by key stakeholders and; 3) decision review processes. \nThere should be mechanisms in place to ensure that ethical decision-making is sustained throughout the crisis.\nDecisions should be made explicitly with stakeholder views in mind and there should be opportunities for stakeholders to be engaged in the decision-making process. For example, decision-making related to staff deployment should include the input of affected staff.\nDecisions should be publicly defensible. This means that the process by which decisions were made must be open to scrutiny and the basis upon which decisions are made should be publicly accessible to affected stakeholders. For example, there should be a communication plan developed in advance to ensure that information can be effectively disseminated to affected stakeholders and that stakeholders know where to go for needed information.\nDecisions should be based on reasons (i.e., evidence, principles, values) that stakeholders can agree are relevant to meeting health needs in a pandemic influenza crisis and they should be made by people who are credible and accountable. For example, decision-makers should provide a rationale for prioritising particular groups for antiviral medication and for limiting access to elective surgeries and other services.\nThere should be opportunities to revisit and revise decisions as new information emerges throughout the crisis as well as mechanisms to address disputes and complaints. For example, if elective surgeries are cancelled or postponed, there should a formal mechanism for stakeholders to voice any concerns they may have with the decision. \nDuty to Provide Care The duty to provide care and to respond to suffering is inherent to all health care professionals' codes of ethics. In an influenza pandemic, demands on health care providers and the institutions in which they work will overwhelm resources. Health care providers will have to weigh demands from their professional role with other competing obligations to their own health, to family and friends. Health care workers will face significant challenges related to resource allocation, scope of practice, professional liability, and workplace conditions. Decision makers should:\n• Work collaboratively with stakeholders and professional colleges in advance of an influenza pandemic to establish practice guidelines • Work collaboratively to develop fair and accountable processes to resolve disputes • Provide supports to ease this moral burden of those with the duty to care • Develop means through which institutions will handle appeals or complaints, especially with regards to work exemptions, or the vaccination/prophylaxis of staff Health care workers who are at increased risk because they are caring for patients with influenza must weigh familial obligations, and obligations to self with their professional duty to care. In addition, they may also have to comply with vaccination or antiviral regimens for prophylaxis which may conflict with their individual liberty.\nThe principle of equity holds that, all things being equal, all patients have an equal claim to receive needed health care. During influenza pandemic, however, tough decisions will need to be made about which health services to maintain and which to defer because of extraordinary circumstances. Measures taken to contain the spread of a deadly disease will inevitably cause considerable collateral damage. In an influenza pandemic, this will extend beyond the cessation of elective surgeries and may limit the provision of emergent or necessary services.\nIn allocating scarce resources, the value of equity could guide in developing fair criteria for allocation while consideration is given also to compensation for those who will not meet inclusion criteria yet are entitled to receive care.\nIndividual liberty is a value enshrined in health care practice under the principle of respect for autonomy. Under usual circumstances, health care providers balance respect for individual autonomy with a duty to protect individual patients from harm. In a public health crisis, however, restrictions to individual liberty may be necessary to protect the public from serious harm. Patients, staff, and members of the public may all be affected by such restrictions.\n• Be proportional to the risk of public harm • Be necessary and relevant to protecting the public good • Employ the least restrictive means necessary to achieve public health goals • Be applied without discrimination Social distancing strategies that employ visitor restrictions in hospitals must be necessary for the protection of the public and must be proportionate to the threat being allayed.\nIndividuals have a right to privacy in health care. In a public health crisis, it may be necessary to override this right to protect the public from serious harm. A proportionate response to the need for private information requires that it be released only if there are no less intrusive means to protect public health.\n• Disclose only private information that is relevant to achieve legitimate and necessary public health goals • Release private information only if there are no less intrusive means to protect public health • Determine whether the good that is intended is significant enough to justify the potential harm that can come from suspending privacy rights, (e.g. the harm from stigmatization of individuals or particular communities) • Provide public education to correct misconceptions about disease transmission and to offset misattribution of blame to particular communities\nThe need to conduct contact tracing of possibly infected people might require that particular groups or even individuals are identified publicly. The need to do so must be weighed against the potential harm of exposing communities and individuals to stigmatization.\nProportionality requires that restrictions to individual liberty and measures taken to protect the public from harm should not exceed what is necessary to address the actual level of risk to, or critical need of, the community. Decision makers should: • Use least restrictive or coercive measures in limiting or restricting liberties or entitlements • Use more coercive measures only in circumstances where less restrictive measures have failed to achieve appropriate public health ends.\nThe decision to close an emergency room must consider if the potential harm in keeping the emergency room open is significant enough to warrant its closure.\nA foundational principle of public health ethics is the obligation to protect the public from serious harm. This principle requires that citizens comply with imposed restrictions in order to ensure public wellbeing or safety. To protect the public from harm, hospitals may be required to restrict public access to service areas (e.g. restricted visiting hours), to limit availability of some services (e.g. elective surgeries), or to impose infectious control practices (e.g. masks or quarantine).\n• Weigh the medical and moral imperative for compliance • Ensure stakeholders are made aware of the medical and moral reasons for public health measures • Ensure stakeholders are aware of the benefits of compliance & the consequences of non-compliance • Establish mechanisms to review these decisions as the public health situation changes and to address stakeholders concerns or complaints When making the decision to quarantine individuals, protection of the public from harm must be weighed against individual liberty. Note that while the ethical value of individual liberty is often in tension with the protection of the public from harm, it is also in individuals' interests to minimize harm to others.\nReciprocity requires that society supports those who face a disproportionate burden in protecting the public good and takes steps to minimise their impact as far as possible. In an influenza pandemic, measures to protect the public good are likely to impose a disproportionate burden on health care workers, patients, and their families. Health care workers may face expanded duties, increased workplace risks, physical and emotional stress, isolation from peers and family, and in some cases, infection leading to hospitalization or even death. Similarly, quarantined individuals or families of ill patients may experience significant social, economic, and emotional burdens.\n• Easing the burdens of health care workers, patients, and patient's families in their hospitals and in coordination with other health care organizations • Ensuring the safety of their workers, especially when redeploying staff in areas beyond the usual scope of practice\nThe provision of antiviral medication and/or vaccination to hospital staff for prophylaxis is one way hospitals can ensure the safety of their workers who may be exposed to greater than usual risks in discharging their duty to care.\nSARS heightened the global awareness of the interdependence of health systems and the need for solidarity across systemic and institutional boundaries in stemming a serious contagious disease. An influenza pandemic will not only require global solidarity, it will require a vision of solidarity within and between health care institutions. Solidarity requires:\n• Good, open and honest communication • Open collaboration, in a spirit of common purpose, within and between health care institutions • Sharing public health information • Coordinating health care delivery, transfer of patients, and deployment of human and material resources Territoriality between hospital departments and between health care institutions needs to be overcome with good communication and sense of common purpose in order to provide equitable care across jurisdictions\nIn our society, both institutions and individuals will be entrusted with governance over scarce resources, such as vaccines, antivirals, ventilators, hospital beds and even health care workers. During a pandemic influenza outbreak, difficult decisions about how to allocate material and human resources will have to be made, and there will be collateral damage as a result of these allocation decisions. Those entrusted with governance roles should be guided by the notion of stewardship. Inherent in stewardship are the notions of trust, ethical behaviour, and good decision-making. Decision makers have a responsibility to: • Avoid and/or reduce collateral damage that may result from resource allocation decisions • Maximize benefits when allocating resources • Protect and develop resources where possible • Consider good outcomes (i.e. benefits to the public good) and equity (i.e., fair distribution of benefits & burdens)\nA hospital's decision to stock-pile antiviral medication must consider whether this is an effective way of protecting staff from infection, where the money for stockpiling will come from, and whether that money could be put to better use elsewhere.\nTrust is an essential component in the relationships between clinician and patient, between staff and the organization, between the public and health care providers, and between organizations within a health system. In a public health crisis, stakeholders may perceive public health measures as a betrayal of trust (e.g. when access to needed care is denied) or as abandonment at a time of greatest need. Decision-makers will be confronted with the challenge of maintaining stakeholders' trust while at the same time stemming an influenza pandemic through various control measures. It takes time to build trust.\n• Take steps to build trust with stakeholders before the crisis hits not while it is in full swing • Ensure decision making processes are ethical and transparent to those affected stakeholders Early engagement with stakeholders may go some distance to justify stakeholder confidence in decision-makers' trustworthiness. In part, the value of trust is respected and promoted by following the ethical processes outlined above. \nWhether or not an ethical framework is used to inform decision-making in a health care institution depends to a large extent on people in senior positions of an organisation seeing its relevance to the decision-making process.\nIn part, this is dependant on how robust the framework is, but it also requires the willingness to frame (at least some) pandemic planning issues as normative in nature.\nSome may argue that the values in the framework are too stringent or impractical to implement under crisis conditions, especially those found in the Ethical Processes part of the framework (see Table 1 ). Certainly, crisis conditions may place constraints on the extent to which each principle can be acted upon. However, efforts should be made to put them into action to the fullest extent possible under the circumstances and in our experience this is only possible with the support of senior administrators.\nThe senior administration at S & W (many of whom were part of the Pandemic Planning Committee) had previous experience with the accountability for reasonableness framework for decision-making, and thus their pandemic influenza planning committee was already familiar with the Ethical Processes part of the framework, and they were receptive to the idea of being guided by an ethical framework. Senior administrators may also have been receptive to the ethical framework because, as they learned from SARS, organisations that did not have decision-making processes that honoured the values for ethical process during SARS have been dealing with a legacy of collateral damage to staff and patients in the form of distrust and low morale [12] . For these reasons, the senior administrators at S & W played an important role in vetting the ethical framework. Ensuring that institutional \"sponsors\" are in favour of adopting an ethical framework is important for gaining widespread support for using an ethical framework in decision-making, and for ensuring that the ethical framework does not become something that looks good but remains unused.\nIn order to obtain support for, or \"buy in\" to an ethical framework, it is important that key stakeholders in an institution vet the framework. This requires careful consideration of who the key stakeholders are in an institution. Not only should this include those with responsibility for decision-making, but also those who will be affected by decisions taken. For the vetting process is not just intended to create \"buy in\" but also to decrease the likelihood that interests and issues that are (morally) relevant to pandemic planning will be neglected or overlooked, thereby enhancing the moral legitimacy of the values in the framework. In addition, a process of stakeholder vetting increases the likelihood that the values instantiated in the framework resonate with the stakeholder community.\nIt has been our experience that the values in the framework did resonate with the pandemic planners with whom we have shared this ethical framework. The primarily pragmatic justification for the selection of the values in the framework means that the framework is provisional so it ought to be subject to revision in light of compelling argument, empirical evidence and further stakeholder feedback. It is important to note, however, that the iterative and inclusive process through which the values in the framework were deliberated amongst the various stakeholder groups lends them a form of discursive ethical legitimacy and helps to justify their inclusion in the ethical framework. We intend that the framework invite further dialogue about its legitimacy and its adequacy. We will return to this issue in the final section of this paper.\nIdeally, the vetting process would include people who can represent the interests of patients, families and volunteers who are part of the hospital's constituency. Although patient relations, human resources and occupational health representatives from S & W provided guidance and feedback in the development of the framework, direct input from patients and family representatives was not obtained. One limitation of our framework is that is has yet to be vetted by these important stakeholders.\nThe importance of solidarity to the management of a public health crisis would also suggest that the public and other health care organisations be considered stakeholders in hospital pandemic planning. While it may not be pragmatic for hospitals to undertake broad public consultation and vetting processes for their pandemic plans in general, and their ethical frameworks in particular, solidarity and equity suggest that these broader stakeholder interests are relevant to pandemic planning. Consequently, opportunities for broader ethical dialogue about pandemic planning need to be encouraged.\nIn order to ensure that the support of key stakeholders is maintained through an outbreak, there need to be effective communication mechanisms in place. An important aspect of responsive decision-making processes is ensuring that there are formal opportunities to revisit and revise decisions as new information emerges. As part of our ethical framework, we formulated a template for decision review processes, (adapted from, Gibson, JL: Formal decision review process template. Unpublished; 2003) that aids organisations in identifying existing and establishing new mechanisms that can be used for the formal reviews of decisions. We believe decision review mechanisms are an essential part of ethical decision-making in a public health crisis, and are one way to put the values in the ethical framework in to action.\nFormal mechanisms for reviewing decisions are needed in order to capture feedback from stakeholders on key decisions, and to resolve disputes and challenges. These processes are important for ensuring that decisions are the best possible under the circumstances given changing information and for engaging stakeholders constructively around the difficult decisions that must be made. Given the unpredictable nature of public health emergencies and the difficulty this poses for those in charge of planning and decision-making, it is reasonable to assume that decisions will be revised throughout the pandemic influenza crisis. Disputes or challenges may arise from the restrictions or requirements imposed on staff, patients and families during a pandemic influenza outbreak. Thus, decision review processes are essential. Again, while some may argue that this is too stringent a measure for a time of crisis, we argue that reviews of decisions will be taking place regardless (most likely in an ad hoc manner), and that to formalize this process is to increase its fairness and moral legitimacy. Indeed, there may be existing mechanisms which can handle these kinds of reviews.\nIt is important to distinguish between different types of ethical analyses in order to explain the approach that was taken to the development of the ethical framework discussed herein. Callahan and Jennings draw a useful distinction between applied ethics and critical ethics [7] . Our ethical framework is an example of applied ethics because the framework identifies and relies on \"general principles that can be applied to real-world examples of professional conduct or decision-making\" [7] and because it is \"designed to give professionals guidance and to give clients and the general public standards to use in assessing professional conduct\" [42] . While there is certainly a need for critical ethical analysis that pays attention to problems that are the \"result of institutional arrangements and prevailing structures of cultural attitudes and social power\" [7] , one would not expect a ethical framework designed to guide clinical decision-making to explicitly address these kinds of issues.\nThis is not to say that this ethical framework cannot address the kinds of issues that a critical ethical analysis might address. For example, the framework promotes values and processes that seek to redress the power disparities within institutions. The section of the framework that deals with ethical processes in particular is a challenge to how institutional decisions are typically made. For example, the value of \"inclusiveness\" as a process principle is essential for redressing power differences amongst key stakeholders [27] . Thus, while the ethical framework is the product of applied ethical analysis, and should be evaluated in light of this, one of its strengths is that it can also redress what Callahan and Jennings would characterize as \"critical\" ethics problem of power disparities within institutions.\nWithin pluralistic societies, there are many different ethical perspectives that exist simultaneously on issues about global, public and individual health. An ethical framework to guide decision-making is robust to the extent that it reflects the values and beliefs of the decision-makers who refer to it and the values and beliefs of those affected by the decisions being taken. Our framework relied heavily on the Toronto experience with SARS to surface and examine the ethical values that are important for a public health crisis. An influenza pandemic is likely to present us with particular ethical challenges that are different from SARS due to the predicted severity of the contagion and its spread to the community. It would therefore be important not to uncritically adopt such a framework but rather to use it as a basis for continued reflection and re-evaluation to ensure its relevance and responsiveness during the unfolding health crisis. It is also important to consider the extent to which an ethical framework is reflective of the community in which it is to be used. Lessons from SARS as it was experienced in China would likely surface some different ethical values, or emphasise different aspects of our framework. As Callahan and Jennings have argued:\nWe submit that a rich discourse on ethics and public health cannot be advanced without relating it to the background values of the general society, and the particular communities, in which it will be carried out. [7] Indeed, as previously maintained, there are many issues related to pandemic influenza planning -particularly those raised by a critical ethical analysis -that require broad public debate. While these kinds of issues require public debate that takes place at the societal level, ethical pandemic planning requires that organisations and agencies foster internal dialogue about the values instantiated in an ethical framework. For it is imperative that the values outlined in a framework resonate with the members of an organisation, and the community it serves. The procedural aspects of the framework provide a means to ensuring that the values of the community are reflected in decision-making through the procedural principles of inclusiveness and responsiveness.\nIt is important, too, to recognise that values are not static, and that circumstances will evolve rapidly during a pandemic influenza outbreak. Ethical frameworks will also require re-evaluation and revision. The challenge will be to continue to recognise the importance of moral reflection under circumstances that are not conducive to it and to encourage a process of re-evaluation that strives to assess whether resulting decisions are consistent with those values the framework is intended to promote. For this reason, it is imperative to start the ethical dialogue in advance, and to find ways to encourage consideration of ethical issues at all stages of decision-making. We hope that this paper will go some way towards advancing this objective, and that this paper stimulates discussion of the ethical issues and values that pervade pandemic planning.\nWe believe that this framework is unique in its blending of clinical, public health, and organizational ethics. One of its strengths is that it draws on lessons from the recent public health crisis of SARS in Toronto, and it is to some extent empirically grounded. Another strength is that it is the product of an inclusive process of development that included stakeholder vetting. It is also unique in its attempt to provide guidance to decision-makers facing a public health crisis. We hope that the framework's acceptance by hospitals and the provincial government in Ontario signals a change in the way that decisions are taken by institutions that are charged with making decisions that have life and death consequences for the public.\n• Good pandemic planning requires reflection on values because scientific information alone cannot drive decision-making.\n• The development of an ethical framework for hospital pandemic planning calls for expertise in clinical, organisational and public health ethics.\n• Stakeholder engagement is essential for the ethical framework to be relevant and legitimate.\n• The ethical framework contains procedural and substantive ethical values to guide decision-making.\n• Three key elements of integration of ethics in to pandemic planning are 1) sponsorship from senior hospital administration; 2) vetting by stakeholders and; 3) decision review processes.\n• An ethical framework is robust to the extent that pandemic influenza planning decisions are seen to be ethically legitimate by those affected by them.\n• In order to increase the robustness of pandemic planning in general, timely public debate about the ethical issues is essential." }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: The clinical definition of acute respiratory distress syndrome (ARDS) includes the acute onset of bilateral pulmonary infiltrates, a ratio of arterial partial pressure of oxygen (PaO 2 ) to fraction of inspired oxygen (FiO 2 ) of 200 mmHg or less, and no evidence of left atrial hypertension [1] . Many risk factors, such as pneumonia, sepsis, and aspiration, are associated with the development of ARDS. However, other diseases and conditions, such as bronchiolitis obliterans organizing pneumonia (BOOP), adverse reaction to drugs, diffuse alveolar hemorrhage (DAH), and hypersensitivity pneumonitis (HP), can also cause ARDS; despite similar clinical presentations, etiological diagnosis can be difficult especially for early-stage ARDS. Although the mortality rate of patients with ARDS ALI = acute lung injury; ARDS = acute respiratory distress syndrome; BAL = bronchoalveolar lavage; DAD = diffuse alveolar damage; FiO 2 = fraction of inspired oxygen; HRCT = high-resolution computed tomography; ICU = intensive care unit; OLB = open lung biopsy; PaO 2 = arterial partial pressure of oxygen; PEEP = positive end-expiratory pressure.\nimproves recently [2] , the rapid clinical deterioration of such patients, who often progress to multiple organ failure, remains a significant challenge for intensivists in the intensive care unit (ICU). To halt the disease progression of early-stage ARDS, accurate diagnosis is critical.\nIt can be difficult to differentiate between infectious and noninfectious etiology as the cause of ARDS in its early stages. Current microbiological sampling techniques are insufficiently sensitive to determine the causes of ARDS in all patients [3] [4] [5] . In patients with negative microbiological cultures, separating a true infection from an inflammatory response with clinical data remains problematic. Empiric broad-spectrum antibiotics are typically prescribed to these critically ill patients immediately after admission. However, unnecessary antibiotic therapy for non-infectious patients can enhance the occurrence of antibiotic-resistant strains of bacteria and increase the potential for subsequent nosocomial infections.\nThe therapeutic benefit of prolonged glucocorticoid therapy during the fibroproliferative stage of ARDS emphasizes the need for the elucidation of the underlying lung pathologies [6] . Additionally, the specific diseases such as BOOP, drug reaction, DAH and HP can cause an ARDS response to steroid therapy. However, inappropriate steroid therapy for patients with ARDS may be associated with complications such as gastrointestinal bleeding, hyperglycemia and increased susceptibility to infection.\nSome previous studies have demonstrated that open lung biopsy (OLB) is a useful and acceptably safe diagnostic technique for patients with ARDS [7] [8] [9] . In the study by Papazian and colleagues [7] , the results of OLB directly altered the therapeutic management for 34 of 36 patients with ARDS (94%), and the OLB complication of an air leak occurred in five patients (14%). The OLB results obtained by Patel and colleagues [8] led to a change in management in the majority of 57 patients with ARDS, the addition of specific therapy for 34 patients (60%), and the withdrawal of unnecessary therapy in 24 patients (37%); major complications occurred in four patients (7%). However, in both studies the duration from intubation to OLB was long: in Papazian and colleagues' study [7] the range was 5 to 89 days, and in Patel and colleagues' study [8] it was 0 to 25 days. This retrospective study attempted to evaluate the utility and safety of OLB in patients with clinically suspected non-infectious early-stage ARDS.\nThe records of patients with ARDS who received OLB in two ICUs at a tertiary care referral center over a five year period between January 1999 and April 2005 were examined. Charts with a discharge diagnosis code 518.82 of the International Classification of Diseases, Ninth Revision, Clinical Modification, suggesting ARDS not related to surgery or trauma, were reviewed for possible inclusion in this study. A total of 819 patients with ARDS were identified and OLBs were performed in 68 patients (8.3%). Forty-one OLBs were performed during early-stage ARDS (one week or less after intubation). Patients supported with noninvasive positive-pressure ventilation or intubated for more than seven days at the time of biopsy were excluded.\nAll patients met ARDS criteria defined by the American-European consensus conference [1] . Decisions to perform OLB were made by senior intensivists in charge of the respective ICUs. OLB was indicated when ARDS was suspected to be noninfectious in origin, with no obvious etiology and with a possible indication for corticosteroid treatment based on clinical presentations with rapid progression, relative symmetric distribution on chest X-ray, and predominant ground-glass attenuation in high-resolution computed tomography (HRCT) of the chest. Informed consent for OLB was obtained from each patient's family.\nChest HRCT was performed before bronchoscopic sampling and OLB. The location for bronchoalveolar lavage (BAL) sampling was selected on the basis of HRCT findings, or on a chest X-ray when HRCT was unavailable. BAL was performed by introducing 200 ml of sterile warm (37°C) saline solution into a lung subsegment and aspirating it back in four 50-ml aliquots. The first aliquot returned (bronchial fraction) was discarded. Each specimen was sent for bacterial examination for Legionella, Mycoplasma pneumoniae, Pneumocystis carinii, and Mycobacteria, and for fungal and virological (cytomegalovirus, influenza virus, parainfluenza virus, adenovirus, herpes simplex virus, respiratory syncytial virus, and coxsackie virus) analyses. Specimens were also sent for cytology and iron stain analysis. BAL results were deemed positive when at minimum one microorganism grew to a concentration of more than 10 4 colony-forming units/ml. All procedures were performed within 24 hours of OLB.\nOLB was performed in an operating room or at the bedside in an ICU by an experienced thoracic surgeon. Bedside OLB was indicated when the FiO 2 used reached 1 with an applied positive end-expiratory pressure (PEEP) of at least 12 cmH 2 O. With regard to mechanical ventilator settings to prevent air leakage, PEEP was immediately reduced 2 cmH 2 O from the baseline level after surgery. Pulmonary tissue was harvested from a site considered new or from a progressive lesion identified by chest HRCT or chest X-ray.\nEach tissue specimen was cultured and examined by a pulmonary pathologist.\nMedical records from these 41 patients were reviewed and analyzed for the following data: age; gender; Acute Physiology and Chronic Health Evaluation (APACHE) II scores at admission to the ICU; acute lung injury (ALI) scores, PEEP, and PaO 2 /FiO 2 ratio at ARDS diagnosis; dates of ARDS onset, respiratory failure, intubation, and biopsy; underlying diseases; diagnostic tests before biopsy; and medications at time of biopsy. Results regarding complications of biopsy, pathological diagnosis, and postoperative therapeutic changes (addition or removal of drugs) were also analyzed. Outcome parameters, including ICU and hospital survival rates and cause of death, were also evaluated.\nFor normally distributed data, values are reported as means ± SD. Student's t tests were used to compare normally distributed continuous variables. Differences between subgroups were compared by using the χ 2 test or Fisher's exact test when the expected number of events was less than five. The significance level (α) for all statistical tests was set at 0.05, and p < 0.05 was considered statistically significant.\nSixty-eight patients underwent OLB for ARDS evaluation during the study period, of whom 27 were excluded because the duration between intubation and OLB exceeded seven days. A total of 41 patients were enrolled. Table 1 lists the baseline characteristics of the patients studied. Twenty-four patients (59%) were immunocompetent and 17 patients (41%) were immunocompromised. Causes of immunocompromise status were hematological malignancy in 10 patients and solid tumors in four patients (three had bronchogenic cancers and one had breast cancer), HIV infection in two patients and renal transplantation in one. The duration from intubation to OLB for these 41 patients was 3.0 ± 1.9 days (mean ± SD; range 1 to 7).\nBAL was performed 24 hours before OLB. Findings of BAL were compatible with pathological diagnosis for only four patients with diagnoses of bacterial pneumonia, mycobacterial tuberculosis, cytomegalovirus pneumonitis, and Pneumocystis carinii pneumonia. Twenty-two patients (54%) had chest HRCT before OLB to identify an appropriate biopsy site. For the remaining 19 patients who did not undergo chest HRCT, OLBs were performed from the right middle lobe in 12 patients and from the lingular lobe in seven patients.\nOf the 41 patients, 26 (63%) underwent OLB in an operating room and 15 (37%) received bedside OLB in an ICU. Videoassisted thoracotomy was performed in eight patients, and the remaining patients underwent limited anterior thoracotomy. No intra-operative complication occurred, and eight patients (20%) had postoperative complications (less than seven days after the operation). Two patients developed transient hypotension after OLB and regained normal status after fluid resuscitation and vasopressor treatment for 12 hours. Two patients had pneumothorax diagnosed by chest X-ray and required a chest tube with low-pressure suction (10 cmH 2 O) drainage for 24 hours after OLB. Two patients had subcutaneous emphysema localized in the chest area after OLB, which resolved spontaneously in two days. Additionally, two patients had bronchopleural fistula with persistent air leaking from the operative chest tube for at least one day and did not need further surgery. Although six of these eight patients (two with transient hypotension, one with pneumothorax, one with subcutaneous emphysema and two with bronchopleural fistula) died, no surgical complication resulted directly in death. The incidence of postoperative complication was 15% (4/26) and 27% (4/15) for patients undergoing OLB in an operating room or at the bedside in an ICU, respectively. Complication rates were not significantly different between these two groups (p = 0.3799).\nAll biopsies provided sufficient data for pathological diagnosis (diagnostic yield 100%). The specimens obtained during OLB were sent for tissue culturing (for both bacteria and viruses); all culture results were negative. Pathological diagnoses were subdivided into specific and nonspecific categories. Eighteen patients (44%) had specific diagnoses established by OLB, and 23 (56%) had nonspecific diagnoses (Table 2) .\nOverall, OLB findings led to alteration therapy for 30 of 41 patients (73%). After OLB, 18 patients were administrated high-dose corticosteroid therapy (1 g/day methylprednisolone in divided doses for three days) and seven patients were treated with low-dose corticosteroid therapy (2-3 mg/kg per day methylprednisolone in divided doses). Three patients received co-trimoxazole for Pneumocystis carinii pneumonia. Antibiotics were changed in one patient and discontinued in one patient on the basis of pathological findings. Treatment was not changed in 11 of 41 patients (27%). Table 3 presents comparative results for patient characteristics, complication rates, alterations in treatment, and survival rates of patients with specific and nonspecific pathological diagnoses by OLB. The rate of treatment alteration was higher in the nonspecific pathological diagnosis group than in that with a specific diagnosis (56% versus 87%; p = 0.0243). No other significant differences between these two groups were noted.\nTwenty-one patients died in the ICU, resulting in an ICU survival rate of 49% (20/41). The hospital survival rate was the same as the ICU survival rate. Multiple organ dysfunction syndrome was the leading cause of death in 10 patients, followed by septic shock in nine patients, hypovolemic shock in one patient and acute myocardial infarction in one patient. Table 4 presents comparative results of patient characteristics and outcomes for survivors and nonsurvivors. No significant differences were observed between survivors and nonsurvivors for baseline data, such as age, gender, severity of illness, complication rate, and treatment alteration rate, between these two groups. Significantly more immunocompromised patients were in the survivor group than in the nonsurvivor group (60% vs 24%; p = 0.0187).\nComparisons between immunocompromised and immunocompetent patients (Table 5 ) showed that immunocompromised patients were younger (p = 0.0004) and had lower ALI scores (p = 0.0045). Furthermore, immunocompromised patients had better hospital survival rates than immunocompetent patients (71% versus 33%; p = 0.0187).\nThis study showed that OLB is an acceptably safe and useful procedure for some selected patients with early-stage ARDS. The treatment alteration rate was higher in patients with ARDS with nonspecific pathological diagnoses than in those with specific diagnoses.\nIn recent studies of patients with ARDS [7, 8] , OLB was employed relatively late, and the time from intubation to OLB was considerable (5 to 89 days in the study by Papazian and colleagues, and 0 to 25 days in the study by Patel). In the present study, OLB was performed within one week of intubation (3.0 ± 1.9 days), substantially earlier than in the previous two studies. Patel Specific diagnosis rates based on OLB findings vary among studies of patients with different disease entities. The specific diagnostic rates in a review by Cheson and colleagues were 21 to 68% in immunocompetent patients and 37 to 95% in immunocompromised patients [11] [12] [13] [14] . In this study, specific and nonspecific diagnostic rates were 44% (18/41) and 56% (23/41), respectively, and specific diagnostic rates for immunocompetent and immunocompromised patients were 33% (8/24) and 59% (10/17), respectively. Although not statistically significant (p = 0.1052), the specific diagnostic rate between immunocompetent and immunocompromised patients was similar to that in previous studies, indicating that OLB obtains a high percentage of specific pathological diagnoses for immunocompromised patients.\nIn this study, the rate of therapy alterations after OLB was 73% (30/41) and was not lower than those in previous reports (range 59 to 75%) [10, 14, 15] . For groups with nonspecific and specific pathological diagnoses, the rate of changed therapy was higher in the nonspecific group (87% versus 56%; p = 0.0243). This analytical finding resulted from a large number of patients with nonspecific pathological diagnoses undergoing corticosteroid treatment as a rescue or anti-inflammatory therapy after excluding potential active infection, such as the fibroproliferative stage of DAD [16] [17] [18] , interstitial pneumonitis, nonspecific interstitial pneumonitis, and organizing pneumonia. Early OLB can achieve diagnoses other than fibrosis that are potentially treatable with corticosteroid. Furthermore, the recent study by the ARDS Clinical Trials Network [19] did not support the routine use of methylprednisolone in patients with persistent ARDS (at least seven days after the onset) and\nsuggested that methylprednisolone therapy might be harmful when initiated more than two weeks after the onset of ARDS. The duration of ARDS before corticosteroid treatment interacted significantly with survival.\nFor immunocompromised patients, some studies [11, 20] suggested that OLB is advantageous for diagnosis and for treatment alteration but that its benefit to survival remains unclear.\nMcKenna and colleagues [21] found that for immunocompromised patients, early OLB (average 3.6 days after admission) benefited the histological diagnosis of interstitial pneumonitis treated with steroids; however, OLB did not improve clinical outcome for all patients. The overall mortality rate was 51% (21/41) in the present study, which is similar to that obtained in previous reports (range 47 to 50%) [7, 8] . More immunocompromised patients were in the survivors group and had a better survival rate than the immunocompetent patients (60% versus 24%; p = 0.0187); the young age and low ALI scores of immunocompromised patients probably accounted in part for their better outcome. Furthermore, the enhanced survival rate of immunocompromised patients might be attributed to more immunocompromised patients (9/13; 69%) than immunocompetent patients (9/17; 53%) receiving high-dose corticosteroid therapy after active infection had been excluded by OLB. Various pulmonary conditions such as infection, disease progression, therapeutic reaction, new and unrelated pathologies, or a combination of these can be present in immunocompromised patients [21, 22] . For diagnostic yield and adequate treatment, early OLB has been considered to be a reliable diagnostic modality, providing an early and accurate etiological diagnosis in immunocompromised patients. Operative complication rates reported for OLB in patients with ARDS have ranged from 17 to 39% [7, 8, 10] . In this study, the overall rate of OLB postoperative complications was 20% (8/ 41). In the late fibrotic stage, lung parenchyma is stiffer than in the earlier exudative or fibroproliferative stages of ARDS. Although operative complications are multifactorial, early OLB in non-stiff lungs (less fibrosis in the present study than in other reports) may account for the low surgical complication rate in this study. Of the 41 patients in the present study, 15 could not be transported to an operating room because they were being administered 100% O 2 and a high PEEP; consequently, OLB was performed at the bedside in the ICU. No intra-operative complications or exacerbation of oxygenation and hemodynamics occurred, even in patients with ARDS with severe hypoxemia. Of these 15 patients, four developed postoperative complications of hypotension, pneumothorax, subcutaneous emphysema, and bronchopleural fistula, respectively. No death was attributable to OLB. The risk for complications due to OLB in early-stage ARDS was therefore acceptable, even for the most critically ill patients with severe hypoxemia.\nSeveral limitations of this study should be considered. First, because of its retrospective nature our study cannot directly address the question of whether early OLB has a survival benefit. However, understanding of a specific etiology would permit the initiation of specific therapy assuming that such a therapy is available. Many of the diagnoses found in this study (such as metastatic malignancy, infectious pneumonia and hypersensitivity pneumonitis) may have an established positive therapeutic effect on outcome. Second, the result of this study cannot be generally applied to all patients with ARDS. The decision to perform OLB was not made at random and the patients referred for OLB were unlikely to be a representative sample of our ARDS population. This selection bias of patients and intensivists would be expected to increase the possibility of an alternative intervention. A third limitation is that some specific diagnosis such as viral pneumonitis may be underdiagnosed because its identification depends on the availability of laboratory facilities. A standardized comprehensive microbiological examination of BAL before OLB should be established.\nThis retrospective study demonstrates that OLB had a high diagnostic yield rate and an acceptable complication rate for some selected patients with early-stage ARDS. The rate of treatment alteration was higher in patients with nonspecific pathological diagnoses than in those with specific pathologically diagnosed ARDS. Further prospective, randomized and control studies should investigate the appropriate indication and effect of OLB on outcome in patients with ARDS.\n• Open lung biopsy is an acceptably safe diagnostic procedure for some selected early-stage patients with acute respiratory distress syndrome.\n• In patients with early-stage acute respiratory distress syndrome of suspected non-infectious origin, open lung biopsy may have a high diagnostic yield rate.\n• The role of open lung biopsy in patients with acute respiratory distress syndrome needs to be investigated in prospective, randomized and controlled clinical trials." }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: Pathogens have highly variable host ranges: in natural conditions some infect only one or a few related species (i.e., specialist pathogens) while other can infect a wide range of hosts belonging to different taxonomic groups (i.e., multi-host or generalist pathogens). A large fraction of described pathogens of humans, animals and plants are generalists [1] [2] [3] . The ability to infect different hosts conditions the epidemiology and pathogenicity of generalist pathogens and, therefore, is highly relevant for pathogen management and disease control [1, 4] . The distribution of multihost pathogens over their host range, i.e. the frequency of infection in the various host species within an ecosystem, may vary largely, which could determine the population dynamics and structure of the pathogen. The distribution of a pathogen species over its host range may also determine important aspects of its biology in hosts significant from an anthropocentric viewpoint (i.e. target hosts), such as reservoirs and inoculum sources, emergence and reemergence, population thresholds for disease invasion or critical community size for disease persistence [e.g., 1, [4] [5] [6] [7] .\nAnimal or plant species may be hosts for a range of pathogens, and most host populations encounter a large number of different pathogen species [8] . For significant host species, there is abundant evidence of differences in the infection frequency of the various pathogen species present in an ecosystem. The distribution of pathogens over their hosts, and the distribution of different pathogens within a host species, will affect the frequency of multiple infection of an individual host by different pathogens. Multiple infection may have important consequences for the infected hosts, for the pathogens, and for host-pathogen coevolution [8, 9] . In the host, frequent co-infections may lead to heterozygote superiority against multiple pathogens and contribute to the persistence in host populations of alleles conferring susceptibility to disease [10] . In multiple infected hosts, pathogens can cooperate or can compete for host resources, which will affect each other's fitness. Hence, multiple infections will be a factor in pathogen evolution. Theoretical analyses predict that the withinhost dynamics of microparasites in multiple infected hosts may have important consequences in the evolution of their virulence [11] [12] [13] [14] , and there is evidence that multiple infection may result in either increased or reduced virulence [e.g., [15] [16] [17] . Multiple infection of a host may also directly affect the genetic diversity of the pathogen population, as co-infection is a prerequisite for genetic exchange between different pathogen species or strains. Also, infection by one pathogen may result in an increased host susceptibility to a second pathogen, a common phenomenon named facilitation or predisposition by animal and plant pathologists, respectively [8, 18] .\nIn spite of its potential impact on pathogenicity, evolution, epidemiology and control, the distribution of pathogens over their host range and the occurrence of co-infections have been largely overlooked, and most research on pathogen ecology and epidemiology has dealt with specific pathogen-host interactions [8] . To our knowledge, it has not been analysed whether the distribution of pathogens over their host range is random or, alternatively, associations between pathogens and hosts occur, neither has been addressed whether host co-infection by different pathogens is random or associations between pathogens occur in particular hosts. Here we address these issues.\nFirst, we propose indices for the observed patterns of host infection by different pathogens, and the observed patterns of coinfection, and tests to analyse if they conform to the null hypothesis of randomness or reflect associations. Second, we apply these tests to data on the prevalence of five insect-borne virus species in wild plant species within an agroecosystem in Central Spain. Results of these analyses uncover patterns that, if general, would be highly relevant to understand the ecology and evolution of pathogens. [19] . Except for TSWV, which has a single-stranded RNA genome of negative and ambisense polarity, all other viruses have single-stranded RNA genomes of messenger polarity. AMV, CMV and WMV are transmitted by aphids in a non-persistent manner, i.e. the virus is retained in the distal structures of the aphid mouth parts for short period of time. BWYV is transmitted in a circulative, non-propagative manner, i.e., the virus penetrates through the gut wall into the haemocoel of the insect vector, and circulates with the haemolymph to reach the salivary glands, from where it is inoculated into new plants. TSWV follows a similar path within the thrips body, but infects and multiplies in the insect cells [19] . All five viruses cause important diseases in vegetable crops worldwide, including the studied region in Central Spain, but infection in the analysed wild hosts was asymptomatic. Table 1 shows the number of samples analysed and the number of infected plants by each of these five virus species, in single or multiple infection, in the 21 most frequently found plant species in three monitored habitats (see Methods) for the analysed period.\nTo this data set tests for association between hosts and pathogens (see Methods) were applied. The index of selectivity of pathogen (ISP), and its significance, is shown in Table 2 for the five viruses. The distribution of three of five analysed viruses over their hosts was significantly non-random, i.e. some of the available hosts were preferentially infected. Fig. 1 shows the relationship between prevalence and the ISP for the five viruses. A positive correlation was found for both parameters (r = 0.9347, P = 0.0189 in a Spearman rank correlation test), i.e., the more host-selective viruses were those with a highest prevalence in the analysed ecosystem. Similarly, the index of selectivity of the host (ISH), and its significance, was calculated for the 21 host plant species in Table 1 , and values are shown in Table 3 . For about half (9/21) of the analysed hosts (Amaranthus spp., Cirsium arvense, Convolvulus arvensis, Diplotaxis erucoides, Lactuca serriola, Medicago sativa, Portulaca oleracea, Solanum nigrum and Taraxacum spp.) differences in the prevalence of the five viruses departed significantly from random. Fig. 2 shows the relationship between virus prevalence and the ISH for the 21 host species. Again, a positive correlation between both parameters was found (r = 0.5161, P = 0.0166, in a Spearman rank correlation test), i.e., the more virus-selective hosts were those with a higher prevalence of virus infection. The relationships between prevalence and selectivity for viruses and hosts were not due to a coincidence in the frequency of infection among hosts by different viruses, as shown by a contingency analysis of counts of infected hosts by the different viruses (P,10 24 ).\nFor 16 of the 21 plant species in Table 1 , co-infection with more than one of the five viruses occurred. For these 16 plant species, 102 plants were infected by at least one virus out of 1060 analysed plants ( Table 4 ). The above described test of association between pathogens was applied to this set. The data in Table 4 showed a tendency of the analysed viruses to associate positively: the distribution of the association index (AI) was skewed towards positive values (Fig. 3 ) so that out of 68 AIs computed for the five viruses in 16 plant species, 47/68 (more than two thirds) were positive and 21/68 were negative. Moreover, there was a conspicuous tendency of the positive AI values to have smaller probabilities (r = 20.6575, P,10 24 , in a Spearman rank correlation test). When the pooled sample from the sixteen plant species was considered, the AI was positive and significantly different from zero for each of the five viruses, i.e. each of the five viruses was found in co-infection with a frequency significantly higher than expected from the null hypothesis of independence of infection. However, this was not so when the data for each of the sixteen plant species were analyzed separately. Hence the association analysis uncovered two patterns that were not obvious: i) a general tendency of the analysed viruses to associate positively, ii) association depended on both the plant and the virus species.\nMost efforts to understand the population biology of pathogens have focussed on specialist pathogens, and population biologists have successfully developed a formal understanding of the dynamics and evolution of single-host pathogens. However, most pathogens of humans, animals and plants are multi-host pathogens [1] [2] [3] 20] . As stated by Woolhouse et al. [1] ''understanding the more complex population biology of multi-host pathogens will be one major challenge in the 21st century ''. There is evidence that within an ecosystem the prevalence of multi-host pathogens may differ largely for the different species of their host range [e.g., [21] [22] [23] [24] [25] ]. Similarly, there is evidence of large differences in the prevalence on a host species of the various pathogens that are able to infect it [e.g., [26] [27] [28] [29] ]. However, no attempt has been made, to our knowledge, to analyse if differences in the distribution of multihost pathogens over their hosts are random or if there are associations between hosts and pathogens. The uncovering of associations between hosts and pathogens would be highly relevant to understand and model the population biology of multi-host pathogens, and for understanding the phenomenon of generalism itself.\nWe present here indices and tests to analyse if there is association between multi-host pathogens and their hosts. The proposed indices of selectivity for the pathogen and for the host measure the degree of association between hosts and pathogens.\nThe tests analyse the homogeneity of distribution of a pathogen over different host species or populations, and of different pathogens on a host, and analyse how significantly the values of the indices departs from zero (i.e. no association). The literature on pathogen ecology does not abound with data on the prevalence of various pathogens on various hosts. Hence, we have applied these indices to our unpublished data on the prevalence of five insectborne plant viruses on 21 species of wild plants in an agroecosystem in central Spain over a three year period.\nThe analysis of the prevalence of the different viruses in each host species by the homogeneity test that we propose, shows that half of the analysed plant species showed an index of selectivity of the host (ISH) significantly different from zero. The distribution of the host species showing virus selectivity was not related to taxonomy, habitat (fallow fields, edges or wastelands), seasonality or vegetative cycle (annual vs. perennial) (not shown). Interestingly, there was a positive correlation between the ISH and the average virus prevalence for these 21 host plant species, showing that the more selective hosts are more prone to be virus-infected, obviously by the virus(es) that better infects them. This phenomenon suggests that in spite that each host encounters a wide array of pathogens, mechanisms of escape and/or resistance [30] to some of them would operate, which could explain their selectivity. In fact, contingency analysis of counts of infected hosts by different viruses, suggest that different viruses specialise on different hosts. The analysis of the homogeneity of prevalence of a virus over its host species showed that for three of the five analysed viruses there was a significant host association, i.e., the value of the index of selectivity for the pathogen (ISP) significantly departed form zero. One major and unexpected finding of the analysis was that there was a positive and highly significant correlation between the value of the ISP and the prevalence of the viruses. The value of the ISP was not conditioned by the number of host plant species infected by each virus, as there was no correlation (r = 0.60, P = 0.173 in a Spearman rank correlation test) between ISP and the number of plant species that each virus infected in the analysed system i.e., the more selective viruses were not those infecting a smaller number of plant species. Thus, the more host-selective viruses were those that did best in the analysed ecosystem. This result could be highly relevant for understanding the evolution of generalism in pathogens. Although most described pathogens are generalists, the advantages of generalism are poorly understood. A generalist strategy provides the pathogen with more opportunities for transmission and survival, but it is predicted that evolution would favour specialism, because pathogen-host co-evolution could result in functional trade-offs that would limit the generalist fitness in any one host [1, [31] [32] [33] [34] . Our results are compatible with the hypothesis that specialism is advantageous for pathogens, as host selectivity is the rule for the analysed set of generalist viruses, and the more host selective is the virus, the more successful its strategy. Hence, our results could suggest that for generalist pathogens a degree of host specialisation, i.e. host-selectivity as defined here, is a successful strategy. Host specialisation in generalist pathogens would also be relevant for important issues of host and pathogen biology, as host specialisation will affect hostpathogen co-evolution and co-speciation, would reduce the opportunities for host switches and jumps, thus constraining the evolution of host expansion, and may result in spatial heterogeneity of hosts, thus favouring the stable maintenance of pathogen and host diversity [6, [35] [36] [37] . In addition, host specialisation may affect the opportunity for different pathogens of sharing a host and, thus, the consequences of multiple infection for pathogen and host evolution, as discussed below. We propose here also a simple procedure to estimate association among pathogens, which enables to compute an association index whose significance can be tested against the null assumption of independence of infections that follow a binomial distribution. The test was applied to the same data set as above, and the second major contribution of our analysis is the finding that co-infection was mostly non-random and that associations among the five analysed viruses were mostly positive. This result is relevant because co-infection of different pathogens may have important consequences for the pathogens, the infected hosts, and for hostpathogen co-evolution [8, 9, 14] . For viruses, co-infection of a host may result in the generation of new genotypes by recombination or by reassortment of genomic segments between different viral species or strains, often with dramatic changes in host range or pathogenicity. The classical example is the reassortment of avian and human strains of influenza A resulting in novel viruses with pandemic potential [38] [39] [40] [41] , but examples abound for both animal and plant viruses [e.g., [3, [42] [43] [44] [45] [46] [47] [48] ]. In the individual host, coinfection may lead to aggravated disease, often resulting from extracellular cooperativity of independently replicating viruses, by which one virus modulates the host response to infection to the benefit of the other [49, 50] . In addition, direct interactions of different viruses in co-infected cells may result in complementation of highly pathogenic defective genotypes, in increased virus replication or in modified cell and tissue tropisms [e.g., [51] [52] [53] [54] [55] [56] [57] ]. Alternatively, there is also evidence that mixed infections of pathogens result in reduced pathogenicity and less severe disease [17] . Examples from viruses include mixed infection with satellite or with defective interfering nucleic acids [58] . In our data set, association between viruses depended on each particular virus-host system. Hence, data suggest that in some hosts, but not in all, coinfection would be advantageous for some viruses, though the underlying mechanism remains to be analysed. The analysis here reported of plant virus infection on weeds has uncovered two major features that should be relevant to understand the population biology of viruses: i) the more hostselective viruses do better on the analysed ecosystem, ii) viruses tend to associate positively in co-infected hosts. It would be of high interest to know how general are these features and in which types of pathogens would they occur. The indices and tests that we propose here could be of general use in the analysis of the ecology of pathogens, and we hope that our results would prompt research on the ecology of pathogen-host and pathogen-pathogen associations, as these analyses might uncover pathogen properties relevant to the formal understanding of the population biology of multi-host pathogens.\nWe study two factors relative of the distribution of pathogens in different hosts (i.e. different host populations, genotypes, species etc): if there are associations between pathogens and their hosts and if there are associations among pathogens. To analyse these two factors we propose the following tests and indices:\nAssociation between pathogens and their hosts Let us call N k the number of analysed individuals in host k (k~1, 2, :::: , n k ) and X ik the number of these individuals that are infected by pathogen i (i~1, 2, :::: , n i ). The prevalence of pathogen i in host k will be the ratio P ik = X ik /N k .\nThe average prevalence of pathogen i over hosts will be\nConversely, the average prevalence of the different pathogens in host k can be defined as\nHomogeneity of the prevalence of a pathogen among hosts can be tested by means of a 2xn i contingency table with elements X ik and (N k 2X ik ) [59] . Different proportions (i.e. lack of homogeneity) will indicate a property of the pathogen that we will call selectivity. Selectivity will be measured by the Cramer's coefficient of contingency [59] of the contingency table. If x 2 i is the chisquared of the 2xn i table, the index of selectivity of the pathogen will be: Both of these indices range from zero to one, with zero meaning equal prevalence of the pathogen over hosts, or of pathogens over the same host, i.e. no selectivity for the pathogen or the host.\nAssociation between different pathogens Let us call Xs ik and Xa ik the number of analysed individuals of host k that are infected only by pathogen i (single infections) and by pathogen i and at least another one (associated infections), respectively, (X ik = Xs ik +Xa ik ).\nThe frequency of pathogen i in host k can be estimated as:\nwhich equals the above defined prevalence. Under the null hypothesis of independence of infection by different pathogens, the probability of a sampled host individual being infected only by pathogen i is:\nThe conditional probability of non-infection by any other pathogen given the presence of pathogen i is:\nps ik~P j=i (1{P jk ), and the conditional probability for the observed multiple infections given the presence of i is:\nSo, under the hypothesis of independence of infection by different pathogens (non-association between pathogens), Xs ik will be distributed as a binomial with X ik trials and probability ps ik . We define the association index (AI) for pathogen i in host k as the difference between the proportion of samples that being infected by pathogen i are infected also by at least another pathogen (Xa ik /X ik ), minus the expectation of this proportion under the null hypothesis (pa ik ). This index has a range from one to minus one and an expected value, under the null hypothesis, of zero. The significance of the observation can be estimated as a onetail test from the binomial above.\nTo test for association of different pathogens within a host, or for a given pathogen across different hosts, we follow the same process, as the expectation of a sum of observations will be equal to the sum of their expectations, and the corresponding sums of observations will be binomially distributed given the X ik .\nTo single out significant tests in a group, raw significance probabilities were corrected by the sequential Bonferroni method for multiple independent tests as indicated in [60] .\nPlants were sampled monthly for three years in a horticultural area in central Spain within three habitats characterised by different degrees of human intervention: fallow fields, edges between fields, and wastelands. Plants were sampled systematically along fixed itineraries, with no consideration of symptom expression, as described in Sacristán et al. [21] . Infection by AMV, BWYV, CMV, WMV and TSWV in the sampled plants was analysed by double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), using commercial antisera (Bio-Rad, Marnes-La-Coquette, France), according to the manufacturer's instructions.\nThe distribution of the host species showing virus selectivity according to taxonomy, habitat (fallow fields, edges or wastelands), seasonality or vegetative cycle (annual vs. perennial) was analysed by chi-squared tests of 2x N contingency tables, and their significances assessed, as in the rest of tests of this work, by simulation following model III." }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: Control of epidemics of (emerging) infectious diseases, such as SARS, pandemic influenza, or foot-and-mouth disease, always faces the difficulty that some infectives are not yet observed. By concentrating control measures only on observed cases (treatment, isolation, culling), resources are used efficiently but control is often not effective enough. On the other hand, by directing control to the whole population (mass vaccination, prophylactic treatment, preventive culling), epidemics are most likely contained, but at high cost.\nContact tracing of symptomatic infecteds works on an intermediate level: treatment or quarantine of contactees (by contact we mean the possible transmission event, by contactee the individual that is contacted) may be effective because unidentified infecteds are most likely to be found among contactees, and efficient because the resources can be directed towards individuals at risk only. Tracing and quarantine has been successfully applied for smallpox control, where the term 'Leicester method' refers to exactly this strategy, with the establishment of specific smallpox hospitals [1] . It was also successful during the more recent SARS epidemic [2] , but not during the British foot-and-mouth epidemic [3] . Here we wish to investigate how these differences can be accounted for by the tracing process, thereby distinguishing tracing and quarantine as having separate effects which can be explained as follows.\nSymptoms and detection divide the reproduction ratio R (the average number of secondary infecteds per primary infected in a susceptible population) into a part before detection and isolation and a part after [4] :\nwhere c is the reduction factor due to isolation, R 0 is the basic reproduction ratio, when no efforts are made to isolate, and R 0 pre is that part of R 0 occurring prior to detection. Contact tracing will lead to earlier prevention of transmission due to quarantine of traced infecteds, thereby reducing the uncontrolled transmission R 0 pre to R pre , and the reproduction ratio R to R q :\nThe effect of decreasing R 0 pre to R pre is distinct from the effect of isolation and quarantine which reduce c. For tracing to be effective, R q should be smaller than 1, so R pre should be smaller than (12c)/(12cR 0 ), a threshold value determined by c and R 0 [4] [5] [6] . For comparison of contact tracing in different situations, we will choose c = 0 (as in [6] [7] [8] [9] [10] ), because it is easy and as arbitrary as any other value. The value c = 0 is valid for foot-and-mouth disease where traced herds are culled.\nSeveral tracing studies have been published, some focussing on specific infections [10, 11] and some with the objective to obtain more general insight, e.g. in a general Markov-type SIR model [7] , in a model incorporating symptom development [4] , or in simulation models with specific network contact structures [8, 9] . Different assumptions were made with regard to the possibility of only tracing contacts of symptomatic infecteds (single-step tracing [4, 10, 11] ), or of iteratively tracing the contacts of traced infecteds (iterative tracing [6] [7] [8] [9] ). However, no general insight has yet been obtained in reducing R pre in relation to time-related characteristics of the infection and the tracing process, such as the latent and infectious periods, the time of symptom-based detection, and delays in the tracing process.\nIn this paper we consider detection and isolation as an autonomous process most likely governed by detection via clinical symptoms. This allows us to concentrate on contact tracing and quarantine, which are initiated by this autonomous process. Also we only regard transmission prior to control: we will consider tracing effective if R pre ,1.\nWe study an epidemic in its initial phase and use a branching process for its description, which means that the epidemic can be regarded as a growing tree. Each branch (contact) connects two nodes (contactees) with a hierarchical relation, the infector having infected the infectee. If one of the contactees is notified as being infected, by becoming symptomatic or by being traced via a secondary contact, the contact is traced with a probability p c (all parameters and functions of the model are given in Table 1 ). Each contact may be traced only once, either from infector to infectee (forwards tracing) or from infectee to infector (backwards tracing), so its traceability can be determined at the time of transmission. Thus, infection trees with traceable and untraceable contacts arise (Figure 1 ).\nOn these infection trees, two types of contact tracing are distinguished. The first type is single-step contact tracing, in which all traceable contactees of a symptomatic case are quarantined, but tracing only continues when an undiscovered infected, quarantined in this way, is detected itself ( Figure 1A ). The second type is iterative tracing, in which tracing of traceable contactees is continued, and a whole cluster of infecteds that is linked through traceable contacts is quarantined ( Figure 1B ). Such continuation would be possible if a test were available to determine the infection status of traced contactees.\nThe underlying model for infection dynamics is based on the framework of [4] . In our model, t measures time since infection of an individual, which starts with a latent period until t = t lat without transmission of the pathogen. During the infectious period, lasting from t lat to t inf , infecteds give rise to b new infecteds per unit of time, as long as they are not detected. As we are interested in the effectiveness of tracing only, we do not model possible transmission that might occur while being isolated, so transmission 'ceases' after detection of the infected. This leaves us with the basic reproduction ratio prior to detection, defined as the expected number of secondary infections per infected in a susceptible population before detection, R 0 pre . Here we have adjusted the interpretation of the model in [4] , where R 0 is divided in an asymptomatic and a symptomatic part, by adding a detection delay after becoming symptomatic. The part R 0 pre is determined by t lat , t inf , b, and the distribution of the detection time, a Gammadistribution with mean 1 (so time is measured relative to the mean detection time) and shape parameter a. Throughout our analyses, b will be scaled accordingly to achieve a desired value of R 0 pre . This model construction allows a flexible way of exploring different assumptions about the time to detection, the infectious period, and their overlap, and it enables us to evaluate tracing effectiveness for most infections.\nFor full understanding of contact tracing in our model, we analyzed four special cases regarding the infectious period and the detection time distribution. The infectious period was assumed to be either very short (all transmission occurs instantaneously, so t inf = t lat ) or very long (of infinite duration, so t inf = '). (In our model, infinite duration can be assumed because each infected will stop spreading the infection after detection. If some infecteds would never be detected, some large t inf ,' should be taken.) The detection time was assumed to be either fixed (a = ') or highly variable (a = 1, i.e. exponentially distributed). As a control we analyzed intermediate cases (results not shown) and four examples of real infections (influenza, SARS, smallpox, and foot-and-mouth disease), of which the parameter values are listed in Table 2 . These parameter values were obtained from literature [2, 3, [12] [13] [14] , assuming that the time to detection consists of the incubation period (time to symptom onset) plus a symptom-to-detection delay, which we assumed to be distributed as observed in the SARS epidemic (average 3.67 days, [2] ).\nIn our analyses tracing effectiveness will be expressed as the critical tracing probability p c *, defined as the proportion of contacts that need to be traced to achieve R pre = 1. If at least that many contacts are traced, epidemics will certainly die out if transmission during isolation or quarantine is prevented or limited to a small number of health-care workers that do not re-introduce the infection into the general community (see also [5] ). If such reintroductions cannot be excluded, a lower R pre may be aimed for. Because of this threshold of 1, the R 0 pre values (without tracing) were assumed to have some value larger than 1 (otherwise tracing would not be needed at all) and less than published R 0 values for the specific cases (Table 2) . First we study p c * as a function of R 0 pre and t lat (with tracing delay d = 0), and second as a function of t lat and d (with R 0 pre = 1.5).\nIn an epidemic where single-step tracing is applied, asymptomatic infecteds can spread the infection until they are detected and isolated. Detected infecteds are asked to name their contactees, and a proportion p c of all contactees will be reported and quarantined. Only when quarantined infecteds are detected themselves, tracing is continued ( Figure 1A) . We determined the critical tracing probability p c * as a function of the latent period for three values of R 0 pre (1.5, 2, and 3). The results are shown in Figure 2 . If the detection time is fixed (a = '), a too large latent period (larger than the detection time) results in a situation where every infected is detected before transmitting the infection, so tracing need not prevent any transmission; hence the maximum t lat of 1 in panels 2B and 2D. If we locate the approximate position of real infections (parameter values in Table 2 ) in Figure 2 , we observe that the long infectious period will be the best approximation for most infections, because we only regard pre-detection transmission and infecteds will often still be infectious at the time of detection. This does not entirely hold for influenza, which therefore fits best between the short and long infectious period. Because of the detection period distributions, smallpox and FMD are best described by panel 2D, whereas SARS and influenza fit best into panel 2C (influenza also into 2A).\nIn three special cases (panels 2A,B,D), with fixed incubation period and/or short infectious period, the proportion of contacts to be traced is at least 121/R 0 pre . This lower limit 121/R 0 pre is due to forwards tracing, when all infecteds that are traceable via their infector, are quarantined before the end of their latent period. Then, the effective reproduction ratio will be equal to the untraceable proportion of R 0 pre , i.e. R q = (12p c )R 0 pre , resulting in p c * = 121/R 0 pre . This is likely to be case with smallpox (panel 2D). However, if the latent period is short, as seen for influenza and possibly FMD, quarantine will occur too late to prevent all infections and more contacts need to be traced.\nIn the fourth panel (2C), with variable detection time and long infectious period, effective contact tracing requires a proportion of contacts smaller than 121/R 0 pre to be traced, if the latent period is large enough (like SARS). This can be explained as follows. If the detection time is variable and the latent and infectious periods are large, many infecteds will be detected before becoming infectious. If many infecteds are not infectious before being detected, the few that are should be very infectious (''superspreaders'') to attain a given R 0 pre (the average number of secondary infections before detection). Because many of the infectees of these superspreaders will be detected early (variable detection time), the superspreaders will be quarantined after backwards tracing, which adds to the effect of forwards tracing preventing infectees to reach their infectious period. Effectiveness may be very sensitive to the latent period, especially if there is little variation in the detection time. This is most apparent in the sharp transition in panel 2B, where tracing is only effective if all infectors are detected (at t = 12t lat ) before the infectious period (at t = t lat ), so t lat .0.5. The high sensitivity can be a problem for assessing the tracing effectiveness for a specific infection: the conclusion may largely depend on how correct the available estimates for the latent and incubation periods are, the incubation period determining the time to detection. Figure 2 indicates that this might be a problem for FMD.\nIn the previous section tracing was an instantaneous process: detection and isolation of a given case were followed by quarantine of traceable contactees of that case without any delay. The effect of a delay of duration d is that contactees of detected infecteds can continue transmitting the infection for an extra d time units, unless they are detected themselves during this interval.\nThe effect of a delay in each tracing step was studied by determining the critical tracing probability p c * for R 0 pre = 1.5, as a function of the latent period and tracing delay. Figure 3 shows the resulting contour plots for the four special cases with singlestep tracing, and it indicates the approximate positions of the four real infections. Along the t lat axis (d = 0) lie the R 0 pre = 1.5 curves of Figure 2 .\nIt appears that the iso-p c * contours in Figure 3 are linear in three of the four special cases (panel 3A,B,D), and approximately linear in the fourth case, with long infectious period and variable incubation time (panel 3C: t inf = '; a = 1). The slopes of all (approximate) lines are always between 0.5 and 1.5. This means that the effect of tracing delays is comparable to the sensitivity to the latent period as observed in Figure 2 , so plots of the critical tracing probability as a function of the delay will resemble the plots in Figure 2 , only mirrored (as in Figure 4 ).\nIn the contour plots in Figure 3 , tracing is ineffective in the dark grey areas (small latent period or large delay), so smallpox and SARS control are predicted to be able to cope with some delays, whereas it might be more of a problem with FMD and influenza. If forwards tracing is maximally effective and not affected by the delay, p c * = 121/R 0 pre = 0.33 as indicated by the light grey area (smallpox if d,0.5). If the detection time is variable, tracing may be effective even if a proportion less than 121/R 0 pre is traced (SARS if d,0.4, panel 3C). Because backwards tracing is needed to attain this result, it is only possible when the tracing delay is shorter than the infectious period (not shown in the Figures).\nThe contour plots show that for some combinations of latent period and delay, the sensitivity to small changes in the delay is large (contours lie close to one another). Then, small changes in the delay can have a major impact when the time of quarantine is shifted from just before to just after the end of the latent period, similar to the sensitivity to t lat as observed in Figure 2 . This effect is most dramatic if there is little variation in the detection time (smallpox: d<0.7, and FMD: d<0.2). Figure 4 shows the effectiveness of single-step tracing for SARS, smallpox, influenza, and FMD using the parameter values listed in Figure 2 . The effectiveness of single-step contact tracing without tracing delays. Effectiveness is expressed as the minimum proportion of contacts that need to be traced for effective control (critical tracing probability p c *). The plots show p c * as a function of the latent period relative to the mean time to detection (t lat ). There are four special cases: A. Short infectious period and variable time to detection; B. Short infectious period and fixed detection time; C. Long infectious period and variable time to detection; and D. Long infectious period and fixed detection time. The three curves denote p c * for different values of the pre-isolation reproduction ratio R 0 pre . Indicated by dashed lines are the average t lat for four infections, in the panels with closest correspondence to the actual parameter values ( Table 2 ). Influenza appears in two panels with long and short infectious period, because it corresponds to both parameter sets equally. doi:10.1371/journal.pone.0000012.g002 Figure 3 . The effectiveness of single-step contact tracing with tracing delays, with the pre-detection reproduction ratio R 0 pre = 1.5. Effectiveness is expressed as the minimum proportion of contacts that need to be traced for effective control (critical tracing probability p c *). The contour plots show p c * as a function of the tracing delay d and the latent period t lat , measured relative to the mean detection time, for four special cases: A. Short infectious period and variable incubation period; B. Short infectious period and fixed incubation period; C. Long infectious period and variable incubation period; and D. Long infectious period and fixed incubation period. Dark grey shadows indicate areas where tracing is ineffective, light grey shadows indicate areas where p c * = 0.33. Indicated by dashed lines are the average t lat for four infections, in the panels with closest correspondence to the actual parameter values ( Table 2 ). Influenza appears in two panels with long and short infectious period, because it corresponds to both parameter sets equally. doi:10.1371/journal.pone.0000012.g003 Figure 4 . The effectiveness of single-step and iterative contact tracing for control of influenza, smallpox, SARS, and foot-and-mouth disease. Effectiveness is expressed as the minimum proportion of contacts that need to be traced for effective control (critical tracing probability p c *); p c * is plotted as a function of the relative delay (d, proportion of the incubation period) or the absolute delay (days). doi:10.1371/journal.pone.0000012.g004 Table 2 . Panel 4A shows the relation between p c * and the relative tracing delay d, from which it appears that the general cases shown in Figures 2 and 3 are good predictors for assessing tracing effectiveness. Influenza can be placed somewhere between long and short infectious periods (panels 2A,C, and 3A,C): it is hardly effective without delay, and ineffective already if d = 0.1. SARS control requires a tracing probability p c *,121/R 0 pre which is relatively insensitive to delays. Smallpox requires p c * = 121/R 0 pre , and is insensitive to delays up to some point (d<0.6) where tracing becomes quickly ineffective. Finally, FMD can be traced effectively only if d is small, but it is sensitive to delays already if d is small and requires a tracing probability p c *.121/R 0 pre . Measured in real time (panel 4B), effectiveness of tracing appears to be highly dependent on the actual generation time of the infection. Influenza control is hardly possible, FMD control will be difficult, whereas tracing is likely to be successful for smallpox and SARS.\nSuppose a test is available to determine the infection status of traced contactees. We can then continue tracing iteratively before infected contactees are detected due to symptoms, until no further infecteds are found ( Figure 1B) .\nWe evaluated iterative tracing with and without delays for the same cases as single-step tracing, resulting in Figures similar to Figures 2 and 3 (see Supporting Information). As expected, the universal effect of iterative tracing is to lower p c *, although the lower limits 121/R 0 pre as observed in three of the four special cases remain intact. The largest difference between single-step and iterative tracing is observed when single-step tracing is on the brink of being effective, so that is the only situation where iterative tracing will make a difference (see also Figure 4 ). Without delays this difference is rather intuitive: single-step tracing is not effective if the latent period is small, but iterative tracing will always be effective, because if all contacts are traceable, the first detection is immediately followed by quarantine of every infected. This is the case with influenza, although the improvement will probably not be sufficient to making tracing effective for influenza control. On the other hand, when the latent period is large and single-step tracing is effective, iterative tracing only improves on this if there are significant delays, providing one or two more days to reach the required p c * (smallpox, SARS, and FMD).\nWe studied the effectiveness of contact tracing in a model for the start of an epidemic, that is flexible enough to use for most infections. Effectiveness of contact tracing was expressed as the minimum proportion of contacts that need to be traced to obtain a reproduction ratio prior to control of 1 (p c *). Other threshold values may be chosen if more is known about transmission of the infection to the general community while infectious individuals are being isolated or quarantined. For instance, if isolated infecteds still cause an average of 0.3 new cases in the general community after isolation or quarantine, p c * should be redefined such that R pre = 0.7.\nThe first conclusion from our model is that, for a given R 0 pre , the critical tracing probability can take any value depending on all infectious disease characteristics in the model: the latent period, the infectious period, and the detection time distribution. In contrast to some earlier publications on contact tracing [7, 9] , there exists no general expression for p c * as there is for the proportion to vaccinate for effective control in a well-mixed population. For smallpox the relation p c * = 121/R 0 pre holds reasonably well, but for SARS it is smaller, and for influenza and FMD it is larger.\nThe second conclusion is that a variable detection time improves tracing effectiveness, possibly even resulting in p c *,121/R 0 pre . This does not mean, of course, that one should aim at late detection of some infecteds (which would increase R 0 pre ), but that apparent variability is an argument to use tracing. The reason is that the few infecteds that are detected late (or not at all, which is essentially the same) will be discovered by backwards tracing which is an additional effect to forwards tracing that in itself may already result in p c * = 121/R 0 pre . It was earlier found that p c * can be smaller than 121/R 0 pre [9] , but by a different mechanism, namely the presence of shared contacts in a network.\nThe third conclusion is that the sensitivity of tracing effectiveness to the latent period and tracing delay may be large, especially in the case of single-step tracing. If this is the case already with a small delay (influenza and to a lesser extent FMD), reliability of parameter estimates will be crucial to establish whether tracing is advantageous. If it is only the case with larger delays (smallpox, SARS), tracing may be effective as long as it is done quick enough.\nThe fourth conclusion is that in most situations single-step and iterative tracing are almost equally effective. A considerable difference can only be expected if single-step tracing is not or hardly effective, which is also when the sensitivity to the latent period and tracing delay is largest (see above). Thus, influenza control will benefit from iterative tracing already without delays, whereas with the other infections one or two days may be gained ( Figure 4C,D) , thus having more time to achieve the required p c *. In the real world, the choice between single-step and iterative tracing will be based on what is possible. Capacity problems may reduce the effectiveness of iterative tracing if effort is directed towards secondary contactees prior to primary contactees; on the other hand, if not quarantine but vaccination is applied, it might be worthwhile to traced contactees of contactees even without diagnostic tests [6] .\nFor determining the cost-effectiveness of contact tracing, not only the critical tracing probability p c * is required, but also how easily that p c * can be achieved. A key aspect will be the possibility to distinguish relevant contacts: for control of sexually transmitted diseases relevant contacts are easily identified (that is, if people are willing to co-operate), but in case of respiratory pathogens like SARS or influenza the notion of relevant contacts is rather diffuse. Good insight will be obtained by not only regarding the sensitivity of tracing (p c ), but also the positive predictive value: what proportion of traced individuals is actually infected, and if we put more effort into increasing p c , how will it decrease the positive predictive value?\nA relatively easy way to increase the proportion of traced contacts for respiratory pathogens like influenza might be quarantine of households. This can be effective if households are the prime location for spread of the infection; specific models taking into account the contact structures within and between households will be better-suited to study this strategy.\nIn our model we only regarded transmission of the infection before tracing or isolation, allowing us to focus on the characteristics of the contact tracing itself. Regarding the control of animal disease epidemics, e.g. foot-and-mouth disease or avian influenza, it is not unreasonable to assume that there will be no other transmission: all infected and suspected farms will be culled and they really will stop being infectious. However, with human infections it is very likely that isolation and quarantine will be less effective. This results in a complicated situation, because transmission will not only be reduced after detection, the contact structure will also change, with only a limited number of people (health-care workers) having contact with multiple isolated patients. For the case of SARS, a more detailed model on exactly this aspect was studied by Lloyd-Smith et al [5] , who indeed concluded that minimizing within-hospital transmission might be crucial, especially if tracing and isolation occur rather slowly. Further studies are required for this complex problem. The present paper provides a good understanding of the principles of contact tracing, and how infectious disease characteristics determine the effectiveness.\nThe effect of single-step tracing can be measured by the effective reproduction ratio R q , the average number of new infections per infected. Whereas the rate of being traced backwards at time t since infection (due to detection of any infectee) is equal for all infecteds, the rate of being traced forwards (due to detection of the infector) depends on the generation-time distribution, which in turn depends on the number of traceable generations backwards in the transmission tree (traceable ancestors). Hence, infectives need to be typed according to the number of traceable ancestors j, and R q is the largest eigenvalue of the next-generation matrix K with entries k ij being the expected number of type-i infecteds (with i traceable ancestors) produced per type-j infected [15] . Because type-j infecteds only produce type-0 and type-j+1 infecteds, by untraceable and traceable contacts respectively, all entries other than k 0j and k j+1,j are equal to 0.\nEven though the matrix is infinitely large, we conjecture from all observed numerical calculations that the positive elements k j+1,j converge in the sense that |k j+1,j 2k j+2,j+1 |R0 as jR'. Therefore, for numerical evaluation we 'closed' the matrix to an (n+1) 2 matrix with k nn = k n+1,n . For the four special cases, R q could be calculated numerically in MathematicaH, but for other cases considered, the entries of the matrix had to be determined by numerical simulation. Analysis of single-step tracing with delay could be done by calculating R from the next-generation matrix, as for the model without delay (see Supporting Information for all details).\nWith iterative tracing, isolation of a single infected results in quarantine of a cluster of infecteds, all mutually linked by traceable contacts ( Figure 1B) . By recognizing these clusters of infectives, an epidemic of infected individuals can be regarded as an epidemic of traceable clusters. Each untraceable contact in the transmission tree results in infection of a new cluster index case, so the average number of untraceable contact infections caused by a single cluster, the cluster reproduction ratio R c , determines the effectiveness of iterative contact tracing. If Y(p c ) denotes the expected cumulative infectiousness of a cluster at the time of cluster quarantine, then\nAs with single-step tracing, effectiveness of iterative tracing is determined by considering the critical tracing probability (for achieving R c = 1):\nIf p c = 0, then each infective will be a separate cluster, and R c = Y(0) = R 0 pre by definition. For p c .0 the cluster size will be larger than one, but cluster infectiousness need not be larger, as backwards tracing can reduce the infectious period of superspreaders with large detection time as it does in single-step tracing.\nFor two of the four special cases (a = '), and for the case considered by Müller et al [7] (a = 1, t inf = ', t lat = 0), Y(p c ) can be calculated numerically in MathematicaH. For all other cases, including the real infections, stochastic simulations were needed.\nIncorporating delays into iterative tracing does not generally change the concept of R c , but it makes the calculation of Y(p c ) very complicated, because there is no longer a single time of cluster quarantine. Two complications arise: first, delays may cause contactees in the chain emanating from a symptomatic infected to become symptomatic themselves before the tracing process reaches them. They then initiate a new tracing process of their own within the same cluster. Second, if the infection process is faster than the tracing process, the cluster size grows infinitely large and iterative tracing becomes ineffective. We determined p c * by stochastic simulation of clusters until quarantine of the final infected (see Supporting Information for all details).\nSupporting Information Supporting information for the paper \"The Effectiveness of Contact Tracing in Emerging Epidemics\" Found at: doi:10.1371/journal.pone.0000012.s001 (0.60 MB PDF)" }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: The emergence of a pandemic strain of influenza is considered inevitable [1] . Provided the emerged strain is not too virulent, it may be possible to eliminate a nascent influenza pandemic in the source region via various combinations of targeted antiviral prophylaxis, pre-vaccination, social distancing and quarantine [2, 3] . If early elimination in the source region is not achieved, then any delay in a local epidemic that a country can effect will be highly valued. To this end, countries may consider introducing non-pharmaceutical interventions such as border screening, promoting early presentation of cases among arriving passengers, requiring the use of personal protective equipment during travels (e.g. the wearing of masks), and reducing traveler numbers. While the case for believing that measures such as these can not stop the importation of an epidemic from overseas has been argued strongly, whether it be SARS or influenza [4] [5] [6] , the extent to which such interventions delay a local epidemic is currently not well quantified, and hence of considerable interest.\nIn this paper we demonstrate how the delay to importation of an epidemic of pandemic strain influenza may be quantified in terms of the growing infection incidence in the source region, traveler volumes, border screening measures, travel duration, inflight transmission and the delay until an infected arrival initiates a chain of transmission that gathers momentum. We also investigate how the delay is affected by the reproduction number of the emerged strain, early presentation of cases among arriving passengers, and reducing traveler numbers. As noted in previous simulation modeling [7] , many aspects of this delay have a significant chance component, making the delay a random variable. Therefore, the way to quantify the delay is to specify its probability distribution, which we call the delay-distribution. Some issues of the delay distribution, such as the natural delay arising in the absence of intervention and the effect that reducing traveler numbers has on this delay has been studied previously [6] [7] [8] . Specifically, if the originating source is not specified, and homogeneous mixing of the worlds population is assumed, then the most likely time to the initial cases arising in the United States is about 50 days assuming R 0 = 2.0 [7] . The additional delay arising from travel restrictions appears minimal until a.99% reduction in traveler numbers [6] [7] [8] .\nThis paper adds to previous work [5] [6] [7] [8] by simultaneously including a wider range of epidemiological factors and possible interventions, such as elevated in-flight transmission, flight duration, the effect of wearing of mask during flight, early presentation of cases among travelers, and quarantining all passengers from a flight with a detected case at arrival.\nConsider a region in which a new pandemic strain of influenza has emerged, and a region currently free from the infection. We refer to these as the source region and the at-risk country, respectively. Travel between these countries is predominantly via commercial air travel and/or rapid transport which could potentially be subject to border screening and other interventions. We restrict our discussion to air travel. The aim is to assess the effects that a variety of non-pharmaceutical border control measures have, individually and in combination, on the time it takes before the epidemic takes off in the at-risk country. An epidemic is said to have ''taken off'' when it reaches 20 current infectious cases, after which its growth is highly predictable (i.e. nearly deterministic) and the probability of fade-out by chance is very low, if intervention is not enhanced. The source country of origin will undoubtedly have a large impact on the natural delay until importation of an epidemic, although this is difficult to quantify [7] . An alternative is to fix the originating city, for example a highly connected city such as Hong Kong [6] , with the obvious effect that results are highly dependent on the choice. We adopt no specific source region, but assume that the number of international travelers originating from it is reasonably small (see Methods), suggestive of a rural or semirural source region [2] . It is further assumed that the current heightened surveillance for pandemic influenza is continued and that a nascent pandemic with human-to-human transmission is identified and the pandemic is declared when there are 10 concurrent cases in the source region.\nFor an epidemic to take off in an at-risk country, a series of events need to occur. First, the epidemic needs to get underway in the source region. Second, an intending traveler needs to be infected shortly before departure. Third, the infected traveler must actually travel and successfully disembark in the at-risk country. Fourth, the infected traveler, or fellow travelers infected during the flight, must initiate an epidemic in the at-risk country with the infectiousness that remains upon arrival. Finally, the epidemic needs to reach a sufficient number of cases to begin predictable exponential growth.\nInternational spread of the emerged pandemic strain of influenza may occur when a recently infected person travels. By 'recently infected' we mean that their travel is scheduled to occur within ten days of being infected. We assume that the number of individuals traveling from the source region to the at-risk country each day is known. The probability that a randomly selected traveler is a recently-infected person is taken to be equal to the prevalence of recently-infected people in the source region on that day. The incidence of infection in the source region is assumed to grow exponentially initially, with the rate of exponential growth determined by the disease reproduction number (the mean number of cases a single infective generates by direct contact) and the serial interval (the average interval from infection of one individual to when their contacts are infected) ( Figure 1A) .\nThe time since infection of a recently-infected traveler is a key component of the calculations, because it affects the chance of positive border screening, the chance of in-flight transmission and the infectivity remaining upon arrival in the at-risk country. The time since infection at the time of scheduled departure is random and the dependence of its probability distribution on the exponential growth rate of infection is illustrated by Figure 1B (see also Supporting Information). The higher the epidemic growth rate in the source region, the greater the probability than an infected traveler will have been infected more recently.\nIt is assumed that individuals detected by departure screening are prevented from traveling. To be detected by screening an infected traveler must be symptomatic and positively screened. An individual is assumed to become symptomatic 48 hours after being infected (cf. [3] who use 1.9 days). The probability of being symptomatic when presenting for departure screening is computed from the curve in Figure 1B . The distribution of the time since infection immediately after departure screening, given that the infected traveler was not detected, is given by the curve in Figure 1C . It contains an adjustment for the probability of being detected at departure.\nThe instantaneous rate at which susceptible contacts are infected depends on the time since infection, and is described by an infectiousness function ( [9] , page 45). We use a peaked infectiousness function, motivated by viral shedding and household transmission data [2] , which has a serial interval of 2.6 days. The basic reproduction number (R 0 ), namely the reproduction number when there is no intervention in place and every contacted individual is susceptible, is given by the area under the infectiousness function. However, our concern is with the effective reproduction number R that holds when various interventions are in place. We obtain any R by simply multiplying the infectiousness function by the appropriate constant (to make the area under the curve equal to R). This keeps the serial interval the same. In the absence of suitable data we assume for most scenarios that the aircrafts ventilation and filtration systems are functioning properly, and that infected travelers transmit the infection at the same rate during a flight as they would while mixing in the community. We examine the sensitivity of this assumption by increasing the inflight transmission by as much as 10-fold (as could potentially happen if air-circulation and filtration systems malfunction, e.g. see [10] ). The in-flight transmission rate is set to zero under the optimistic scenario that all travelers wear 100% effective masks during transit. In terms of a sensitivity analysis this illustrates what would be achievable in a best-case scenario. The number of offspring that an infected traveler infects during a flight is a random variable, taken to have a Poisson distribution with a mean equal to the area under the infectiousness function over to the flight duration.\nTravelers infected during flights of less than 12 hours duration are asymptomatic at arrival and will not be detected by screening. The probability that an arriving traveler who was infected in the source region is detected on arrival is computed from the distribution of the time since infection on arrival. This distribution is obtained from the curve in Figure 1C by shifting it to the right by an amount equal to the duration of the flight. The distribution of the time since infection for an individual infected in the source region, who passes through arrival screening undetected has a further adjustment for the chance of being detected at arrival ( Figure 1D ). This curve shows that an infected traveler who escapes detection at departure and arrival is highly likely to enter the at-risk country with most, or all, of their infectious period remaining.\nAuthorities are assumed to implement one of two control options when detecting an infected traveler by arrival screening. Under option one (individual-based removal), all passengers who test negative are released immediately and only passengers who test positive are isolated. Under the second option (flight-based quarantining), authorities prevent all passengers from dispersing into the community until the last person has been screened from that flight. Should any one passenger be detected as infected then all passengers will be quarantined, as previously recommended [5] .\nTransmission chains can be initiated in the at-risk country by infected travelers who mix within the community upon arrival. Suppose now that a flight arrives with one, or more, infected passengers who mix within the community. We classify these infected arrivals into those who are 'pre-symptomatic' and those who are 'symptomatic' at entry. It is assumed that the 'symptomatic' infected arrivals do not recognize their symptoms as pandemic influenza and will not present to medical authorities. In other words, they spend the remainder of their infectious period mixing in the community. On the other hand, the 'presymptomatic' infected arrivals, including all individuals infected during flight, are assumed to mix freely in the community only from entry until they present to medical authorities after some delay following the onset of symptoms.\nNot all infected travelers entering the community initiate a 'major' epidemic, even when the reproduction number (R) exceeds one. Quite generally, the distribution of the size of an epidemic initiated by an infected arrival is bimodal, with distinct peaks corresponding to a major epidemic and a minor outbreak ( Figure 1E ). In the latter event the outbreak simply fades out by chance despite there being ample susceptibles in the population for ongoing trans- In (B), the step illustrates the probabilistic removal of travelers who have completed their incubation period. In (D), the distribution of time since infection in (C) will have shifted to the right by an amount equal to the flight duration, and cases incubated in-flight may be detected by symptomatic screening, as will those symptomatic cases that were not detected previously. Screening sensitivity for this illustration is 60% on both departure and arrival. (E) Upon entering the community undetected, an infected traveler may initiate a minor (inconsequential) or major epidemic, depending on the characteristics of the disease and public health policy. doi:10.1371/journal.pone.0000143.g001 mission [11] . The number of cases in an outbreak that fades out is typically very small compared to an epidemic.\nThe probability that a typical infective generates a local epidemic is computed by using a branching process approximation [12] for the initial stages of the epidemic, and equating 'epidemic' with the event that the branching process does not become extinct. This calculation is well known (e.g. [13] , page 473), but is modified here to allow for the fact that the process is initiated by a random number of infected arrivals and some of them have spent a random part of their infectious period before arriving in the at-risk country. The distribution for the random number of individuals infected by an infected individual when all their contacts are with susceptible individuals is needed for the calculation. The lack of data prevents a definitive conclusion for the most appropriate offspring distribution for influenza transmission [14] , and we use a Poisson distribution with a mean equal to R, discounted for individuals who spent only some of their infectious period mixing in the at-risk country. A Poisson offspring distribution is appropriate when the area under the infectiousness function is non-random (i.e. all individuals have the same infection 'potential'). We assume that R is the same in the source region and the at-risk country. For an undetected infected traveler and all their in-flight offspring to fail to initiate an epidemic on arrival, all of the chains of transmission they initiate must fail to become large epidemics (see Supporting Information).\nWe calculate the probability distribution of D, the total delay until an epidemic gathers momentum by noting that it is given by D = D 1 +D 2 , where D 1 is the time until an epidemic is first initiated and D 2 is the time from initiation until the local epidemic gathers momentum. For an epidemic to be first initiated in the at-risk country on day d, it must have not been initiated on all previous days. Hence the probability distribution of the time delay (D 1 ) until the epidemic is first initiated in the at-risk country following identification in the source region is described by:\nPr (D 1~d )~ p p 1 p p 2 p p 3 ::: p p d{1 p d where p d denotes the probability that the epidemic is initiated on day d , and p p d~1 {p d denotes the probability that the epidemic is not initiated on day d (see Supporting Information for calculation of p d ).\nOnce successfully initiated, an epidemic may initially hover around a handful of cases before reaching a sufficient number of cases for its growth to become essentially predictable. As mentioned, 20 concurrent cases is our criterion for an epidemic to have gathered momentum. We determine the distribution of D 2 , the time to this occurrence, from 10,000 stochastic simulations and approximate this empirical distribution by a shifted gamma distribution. Our criterion of 20 concurrent cases is conservatively high, as results from the theory of branching processes shows that the probability of a minor epidemic (and hence no take-off) starting from 20 concurrent cases is about 3610 28 when R = 1.5, and even smaller for higher values of R. Finally, the distribution of the total delay (D = D 1 +D 2 ) from the pandemic being identified in the source region until 20 cases in the at-risk country was calculated by the convolution of the distributions of D 1 and D 2 .\nFor the illustrative purposes, we chose values of 1.5, 2.5 and 3.5 for R, which encompass estimates proposed for previous pandemics [2, 3, 15] . The number of people within the infected source region was assumed reasonably small (5 million), and there was one flight per day traveling from the source region to the at-risk country carrying 400, 100 or 10 passengers. A higher number of travelers affects the delay only marginally, assuming the epidemic takes off in the source region (see Results). We assume a typical travel duration between attempted departure and possible arrival of 12 hours, but also examine the effect of varying this from 0-48 hours. The time to presentation following symptom onset is varied from 'immediately' to 'never presenting', with a time of 6 hours considered likely in the presence of an education campaign. The sensitivity of symptomatic screening is varied from 0-100%, with results presented for 0, 50 and 100% sensitivity.\nThe probability that a recently infected traveler evades screening is substantial even if screening reliably detects symptomatic travelers (Figure 2A) , because the typical travel duration is shorter than the 2-day incubation period. In addition, during the early stages of the epidemic a high R in the source region acts to increase the probability that an infected traveler has been infected quite recently and hence will escape detection due to being asymptomatic during their travels (Figure 2A ). For example, assuming 100% sensitivity for detecting symptomatic infection, we calculate that during the early stages of the epidemic the proportion of infected travelers that evade both departure and arrival screening after 12 hours of travel is 0.26, 0.45 and 0.59 for disease reproduction numbers 1.5, 2.5 and 3.5, respectively.\nAs the duration of travel approaches the disease incubation period, effective symptomatic screening substantially reduces the likelihood that a traveler evades screening and initiates an epidemic ( Figure 2B ). Reducing the time from the onset of symptoms to presentation (and subsequent isolation) for each infected arrival also reduces the probability that a major epidemic is initiated, however the best case scenario of infected travelers and all their in-flight offspring presenting immediately following the onset of symptoms still poses a substantial risk of epidemic initiation arising from pre-symptomatic transmission ( Figure 1C ).\nThe delay contains a fairly substantial natural component, primarily due to the time it takes to increase the number of infectives in the source region sufficiently to make the chance of a recently infected traveler appreciable ( Figure 3A ), and the time (D 2 ) it takes for a local epidemic in the at-risk country to gather momentum following successful seeding ( Figure 4A ). In the absence of any interventions, the number of infected individuals who successfully enter the community of the at-risk country initially increases exponentially ( Figure 3A ). With individual-based removal of infected travelers, the number of individuals entering the at-risk country undetected by screening is proportionately reduced over the course of the epidemic ( Figure 3A ). With flightbased quarantining, the number of infected individuals entering the at-risk country undetected is dramatically reduced over the course of the epidemic, even for relatively insensitive screening ( Figure 3A ). With flight-based quarantining, the number of infected passengers slipping through undetected is bimodal, with the first peak occurring when the number of infected travelers attempting to travel is still in single figures.\nWithout screening, the daily probability that an epidemic is initiated (p d ) increases, and becomes near certain once the number of infected travelers arriving undetected exceeds about 10 ( Figure 3B, solid line) . With screening and individual-based removal of infected individuals, p d follows a similar pattern only reduced somewhat. With screening in combination with flightbased quarantining, this probability is changed dramatically. After an initial rise it dips, to become essentially zero during the height of the epidemic in the source region ( Figure 3B , dotted line). This arises because once a flight has several infected travelers, the probability that at least one is detected approaches one (even if screening is imperfect), and all passengers on such a flight are quarantined. Once the epidemic starts to wane in the source region (assuming the unlikely event of the pandemic strain is restricted to the source region), the probability of initiation rises once again. The corresponding distribution of D 1 , the delay until The effects of R and the time from symptom onset to presentation on the probability that an infected traveler, having entered the wider community following arrival, will initiate an epidemic. There is no screening. doi:10.1371/journal.pone.0000143.g002 the epidemic is first initiated in the at-risk country, is bi-modal in the presence of screening ( Figure 3C) .\nAlthough flight-based quarantining is effective in preventing the entry of infected travelers during the height of the epidemic, a substantial cumulative risk of initiation has already occurred before this from the handful of infectives that have slipped through undetected ( Figure 3B ). Hence, whilst the effect of border screening, particularly in conjunction with flight-based quarantining, on the daily probability of initiation is dramatic, its effect on the delay to initiation is much less pronounced ( Figure 3C ). Border screening, even with perfect sensitivity for detecting symptomatic cases, tends to increase D 1 , the time to an epidemic being initiated, by a matter of days to weeks. The time (D 2 ) from initiation (the arrival of the index case) to an epidemic reaching 20 concurrent cases within the at-risk country is adequately modeled using a shifted Gamma distribution ( Figure 4A ). The convolution of this right-skewed Gamma distribution with the left-skewed delaydistribution of D 1 (Figure 3C ) yields the distribution for D, the total delay until the epidemic reaches 20 cases in the at-risk country ( Figure 4B ). The distribution of D is approximately symmetrical. The effect of border screening on the total delay D is quite modest, though sensitive to how screening is implemented. For example, with R = 1.5 and 400 travelers per day, 100% sensitive screening with individual-based removal increases the median delay from 57 to 60 days ( Figure 4B ). Flight-based quarantining would extend the median delay to 70 days. In general, the added delay arising from flight-based quarantining is about four-fold that arising from individual-based removal.\nThe natural component of the delay is highly sensitive to the disease reproduction number ( Figure 5A ). For example, with 400 passengers per day departing the source country and in the absence of any interventions, the median delay ranges from a low of 17 days for R = 3.5 to 57 days for R = 1.5 ( Table 1 ). The delay is less sensitive to the number of intending travelers, with little appreciable increase in the median delay occurring until traveler numbers become very low ( Figure 5B ). For example, if R = 1.5, with no other border control measures, decreasing the number of intending travelers departing the source region from 400 to 100 per day increases the median total delay D from 57 to 66 days. A further decrease in the number of intending travelers to 10 per day increases the median delay to 83 days ( Table 1) .\nThe delay is quite insensitive to the rate of transmission in-flight. For example, with R = 1.5, a 12-hour flight, 400 travelers per day and no other interventions, preventing in-flight transmission altogether increases the median delay from 57 to 58 days. Conversely, doubling the rate of in-flight transmission reduces the median delay from 57 to 56 days. A 10-fold increase in the rate of transmission in-flight only decreases the median delay from 57 to 53 days. Encouraging the early presentation of cases among travelers following the onset of symptoms has a limited effect on the delay distribution ( Figure 5C ). For example, for R = 1.5, 400 intending travelers per day and no other interventions, reducing the time to presentation from 'never presenting' to 6 hours increases the median delay from 57 to 61 days. Immediate presentation at symptom onset only increases the median delay a further day in this scenario.\nIn general, the additional delay achieved by introducing nonpharmaceutical border control measures is generally small in comparison with the natural delay ( Figure 5D ). For the scenario with R = 1.5 and 400 intending travelers per day, a combination of 100% flight-based quarantining, 100% compliance with mask wearing during travel and immediate presentation at symptom onset extends the estimated median delay from 57 to 79 days ( Figure 5D ). This added delay diminishes in absolute terms as R increases. For example, if the same interventions are applied with R = 3.5, the median delay is extended from 17 to just 20 days ( Figure 5D ). The one exception to this generalisation is when travel numbers are reduced dramatically. The added delay achieved when a drastic reduction in travel numbers is combined with other border control measures appears to be greater than adding the delays each achieves on its own. For example, if R = 1.5, and we reduce the number of intending travelers from 400 to 10 per day, implement 100% flight-based quarantining, implement compulsory mask wearing during travel and presentation at 6 hours following symptom onset then there is a substantial probability (0.74) that the pandemic strain will never be imported (assuming the epidemic is confined to the source country). The estimated quartile delay (the median in this case is undefined) to the start of a major epidemic in an at-risk country is extended from 50 to 125 days. Again, the added delay decreases rapidly as R increases, and if the above interventions were applied with R = 3.5, the estimated median delay is extended from 17 to 26 days, and the importation of the epidemic is certain ( Figure 5D ).\nWe have formulated a model of the importation of an infectious disease from a source region to an at-risk country that permits a comprehensive analysis of the effect of border control measures. Our results are most relevant to the early stage of a pandemic when most cases are contained within a single source region. Once the pandemic has spread to several countries, models with greater complexity and ability to more realistically model global mixing patterns [6] [7] [8] are required. Our model is developed with a pandemic-strain of influenza in mind, but could apply to any emerging infectious disease that is transmitted from person to person. We have assumed a Poisson distribution for the number of secondary infections, which a natural choice when each infected individual has the same infectivity profile. A distribution with a larger variance is appropriate when individuals vary substantially in their infectiousness. Our results are conservative in the sense that they give an upper bound for the probability that an infected traveler manages to initiate an epidemic, compared to an offspring distribution with a greater variance but the same reproduction number [14] .\nThe nature of the next pandemic influenza virus, and particularly its reproduction number, is uncertain. If its reproduction number is low (R,2.0), our results indicate that at-risk countries receiving a reasonably small number of travelers (say 400 per day) from the infected source region can expect a natural delay until importing an epidemic of the order of 2 months. This is quite variable and under favourable conditions it could be 4 months. However, the natural delay decreases rapidly as R increases.\nThe additional delay from isolating individuals detected by border screening is merely a few days under most plausible scenarios, even if both departure and arrival screening is introduced and screening detects every symptomatic traveler. While the extra delay is more than quadrupled if flights with a detected case(s) are quarantined, the effect remains modest (weeks at most) and it is questionable whether the extra delay achieved warrants the disruption created by such a large number of quarantined passengers.\nIn-flight transmission is a commonly raised concern in discussions about the importation of an infection, so inclusion of in-flight transmission is an attractive feature of our model. Events of substantial in-flight transmission of influenza have been documented [10, 16] and modeling of indoor airborne infection risks in the absence of air filtration predicts that in-flight transmission risks are elevated [17] . However, it difficult to estimate the infectiousness of influenza in a confined cabin space, as there is undoubtedly substantial under-reporting of influenza cases who travel and fail to generate any offspring during flight. Provided the aircraft ventilation system (including filtration) is operational, it is considered that the actual risk of in-flight transmission is much lower than the perceived risk [18] . Our results indicate that the delay is relatively insensitive to the rate of in-flight transmission, making in-flight transmission less of an issue than commonly believed. A highly elevated transmission rate inflight will hasten the importation of an epidemic only marginally. Consistent with this, eliminating in-flight transmission by wearing protective masks increases the delay only marginally.\nEarly presentation by infected arrivals not detected at the borders was found to add only a few days to the delay. To some extent this arises due to our assumption that pre-symptomatic transmission can occur, for which there is some evidence. In contrast, Ferguson et al. [2] assume that the incubation and latent periods are equal, with a mean of 1.5 days. In their model presymptomatic transmission is excluded and infectiousness is estimated to spike dramatically immediately following symptom onset and declining rapidly soon afterwards. Under their model assumptions, immediate presentation at onset of symptoms would reduce transmission effectively. However, as presentation occurs some time after onset of symptoms and the bulk of infectivity occurs immediately after onset of symptoms the results on the effect of early presentation of cases are likely, in practical terms, to be similar to those found here. Given the variable nature of influenza symptoms, there is likely to be a difference between the onset of the first symptoms as measured in a clinical trial (e.g. [19] ) and the time that a person in the field first suspects that they may be infected with influenza virus. To fully resolve the issue of how effective very early presentation of infected travelers is in delaying a local epidemic we need better knowledge about the infectiousness of individuals before and just after the onset of symptoms. It is assumed that the pandemic is identified and declared when there are 10 concurrent cases in the source region attributed to human-to-human transmission, and that screening is applied at both departure and arrival. The time between screening events is assumed to be 12 hours and infected travelers are not isolated following the onset of symptoms. Of the border control measures available, reducing traveler numbers has the biggest effect on the delay and even then it is necessary to get the number of travelers down to a very low number. An equivalent control measure is to quarantine all arriving passengers with near perfect compliance.\nOur results indicate that short of virtually eliminating international travel, border control measures add little to avoiding, or delaying, a local epidemic if an influenza pandemic takes off in a source region. All forms of border control are eventually overwhelmed by the cumulative number of infected travelers that attempt to enter the country. The only way to prevent a local epidemic is to rapidly implement local control measures that bring the effective reproduction number in the local area down below 1, or to achieve rapid elimination in the source region, in agreement with other recent studies [6] [7] [8] . Preventing the exponential growth phase of an epidemic in the source region appears to be the only method able to prevent a nascent influenza pandemic reaching atrisk countries.\nText S1 Estimating the daily probability of epidemic initiation Found at: doi:10.1371/journal.pone.0000143.s001 (0.08 MB PDF)" }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: Editor's note: The potential efficacy of pre-and post-exposure prophylaxis against Ebola virus infection, as well as the fundamentally important question of whether neutralizing bodies are important for Ebola virus resistance, is addressed by a related manuscript in this issue of PLoS Pathogens. Please see doi: 10 .1371/journal. ppat.0030002 by Feldmann et al.\nPassive transfer of relatively high concentrations of neutralizing antibodies can protect against challenge with a range of viruses in animal models and in humans [1] [2] [3] . Protection in some cases is in the form of sterilizing immunity, i.e., no viral replication is observed following challenge [2, 4, 5] . In other cases (e.g., [5, 6] ), some replication is observed but protection from disease is achieved, presumably because neutralizing antibody sufficiently blunts infection for T cell and innate immunity to resolve infection [7] . It might be expected that passive neutralizing antibody would be most effective against challenge with acute viruses. Many acute viral infections are resolved even in the absence of neutralizing antibody, and the blunting effect of passive antibody would provide more time for the development of effective cellular immune responses. In contrast, chronic viruses may present a greater challenge to passive antibody, since, in the absence of sterilizing immunity, there is a window of opportunity for the virus to establish a chronic infection before cellular immunity can be mobilized.\nEbola virus (EBOV) causes a severe acute infection in humans [8] . Infection with the Ebola Zaire strain, Zaire ebolavirus (ZEBOV), produces mortality in the range of 60%-90% [9] with death generally occurring around 7-11 d following the appearance of symptoms [8] . There is a single report describing the use of convalescent sera to treat EBOV infection [10] . However, the patients in this report may have already been through the worst stages of the disease, and it is not clear that serum antibodies were responsible for their recovery [10] . Further, neutralizing antibody titers in survivors of EBOV infection tend to be rather low, although we have isolated a neutralizing human monoclonal antibody (mAb), KZ52, of good potency from a convalescent individual [11] .\nThe ability of passive antibody to protect against EBOV has been investigated in a number of animal models. The guinea pig and mouse models use EBOVs that have been serially passaged to adapt to replication in the respective animals and are highly lethal. Protection has been demonstrated in the guinea pig model using neutralizing horse, sheep, and goat immunoglobulin G (IgG) against EBOV [12, 13] and the human anti-EBOV GP mAb, IgG KZ52. This antibody neutralizes ZEBOV (1995, Kikwit) with a 50% inhibitory concentration (IC 50 ) of 0.05-0.3 lg/ml and an IC 90 of 0.5-2.6 lg/ml in Vero cells [11, 14] and an IC 50 of approximately 0.05-1 lg/ml and a IC 90 of 0.5-2 lg/ml in primary human monocytes/macrophages [14] . We showed that when administered subcutaneously at a dosage of 25 mg/kg up to 1 h after challenge, the antibody protects against robust ZEBOV challenge (10,000 plaque-forming units [pfu] ) in the guinea pig model [6] .\nMacaques provide a model of EBOV infection that is likely closer to human infection. The human virus can be used directly in macaques without need for adaptation and the course of disease mirrors that seen in humans [8] . In cynomolgus macaques (Macaca fascicularis), ZEBOV infection produces a mortality rate of 100% with death occurring 6-8 d following infection with 1,000 pfu [15] , while in rhesus macaques (Macaca mulatta) ZEBOV produces about 100% mortality with death occurring 7-10 d after infection with 1,000 pfu [16] . In contrast to the guinea pig experiments, the passively transferred polyclonal equine neutralizing IgG described above provided only some minor benefit in the form of a slight delay in the onset of viremia from day 5 to day 7 [13] following ZEBOV challenge of cynomolgus monkeys. No significant reduction in mortality was observed. However, protection against EBOV in primates has been observed in a low dose challenge model. Thus, neutralizing equine IgG protected baboons from ,30 LD 50 (50% lethal dose) ZEBOV challenge when the IgG was given up to 1 h after infection and the serum contained high neutralizing antibody titers (1:128 to 1:512) [17, 18] , and, similarly, neutralizing ovine serum protected baboons against 0.6 LD 50 ZEBOV challenge [19] .\nHere, we studied the ability of passively transferred neutralizing human mAb KZ52 to protect against ZEBOV challenge in rhesus macaques. This passive transfer failed to protect the macaques against challenge with ZEBOV, and, furthermore, had a minimal effect on the explosive viral replication following infection. We showed by ELISA that antibody was present at high levels in serum of the monkeys and that neutralization escape was not responsible for the resistance of virus to antibody prophylaxis.\nTo evaluate whether IgGl KZ52 could protect against Ebola virus infection in a nonhuman primate animal model, antibody was passively transferred to rhesus macaques followed by challenge with the 1995 ZEBOV (Kikwit) isolate 24 h later. Protection against virus challenge by neutralizing antibodies in naive animals often requires high doses of antibody [2] . Therefore, we used a high dose of 50 mg/kg KZ52, which was close to the maximum practically achievable. In addition, we gave a second bolus of 50 mg/kg of KZ52 on day 4 following infection. The results for the four antibodytreated animals show a steady increase in plasma viremia up to 10 5 -10 7 pfu/ml on day 7 ( Figure 1 ). These levels of plasma viremia closely parallel those seen in the control animal and typically seen in historical controls [15] . The second bolus of antibody given on day 4 did not appear to have any impact upon the rate of increase of plasma virus ( Figure 1 ). Three of the treated animals were euthanized when moribund at day 9 or 10 post infection. The fourth treated animal showed a decrease in plasma viral load after the peak and survived to day 28 before becoming moribund when it too was euthanized. Although monkey CH46 had less severe symptoms than the other animals in the study, it was concluded that this animal, too, was suffering from disease due to ZEBOV, as evidenced, for example, by copious Ebola virus antigen in the lungs (see below).\nSerum antibody (KZ52) loads were measured 1 d before virus challenge (day À1) and 4 d after challenge but before antibody boosting (day 4) by an ELISA designed to detect KZ52 as a human antibody that has bound to immobilized ZEBOV glycoprotein. The serum KZ52 antibody levels on day 4 were in the approximate range 200-400 lg/ml ( Table 1 ). The Figure 1 . Plasma Viremia in Macaques Challenged with ZEBOV Shown is the measured viremia, in log 10 pfu per ml, for four antibodytreated monkeys (CH46, CH56, CH57, and CH83) and one untreated control animal (EHD) at days 4, 7, 9, and 10 in plasma by plaque assay as described in Materials and Methods. 50 mg/kg of KZ52 IgG1 human antibody [11] was given intravenously to four rhesus macaques 1 d before and again 4 d after challenge with 1,000 pfu (intramusculary) of the 1995 ZEBOV (Kikwit) isolate. Ab, antibody. doi:10.1371/journal.ppat.0030009.g001\nPLoS Pathogens | www.plospathogens.org January 2007 | Volume 3 | Issue 1 | e9 0063\nAnti-Ebola Antibody Activity In Vivo\nEbola virus is one of the most feared of human pathogens with a mortality that can approach 90% and an extremely rapid disease course that can lead to death within days of infection. Antibodies able to inhibit viral infection in culture, neutralizing antibodies, can typically prevent viral infection in animals and humans when present prior to infection, at sufficient concentration. Such neutralizing antibodies may be provided through passive administration or induced by vaccination. We have previously shown that a human neutralizing antibody can protect guinea pigs against Ebola virus. However, here we show that this antibody does not protect monkeys against Ebola virus and surprisingly appears to have very little impact upon the rapid course of infection, despite being present at very high levels in the blood of the monkeys. We conclude that administering antibody prior to or immediately following exposure to Ebola virus, for example, after an accident in a research setting or a bioterrorist attack, is unlikely to be effective in preventing disease. Recent successes in protecting monkeys against Ebola virus through vaccination may be independent of antibody, or, more likely, critically dependent on the cooperation of antibody and cellular immunity.\ntwo control monkeys, EHD (untreated and challenged), and a negative monkey (neither treated nor challenged), had very similar background levels of reactivity to the glycoprotein and anti-human antibody as each of the treated monkeys before treatment. A 50-mg/kg dose typically produces serum mAb concentrations in animals on the order of 500 lg/ml after injection [6] . Since the neutralization titer of KZ52 (IC 90 ) for ZEBOV is on the order of 0.5-2.5 lg/ml, depending upon the target cell and the presence of complement, the concentrations of KZ52 in the animals at the time of challenge and for the first few days were, as expected, greater than, or on the order of 100 3 IC 90 . These concentrations typically provide sterilizing immunity against challenge by a number of viruses [4, 20] .\nOne formal possibility is that the neutralizing antibody has little effect on the course of infection in the treated monkeys because of the rapid emergence of neutralization escape mutants. Accordingly, virus was isolated from a selection of plasma from day 4 (monkeys CH56, CH57, and CH83) and day 7 (monkeys CH56 and CH57). All of these viruses were sensitive to KZ52 so that essentially 100% neutralization was observed in vitro at 40 and 400 lg/ml KZ52 in a plaque assay (see Materials and Methods) using Vero E6 target cells.\nIn order to gain a better understanding of any differences in pathology between the control and antibody-treated animals, the levels of virus in different organs were surveyed postmortem. Viral levels in the liver, spleen, kidney, adrenal glands, lung, and mesenteric and inguinal lymph nodes were high (10 4 -10 6 pfu/g) in the control and three of the four treated animals (Table 2 ). However, monkey CH46, who survived much longer than the other animals (to day 28) showed some major histopathological differences from the other infected monkeys and from the norm for ZEBOV infection [15] . Relatively low viral levels were observed in most of the organs of CH46, and none in the liver, spleen, and adrenal glands. In addition, large immunoreactive monocytes were found in the blood of monkeys CH56, CH57, and CH83, but not in CH46 (Figure 2) . Typically, smaller immunoreactive monocytes are seen with ZEBOV infection [15] . The presence of large immunoreactive monocytes may simply reflect uptake of antibody-coated virions via Fc receptors and subsequent viral clearance. However, it is interesting to note that previous studies have implicated mononuclear phagocytes as vehicles for transport of filovirus particles to specific organs such as liver and spleen [21] [22] [23] [24] . If virus particles could remain infectious following Fc receptormediated uptake in a subset of cells (compare DC-SIGN mediated uptake of HIV-1 by dendritic cells [25] ), then the course of disease in monkeys CH56, CH57, and CH83 might represent the net result of inhibition by neutralization and enhancement by antibody-mediated cellular uptake. Interestingly, monkey CH46, who fared somewhat better than the others and lacked virus in the liver and spleen, did not show the presence of large immunoreactive monocytes. This is suggestive of lowered Fc receptor-mediated uptake of virus or reduced activity of the mononuclear phagocytic system.\nHere, we describe the case of a potent neutralizing human monoclonal antibody, administered to give a high serum concentration, which is shown to be unable to protect macaques against challenge with a lethal dose of ZEBOV. The antibody appeared to have very little effect on the course of virus replication or disease in three of four treated animals. Neutralization escape does not appear to explain the lack of protection observed by the antibody. In one animal, a more limited infection was observed, but this macaque did also eventually succumb to the effects of viral disease.\nThe challenge dose of 1,000 pfu used corresponds to the amount of EBOV contained in a relatively small quantity of fluid (on the order of 1 ll) from an infected individual given the high titers of virus typically found in such individuals (on the order of 10 6 pfu/ml of blood, for example). Therefore, the challenge dose was not unreasonable in terms of a natural exposure to virus. The negative results with passive antibody contrast strongly with recent successes in preventing EBOV infection in macaques through vaccination [26, 27] . Does this mean that neutralizing antibody is unimportant in vaccine protection?\nThe answer to this question must await further studies. However, a plausible hypothesis to explain all the data would still allow for an important contribution of antibody to vaccine protection. This hypothesis would argue that passive antibody is unable to completely block all EBOV entry to cells, and once a few cells are infected, virus replication is so explosive that it cannot be contained by a de novo generated cellular immune response. Vaccination, on the other hand, will provide CD8þ memory T cells that can be rapidly recruited to become effector cells and limit infection. Certainly, although mAb KZ52 was able to provide protection from disease following ZEBOV challenge in the guinea pig model, immunity was not sterilizing and some viral replication was noted [6] . Since 1 pfu of EBOV is a lethal dose for primates [8] , a failure of passive antibody to achieve sterilizing immunity may be critical. Immunohistochemistry, as discussed above, gives some intriguing hints that uptake of antibody-coated virions by monocytes may possibly have a role to play in the course of infection following antibody treatment. We note, however, that previous in vitro studies using isolated human monocytes/macrophages did not find evidence of infectivity-enhancing antibodies [14] . More detailed in vitro and in vivo investigations will be required before any firm conclusions can be drawn. We also note that our experiments were carried out with a single neutralizing monoclonal antibody. It is possible that a more favorable outcome may have been apparent for a combination of neutralizing antibodies or even a combination of neutralizing and nonneutralizing antibodies [2] . However, these possibilities should be weighed against the very high concentrations of neutralizing monoclonal antibody used in the experiments and the efficacy of the antibody in the guinea pig model.\nIn summary, the inability of high concentrations of neutralizing antibody to even slow viral replication in infected macaques is remarkable and implies a mechanism of infection propagation that is virtually insensitive to antibody. Overall, the results suggest that monoclonal antibody prophylaxis or post-exposure prophylaxis alone are unlikely to be effective strategies in protecting against EBOV, for example, following a needle-stick accident in a research setting or a bioterrorist attack.\nPassive transfer experiment. 50 mg/kg of KZ52 IgG1 human antibody [11] was given intravenously to rhesus macaques (weight, 3.9-4.4 kg) 1 d before challenge (day 0) with 1,000 pfu intramuscularly of the 1995 ZEBOV (Kikwit) isolate and again 4 d later (day þ4). One monkey was not given any antibody treatment. The animals were carefully monitored for signs of disease, and Ebola virus plasma viremia was determined at days 4, 7, 9, and 10 in serum by plaque assay as described below.\nThe investigators adhered to the Guide for the Care and Use of Laboratory Animals when conducting research, using animals [28] . The United States Army Medical Research Institute of Infectious Diseases (USAMRIID) animal facilities and animal care and use program are accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International. All infectious material and animals were handled in a maximum-containment biosafety level 4 facility at USAMRIID under standard operating conditions. Antibody purification. IgGl KZ52 was produced and purified as described by Parren et al. [29] and was .98% pure, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and contained ,1 IU of endotoxin/ml, as determined in a quantitative chromagenic Limulus amoebecyte lysate assay (BioWhittaker, Cambrex, http://www.cambrex.com).\nViremia determined by plaque assay. Plasma viremia and viral loads in organs was determined by virus titration in a conventional plaque assay on Vero E6 cells, as described elsewhere [13, 21] .\nNeutralization assay. Samples were diluted into Eagle's minimal essential medium (EMEM; Invitrogen, http://www.invitrogen.com) with 5% heat-inactivated fetal bovine serum (FBS). In the presence and absence of a constant dilution of 1:10 or 1:100 of 4 mg/ml KZ52 (thus, 0.4 mg/ml or 0.04 mg/ml), plasma were titrated from 10 À1 to 10 À6 dilutions. Viremia was determined by counting pfu on Vero E6 cell monolayers. Cells grown to confluence in 6-well plates were given 0.2 ml of plasma with and without additional KZ52. The titrated samples were incubated in the presence of KZ52 for 1 h at 37 8C in 5% CO 2 . After absorption, the cells were overlaid with 2 ml of EMEM containing 5% FBS, 25 mM HEPES buffer, 50-lg gentamicin per ml, and 1% agarose. After 10 d, plaques were visible and the cells were removed from the humidified 37 8C incubator to visualize plaques with an inverted phase microscope. 2 ml of neutral red (1:6,000 final concentration; Sigma-Aldrich, http://www.sigmaaldrich.com) was added to each well, and after an additional 24-h incubation, the plaques were counted [11, 30] .\nELISA. Nunc-Immuno Maxisorp enzyme-linked immunosorbent assay (ELISA) plates (Nunc, http://nuncbrand.com) were coated with 100 ll/well of 10 lg/ml lectin from Galanthus Nivalis (Sigma-Aldrich) in PBS and incubated overnight at 4 8C. The plates were then blocked in phosphate-buffered saline (PBS) containing 10% FBS for 2 h. The wells were then washed twice with wash buffer, PBS containing 0.2% Tween 20 (Sigma-Aldrich). 293 cells were transfected with a mammalian expression plasmid coding for transmembrane domaindeleted Ebola glycoprotein. Supernatant (100 ll) from these cells, which contains 0.8-1.3 mg/ml total protein, was used to coat each well for 1 h at room temperature (RT) after the blocking solution was removed from the ELISA plates. Plates were then washed six times with wash buffer. Monkey sera were added in 10-fold dilutions from 1:10 to 1:10 5 in dilution buffer (PBS containing 1% BSA and 0.02% Tween) and incubated at RT for 1 h. The wells were then washed six times, and a secondary antibody alkaline phosphatase-conjugated goat anti-human immunoglobulin G (IgG) against the F(ab9) 2 portion of the antibody (Pierce, http://www.piercenet.com) diluted 1:500 was added, and this was incubated for 1 h. Finally, the plates were washed again six times and developed by one tablet of phosphatase substrate Typically smaller immunoreactive monocytes are seen with ZEBOV infection [15] . Immunohistochemistry was performed as described in Materials and Methods. doi:10.1371/journal.ppat.0030009.g002\n(Sigma-Aldrich) in 5 ml of alkaline phosphatase stain buffer (pH 9.8) per plate. The assay was performed as per manufacturer's directions. The plates were read at an optical density of 405 nm on a microplate reader (Molecular Devices, http://www.moleculardevices.com) at 30 min after adding substrate. A panel of normal sera was run each time the assay was performed.\nImmunohistochemistry. Sections were pretreated with Dako Ready to Use Proteinase K (Dako, http://www.dako.com) for 6 min at RT after deparaffinization and rehydration through a series of graded ethanols. Blocking was performed with Dako's Serum-Free Protein Block for 20 min pre-antibody exposure. The tissue sections were then incubated overnight at 4 8C in primary antibody using an equal mixture of mouse monoclonal antibodies to EBOV GP and VP40 (1:5,000). An alkaline phosphatase-labeled polymer (Dako Envision System, alkaline phosphatase) was incubated on the sections for 30 min, and then color was developed by exposing tissue to 6-bromo-2hydroxyl-3-naphtholic acid (HistoMark Red; Kikegaard and Perry Laboratories, http://www.kpl.com) substrate for 50 min in the dark. Counterstaining was done with hematoxylin. Positive controls included archived EBOV-infected cynomolgus tissue, and negative controls included replicate sections exposed to anti-Marburg virus antibodies and uninfected cynomolgus macaque tissue [15] ." }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: International and national events have brought critical attention to the Canadian public health system and how prepared the system is to respond to various types of con-temporary public health threats. Whether those threats result from emerging infectious diseases or bioterrorism, the public health system is responsible for protecting the health of the population. Outbreak response occurs first at the local or regional levels and thus the potential impact that a threat will have on the overall Canadian population can differ significantly depending on the capacity of public health systems to respond [1] . For example, following the SARS outbreak in Toronto, the Canadian National Advisory Committee (NAC) on SARS and Public Health held it as fortunate that the SARS outbreak struck primarily in Toronto and not in other parts of Canada where the capacity to combat public health threats is limited [2] . While a \"general renewal of the public health infrastructure\" and its capacity to respond are in order the effective coordination of all agencies at regional, provincial, and federal levels is necessary to assure a comprehensive surveillance and management of public health threats. As Ralph Klein, the Premier of Alberta, commented: \"Albertans and all Canadians understand a disease outbreak like SARS or West Nile virus in one region affects other parts of the country. Coordinated approaches will help deal with public health threats.\"\nDespite the commitment of federal and provincial agencies in Canada to improve public health system performance and develop overall public health preparedness, little is still known about the current systems-level state of public health preparedness in Canada. While coordination is seen as a necessary element for public health preparedness, few measures of inter-organizational collaboration relevant to public health preparedness have been developed and assessed in relation to other features of the organizational and inter-organizational environment. This study protocol presents the premises, conceptual model and methods used to measure and assess public health preparedness in Alberta, Canada. Although provincial public health agencies are themselves embedded within federal and global public health preparedness systems, we focus primarily on the organizational environment of public health preparedness in Alberta so as to develop contextually-relevant measures and evaluation techniques as well as address issues related to potential cross-Alberta variations in public health preparedness.\nIn Alberta, public health and emergency response procedures are primarily governed under the Disaster Services Act [3] and the Public Health Act [4] . Provincial response to a public health disaster event may thus call into action organizations and units under the jurisdiction of local municipalities, regional health authorities, emergency management districts, and/or the provincial government. There are a number of organizational actors that may be involved in preparation, response, or recovery phases of a public health disaster, including departments or portfolios within agencies. Each of which are potential actors in the provincial public health and emergency response network. While we consider the overall public health and emergency management network to consist of all poten-tial organizational actors involved in responding to public health threats, the precise set of organizations that come together around a particular public health threat depends on the nature of that threat. For example, pandemic influenza and the West Nile virus are both emerging infectious diseases but the nature of their transmission differs and distinctive sets of organizational actors must be in place to confront them. In the case of West Nile Virus, Alberta's Department of the Environment and Sustainable Resource Development would be integrally involved for mosquito surveillance and control whereas it would not be as integral in the event of a pandemic influenza outbreak [5] . To assess potential differences by public health threat in Alberta public health preparedness, we examine and compare two potential public health threats: pandemic influenza and West Nile Virus.\nUsing an embedded multiple-case study design[6] and integrated qualitative and quantitative methods, the project proposes to measure empirically the degree of seamless coordination existing among municipal, regional or district-level and provincial public health and emergency management agencies in Alberta. Although federal authorities and agencies play an important role in provincial-level responses to public health emergencies, for the purposes of this research, the focus will be on the inter-organizational linkages and organizational environments of provincial and sub-provincial actors; relevant federal-level organizations will be identified for future investigation. The seamless coordination of activities and tasks that should characterize the overall federal-provincial-regional linkages should also characterize the intraprovincial linkages. Fluid communication and resource flows among such diverse organizations and administrative authorities require an integrated and coordinated network of actors. Our research is premised on empirical research that has shown that effective and coordinated approaches to public health threats require a well-integrated and responsive inter-organizational system and that the most appropriate method in which to analyze inter-organizational relations and the degree of integration is one based on network analysis [7] . Yet, strong inter-organizational ties are not enough for effective preparedness if the organizations involved do not have the necessary individual capacity to respond to public health threats. In this regard, fluid communication and integrated organizational connections are but one element in an overall systems-level approach to assessing public health preparedness. As shown in Figure 1 , we view public health preparedness to be a product of several interrelated factors: 1) individual organizational attributes, 2) the inter-organizational networks existing among relevant organizational actors, 3) the institutional environments in which organizations and inter-organizational networks are located and in which threats first emerge, and 4) the integrated coordination of specific action-sets. An actionset refers, in this case, to a subgroup of organizations in the network that come together purposefully to address a specific public health threat. We see these four factors together influencing system-level public health preparedness.\nOur target population for the research is all public health and emergency management-related organizations in Alberta. The construction of the project's sampling frame is based on a stratified, multistage cluster design. The strata are four administrative divisions: (1) provinciallevel agencies, (2) sub-provincial administrative agencies, i.e., emergency-management districts (disaster services) and regional health authorities, (3) metropolitan areas, and (4) towns and local areas. Within strata 1-3, the sampling frame includes all public health or emergency management organizations, i.e., agencies, departments, or units, within those strata. The fourth stratum will be substratified according to the Alberta Emergency Management district divisions into 6 geographical areas: (1) northwestern Alberta, (2) northeastern Alberta, (3) northcentral Alberta, (4) central Alberta, (5) south-central Alberta, and (6) southern Alberta. From each of the 6 geographical regions, we will draw a proportionate, random sub-sample of towns. For stratum 4, the sampling frame consists of all public health or emergency managementrelated organizations in those towns randomly selected. The project's frame population will thus consist of those organizations listed in the strata 1-3 sampling frame and those listed in the stratum 4 sampling frame.\nThis study will be conducted in 2 phases: phase 1 qualitative and phase 2 quantitative research. Phase 1 is used in part to help identify the organizations responsible for public health and emergency responses and the relevant relationships among them. Initial qualitative research will consist of the analysis of official documents and reports and interviews with key organizational representatives. Government documents and reports will be analyzed to 1) help identify agencies involved in the response system, 2) determine the formal tasks assigned to those agencies, and 3) initially assess the overall level of preparedness and responsiveness for the three case studies. Interviews with \nAttributes key informants and health officials will be used to determine 1) if there are additional organizations involved in the response system that were not noted in the official documents, 2) the types of relationships characterizing the inter-organizational ties within and among actionsets, and develop 3) relevant measures of the institutional environment and of public health preparedness and responsiveness. While laws mandate that certain organizations be involved in emergency responses to specific public health threats, key organizational representatives may identify agencies that play an important albeit informal role in local emergency response systems.\nIn addition to helping identify organizations that may be absent from official documents, the interviews will also investigate the perceived characteristics of inter-organizational relationships. Mandated relationships, for example, tend to be characterized by lower levels of perceived cooperation among organizations [8] . While such characteristics will be investigated using quantitative methods in phase 2, the qualitative research will reveal the points of conflict and cooperation that emerge in the course of public health and emergency responses and thus provide a basis for the questions that will be used in the organizational questionnaire. The final focus of the phase one interviews will centre on the development of measures of the institutional environment, network effectiveness, and public health preparedness. What are the resources that key agency representatives themselves identify as critical and important to their organization and the network? What processes do the respondents use to evaluate if their organization or the system was prepared?\nIn phase 1, we will draw a non-proportional sample from the project's sampling frame. We will interview 6 organizational representatives from the provincial level (stratum 1), 12 representatives from the sub-provincial level (strata 2 and 3), and 18 representatives from the local level (stratum 4). Phase 1 interview respondents will be organizational representatives who are randomly selected from each of these strata and choose to participate.\nInterviews will be tape-recorded and transcribed so that content analysis, thematic, and inter-textual analyses can be conducted on the interviews. Transcripts will be coded for such indicators as (1) organizations involved in the public health and emergency management preparedness, (2) types of information-sharing among organizations, (3) types of resource flows existing among organizations, and (4) perceptions of local and provincial public health preparedness. Content analysis will be used to identify relevant organizations that were not identified in the documentary research [9, 10] . Thematic analyses will be used to identify the critical themes emerging in the 36 interviews; inter-textual analyses will be used to compare the responses of interviewees across the three strata dimensions of jurisdiction, domain, and geographical location.\nPhase 2 involves the administration of an organizational/ inter-organizational questionnaire to Alberta-based public health and emergency management agencies. Information on organizations and inter-organizational networks will be collected by web-based or telephone questionnaires. For phase 2, we intend to administer questionnaires to representatives of all organizations listed in the project's sampling frame (described earlier). The questionnaire consists of three components: 1) an organizational attribute component, 2) an inter-organizational network component, and 3) an organizational environment and network assessment component. The organizational attribute component consists of questions on the general characteristics of organizations such as staff size, training background of specific occupational roles in the organization, sectoral operations, budget and more emergency-response related questions, such as whether an agency has formally assessed its epidemiology capacity.\nThe inter-organizational network component consists of items that ask representatives to identify those organizations with which their own organization has ties along specific dimensions. The specific content and dimensions of the network to be mapped will be determined following phase one analysis but candidate dimensions used in other organizational network studies include (i) information-sharing, (ii) resource-sharing, including staff and (iii) joint-planning. For each of the critical dimensions identified in phase one, the respondent will be presented with a list of organizations within the relevant action-set and asked, for each one, \"who do you share information with?\" or \"who do you share resources with.\"\nThe third component of the questionnaire will consist of the respondent's subjective assessments of the institutional environment and inter-organizational network, and the impact that each has on perceived public health preparedness. For example, how do representatives assess the environment in which they operate, i.e., do they see resources as easily available or scarce? How prepared do they perceive their own organization and local system to be for a public health threat? Since mandated relations involve \"sequential interdependence,\" an important question is whether organizations perceive the task transitions among organizations as functioning smoothly. The actual questions that will be used in this component of the questionnaire will be determined following phase one data analysis.\nEthical approval of the research has been granted by the Conjoint Health Research Ethics Board of the Calgary Health Region and University of Calgary, Faculty of Medicine (ID#17873) and of the Capital Health Region and University of Alberta (#B-251005).\nThe capacity of any organization to be prepared for a public health threat is influenced in part by the system in which the organization is located, just as the preparedness of the overall system can be influenced by the strengths and weaknesses of any single organization. The cross-case and intra-provincial comparative design allows our research to discriminate among different system elements and examine the independent influence of organizational attributes, institutional environments, and inter-organizational networks on public health preparedness. In addition, the mixed methods approach enables our research to analyse the role of both informal and formal relationships in the emergence of inter-organization networks and the development of prepared and responsive public health systems. As far as we are aware, our project is one of the first to assess public health preparedness at the systemslevel while accounting for the critical role that inter-organizational relationships play in the emergence of a cohesive and responsive system. Moreover, in deciphering the independent influence of inter-organizational ties within the overall system, the research will provide measures that will help researchers and policy-makers evaluate the degree of coordination among organizational actors.\nWhere are the gaps in system linkages? Which organizations need to be made more central to preparedness activities? These are just a few of the questions that our research is designed to answer." }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: The immunoregulatory cytokines [interleukin (IL)-2, IL-10, transforming growth factor (TGF)-b] and allergy-associated cytokines [IL-4, IL-5, granulocyte Á/ macrophage colony-stimulating factor (GM-CSF)] are mediators of the immune system, which are actively involved in regulation of molecular and cellular processes accompanying different types of inflammation.\nIL-2 is the up-regulating cytokine, which stimulates primarily the cell-mediated inflammatory response by promoting growth, proliferation and differentiation of T cells, B cells, natural killer (NK) cells, monocytes and macrophages. It is secreted mainly by activated T cells. IL-2 induces activation and rapid clonal expansion of mature T cells, and cytokine production in T cells, including interferon (IFN)-g and IL-4; 1 growth of B cells and immunoglobulin (Ig) J-chain switching and secretion of IgM in B cells; 2 proliferation, production of IFN-g and cytolytic activity of NK cells; 3 proliferation and differentiation of macrophage precursors; 4 and cytolytic activity of monocytes. 5 In contrast to IL-2, IL-10 (known as the cytokine synthesis inhibitory and macrophage deactivating factor) down-regulates the immune reactions accompanying acute inflammation and limits the duration of inflammatory responses. IL-10 can also promote and regulate chronic inflammatory processes. IL-10 is produced by a variety of cell types, including CD4 ' T cells, activated CD8 ' T cells and activated B cells. The main anti-inflammatory activities of IL-10 */ namely, inhibition of cytokine production in macrophages, neutrophils, T cells and NK cells, 6 Á 9 and inhibition of the macrophage Á/monocyte activation and the antigen presentation abilities of these cells 10 */lead to the resolution of inflammation. However, if the acute inflammatory process has not been resolved, IL-10 can induce humoral inflammatory reactions such as the immunoglobulin isotype switching in B cells 11, 12 and differentiation of B cells into plasma cells, 13 and thus promote switching of inflammation in the chronic stage.\nTGF-b, produced by T cells, platelets and monocytes, plays an important role in regulation of the inflammatory processes and in tissue reparation. The effects of TGF-b depend on the differentiation state of the responsive cells. 14 TGF-b, as well as IL-10, participates in resolution of acute inflammation by inhibiting antigen presentation and cytokine production in macrophages, 15 and suppressing proliferation of T cells and NK cells and cytokine production in activated T cells. 14 Simultaneously, TGF-b recruits monocytes to the site of inflammation and upregulates their pro-inflammatory activity; secretion of cytokines and growth factors, in particular. 14 The contradictory effects of TGF-b on monocyte/macrophage lineage, such as up-regulation of monocyte functions and deactivation of macrophages, demonstrate the highly coordinated role of TGF-b in regulation of inflammatory responses.\nThe immunoregulatory and allergy-associated cytokine IL-4, produced by CD4 ' T cells, mast cells and basophils, is actively involved in regulation of different types of inflammation. IL-4 up-regulates humoral inflammatory responses by inducing growth of B cells, isotype switching in activated B cells and differentiation of B cells into antibody producing plasma cells. 16 IL-4 also stimulates the cell-mediated inflammatory processes by promoting growth of activated T cells 17 and enhancing the development of virus-specific cytotoxic T cells. 18 Simultaneously, IL-4 possesses powerful anti-inflammatory effects. In particular, IL-4 controls numerous molecular processes in monocytes, macrophages and neutrophils, which down-regulate production and secretion of the pro-inflammatory cytokines tumour necrosis factor (TNF)-a, IL-1 and IL-8, 19 Á 21 deactivate inflammatory macrophages and lead to suppression of acute inflammation. IL-4 can also induce switching of acute inflammation in the chronic stage owing to its ability to up-regulate expression of the mannose receptor on activated macrophages. 22 The mannose receptor promotes fusion of activated macrophages and formation of giant multinucleated cells in the zone of inflammation 23 that create the cellular background for manifestation of chronic inflammation.\nIL-5 is an important regulator of the humoral immune response and is produced by CD4 ' T cells as well as NK cells. 24 Being a late regulating factor in differentiation of B cells, IL-5 plays an essential role in cytokine-induced production and secretion of immunoglobulins and supports humoral inflammatory reactions such as production and secretion of IgA. 25 IL-5 is also the allergic cytokine, which by stimulating production, activation, differentiation and migration of eosinophils creates a strong molecular background for eosinophilic inflammation. 26, 27 GM-CSF regulates proliferation and maturation of granulocyte and macrophage precursors and activates mature neutrophils, eosinophils and macrophages, 28 and thus participates in different types of inflammation. 29 Monocytes, T cells, fibroblasts and endothelial cells activated by macrophage cytokines IL-1b or TNF-a produce GM-CSF. The GM-CSF enhances neutrophil phagocytosis, enhances release of chemotactic factors by neutrophils, 30 induces production of the pro-inflammatory cytokines by macrophages and increases their function as antigen-presenting cells, 31 and in this way promotes acute inflammatory reactions. In the presence of IL-4, GM-CSF can initiate a specific differentiation of monocytes into osteoclast-like multinucleated giant cells 32 that create a background for chronic inflammation. GM-CSF can also contribute to allergic inflammation by promoting growth, prolonged survival and activation of eosinophils and basophils. 33 Both groups of cytokines, the immunoregulatory IL-2, IL-10 and TGF-b and the allergy-associated IL-4, IL-5 and GM-CSF, have been identified in otitis media and their presence is the evidence of their participation in regulation of local inflammatory processes. These cytokines can switch the acute phase of middle ear inflammation into the chronic stage and lead to the chronic condition of otitis media with effusion (OME). OME: a brief description OME, the commonest cause of childhood deafness in the developed world, is a chronic inflammatory condition of the middle ear cleft with repetitive recurrences of acute middle ear inflammation. The disease is characterized by a middle ear effusion that persists for months to years, cannot be cleared by the normal mucociliary transport mechanisms and is must usually removed by surgical operation.\nA surgical procedure, which includes myringotomy, aspiration of fluid and the insertion of ventilation tubes (grommets) in the anterior tympanic membrane (Fig. 1) , is the most effective option in treatment of OME. The main problem with ventilation tubes is secondary infection and post-operative otorrhea, which can be managed using antibiotic eardrops. 34, 35 However, increasing antibiotic resistance of bacterial pathogens 36 and possible ototoxicity of topical antibiotics 37 can complicate the antimicrobial therapy in post-surgical management of otitis media. Other non-surgical approaches in treatment and prophylaxis of OME include traditional methods, such as combined steroid Á/antimicrobial therapy 38, 39 and polyvalent pneumococcal vaccination, 40, 41 and relatively new methods based on herbal medicine 42, 43 and homeopathy. 44, 45 The chronic condition of otitis media is associated with proliferative changes in the middle ear tissues, especially in the surface middle ear mucosa, which is presented in OME as a modified pseudostratified epithelium, 46 where basal cells are differentiating into goblet cells, 47 goblet cells are proliferating with enhanced secretory activity 48 and formation of mucus glands occurs 49, 50 (Fig. 1) .\nGoblet cells produce and secrete mucins, which are important glycoproteins in the mucociliary transport system of the middle ear and are the main component of middle ear effusions, responsible for the viscous properties of effusions. 51, 52 However, under disease conditions, alterations that occur in the middle ear and eustachian tube mucin metabolism, 53 in the structure of mucin glycoproteins 54, 55 and in the glycoconjugate expression in cilia and goblet cells 56 promote the dysfunction of the normal mucociliary transport system and the formation of effusion in the middle ear cleft.\nThe expression of several mucins has been detected in OME. 53, 57 However, overproduction of only two mucins has been observed in otitis media; namely, the membrane-bound MUC4 53 and the secreted MUC5B. 53, 55, 58, 59 Another secreted mucin (MUC5AC) is always presented in effusions, but in varying amounts, 53, 55, 57, 60 and its levels maybe linked to levels of the pro-inflammatory cytokines TNF-a, IL-6 and IL-8, which can promote different levels of MUC5AC secretion. 61, 62 The initial step in the pathogenesis of OME is the inflammatory process in the middle ear mucosa. Bacteria, 63, 64 viruses, 65,66 allergic reactions 67,68 and even gastroesophageal reflux 69 Á 71 in tandem with predisposing factors such as eustachian tube dysfunction, 72 cleft palate 73 and obstructive adenoids 74 can stimulate the middle ear inflammation.\nDifferent groups of inflammatory mediators were identified in the human middle ear mucosa, fluids and effusions. A lot of different mediators participate in initiation and early stages of the middle ear inflammation, including arachidonic acid metabolites (prostaglandin E 2 and leukotrienes LT-B 4 , LT-C 4 ), 75, 76 histamine, 77,78 platelet-activating factor, 79 surface cell adhesion molecules (intercellular adhesion molecule-1, vascular cell adhesion molecule-1, endothelial leukocyte adhesion molecule-1, platelet endothelial cell adhesion molecule), 80,81 soluble cell adhesion molecules (soluble intercellular adhesion molecule-1 and soluble vascular cell adhesion molecule-1), 82, 83 chemokine RANTES, 84 complement C3a anaphylatoxin 85 and interferon-g. 86 However, cytokines are the key mediators of the middle ear inflammation. Cytokines regulate different stages of inflammation, are responsible for resolution of inflammation and can initiate local molecular processes leading to histopathological changes in the middle ear mucosa and submucosa, and the chronic condition of otitis media. Different groups of cytokines have been identified in the middle ear effusions and mucosa: the pro-inflammatory TNF-a, IL-1b, IL-6 and IL-8; the immunoregulatory IL-2, IL-10 and TGF-b; and the allergy-associated IL-4, IL-5 and GM-CSF.\nThe pro-inflammatory cytokines and their participation in the pathogenesis of otitis media have been already discussed, 87 and are more related to the acute phase of middle ear inflammation. In the present review we have analysed the immunoregulatory and the allergy-associated cytokines in otitis media, which, by providing the cellular and molecular background for the chronic inflammation in middle ear, promote the chronic condition of OME.\nThe immunoregulatory cytokines IL-2, IL-10, TGF-b and their cellular and molecular networks in OME IL-2 in otitis media IL-2 has been detected in middle ear effusions from children with chronic OME at high concentration (greater than 300 pg/ml). 88 The analysis of peripheral blood immunologic pattern in patients with persistent middle ear effusions 89,90 showed the following: (1) the number of CD4 ' and CD8 ' T lymphocytes was decreased and the T-cell subset ratio (CD4 ' / CD8 ' ) was reduced; (2) proliferative responses of blood T cells were diminished; and (3) generation of IL-2 by peripheral blood lymphocytes was decreased. Another immunologic investigation of children with recurrent otitis media revealed a consistent inability of adenoidal T cells to turn on B cells to mature into immunoglobulin-secreting plasma cells that has been explained by defective production of IL-2 in adenoids. 91 This assumption was confirmed by cytokine assay of IL-2, which has been done simultaneously in nasopharyngeal lymphoid tissues and in peripheral blood of children with recurrent otitis. The adenoidal lymphocytes produced significantly less IL-2 compared with the patient's peripheral blood lymphocytes. 92 It has been assumed that poor production of IL-2 in adenoidal tissues is probably linked to a deficiency of IL-2 production in the middle ear tissues, and that could be associated with the persistence of OME. Thus, general and/or local deficiency of the IL-2 production could induce defective immunoregulation of the middle ear inflammation and promote the persistence of OME.\nThe investigation of IL-2 in experimental models of otitis media showed the involvement of IL-2 in regulation of acute inflammation because: (1) the IL-2 producing cells were predominant in acute OME compared with chronic OME, 93 and (2) recombinant IL-2 trans-tympanically injected into the middle ear of normal guinea pigs caused the inflammatory middle ear effusion within 24 h, which cleared by 72 h after inoculation. 94 However, differing degrees of responsiveness of effusion production were observed following the instillation of IL-2 in the middle ear, 95 varying from pronounced middle ear effusion causing rather severe mixed hearing loss to complete lack of effusion, and IL-2 was detected in all the middle ear effusions in experimental OME with signs of sensorineural hearing loss. 96 By analysis of clinical and experimental data we can propose the following conclusions about the role of IL-2 in OME:\n1. IL-2 is involved in regulation of the middle ear inflammation and, as the main T-cell growth and differentiation factor, IL-2 can support acute and chronic inflammatory processes in the middle ear. 2. IL-2 can regulate local acute inflammatory response by inducing activation, differentiation of T cells and production of the proinflammatory (IL-1b, IL-6) and anti-inflammatory cytokines (IL-10, TGF-b) by activated T cells (Fig. 2 ). 3. The deficiency of local or general IL-2 production can suppress reactions of acute inflammation and promote persistence of OME, which can develop with time into the chronic OME (Fig. 3 ). 4. The excessive production of IL-2 can provoke chronic cell-mediated and (or) humoral inflammatory processes (proliferation of activated T cells, increased production of cytokines IL-4, IL-5 and GM-CSF by proliferated T cells, immunoglobulin-switching and IgM secretion in B cells), which will induce switching of the middle ear inflammation in the chronic stage with subsequent development of chronic OME (Fig. 3) . \nThe clinical investigations demonstrated the involvement of IL-10 in chronic otitis media. 97,98 IL-10 was identified in middle ear effusions from children undergoing tympanostomy tube placements and its mean concentration was 569/58.7 pg/ml. 97 It has been assumed that the presence of IL-10 in middle ear effusions might be one of the causes of a lack of clinical features of acute inflammation and could lead to a chronic inflammatory state. Chronic otitis media has been also associated with dysregulated local production of IL-10. 98 Patients with chronic otitis media showed high levels of IL-10 ( /100 pg/ml) produced by sinus lavage cell culture and simultaneously low production of IL-10 by peripheral blood mononuclear cells. Investigations of cytokine profiles in different experimental models of OME showed a rapid appearance of IL-10 in the early stages of acute otitis media 99 Á 103 and that IL-10 in mucosa was produced predominantly by CD4 ' T cells. 104 Streptococcus pneumoniae induced expression of IL-10 mRNA in middle ear mucosa within 1Á/2 days after inoculation. 99, 100 Lipoteichoic acid, one of the components present within the cell wall layer of most Grampositive bacteria, 101 and non-typeable Haemophilus influenzae (NTHi) 102,103 induced the expression of IL-10 mRNA in middle ear mucosa even faster, within 6 h after inoculation. It is interesting to note that the expression of IL-10 mRNA followed the expression of TNF-a and IL-6 mRNAs, 99,100 and sometimes occurred simultaneously with the pro-inflammatory cytokines. 101, 102 Thus, clinical and experimental data about IL-10 in OME lead to the following conclusions:\n1. IL-10 is involved in regulation of the middle ear inflammatory response induced by bacterial infection. 2. The expression of IL-10 in acute otitis media is up-regulated shortly after or even simul- taneously with the pro-inflammatory cytokines, TNF-a and IL-6. That is, the evidence of initiation of corrective immunoregulation in the middle ear mucosa, because IL-10 as the main anti-inflammatory cytokine, can down-regulate the local population of inflammatory cells, macrophages and neutrophils, and production of the proinflammatory cytokines TNF-a, IL-1b, IL-6, IL-8 and promote resolution of inflammation (Fig. 2 ). 3. If the acute inflammatory response is not resolved correctly, the prolonged presence of IL-10 in the zone of inflammation can provoke the amplification of chronic humoral inflammatory processes, such as increased secretion of immunoglobulins, especially IgA, by activated B cells and possible generation of plasma cells, in the presence of IL-4 ( Fig. 4) . In this way, IL-10 can contribute to switching of the disease into the chronic stage. 4. The deficiency of IL-10 in acute otitis media can also lead to chronic condition of OME, because shortage of IL-10 will promote overproduction of the inflammatory cytokines, especially IL-1b, TNF-a and IL-6, which can induce tissue damage and irre-versible changes in the middle ear mucosa (Fig. 4) .\nGeneral conclusions about the role of IL-2 and IL-10 in an aetiology of OME Both cytokines, IL-2 and IL-10, participate in balanced immunoregulation of the middle ear inflammatory response. IL-2 up-regulates cellular and molecular events, accompanying acute inflammation, whereas IL-10 down-regulates acute inflammatory reactions and promotes resolution of inflammation. Any imbalance in production of these cytokines will induce chronic inflammatory processes (cellmediated or humoral, or both) or stimulate uncontrolled inflammatory-related damage of the middle ear tissues (Figs 3 and 4) . Thus, IL-2 and IL-10 can be considered as the key cytokine mediators, regulating switching of the acute phase of middle ear inflammation in the chronic stage, which will lead to the chronic condition of OME.\nClinical investigations of TGF-b in OME showed its relation to chronic otitis media. 105, 106 The identification of TGF-b in serous and mucoid middle ear effusions was associated with a history of previous tympanostomy tube placements. 105 Investigation of growth factors in tympanic membrane perforations showed that TGF-b could promote development of the fibrotic scar at the perforation margin, explaining the deficient healing pattern of tympanic membranes in chronic otitis media. 106 However studies of cytokines in experimental OME demonstrated the presence of TGF-b also in acute otitis media. 100, 102, 103 The expression of TGF-b mRNA was up-regulated in acute otitis media caused by S. pneumoniae 100, 102 and NTHi infections, 102, 103 and was detected early during the course of acute inflammation, within 24 h after inoculation, together with cytokines IL-1, TNF-a and IL-10. However, the highest mRNA levels for TGF-b were recorded considerably later, on days 6 and 28, for the NTHiinfected and pneumococcus-infected animals, respectively, and its expression continued even after resolution of acute otitis media. 102 TGF-b regulates host response in infectious diseases through the TGF-b-activated kinase 1 107 and TGF-b-Smad signaling pathway. 108 It has been shown that NTHi, a major human bacterial pathogen of otitis media, strongly induced up-regulation of MUC5AC mucin in human epithelial cells. This induction occurred via activation of the Toll-like receptor 2-MyD88-dependent p38 mitogen-activated protein kinase (MAPK) pathway. However, activation of TGF-b-Smad signaling by TGF-b resulted in downregulation of p38 MAPK by inducing MAPK phophatase-1, and subsequent down-regulation of MUC5AC transcription. 108 TGF-b thereby acted as a negative regulator of MUC5AC mucin gene expression. This is a very significant fact in understanding the role of TGF-b in otitis media, because varying levels of the secreted mucin MUC5AC are presented in the middle ear mucosa and effusions in patients with OME. 53, 55, 57 By analysis of these data we can make the following conclusions about the role of TGF-b in OME:\n1. TGF-b participates in regulation of the middle ear inflammation. 2. TGF-b is a negative regulator of acute inflammation in the middle ear. Early activation of TGF-b together with another antiinflammatory cytokine, IL-10, in the course of acute otitis media demonstrates the involvement of TGF-b in the correct resolution of acute inflammatory response. TGF-b as well as IL-10 can inactivate macrophages and inhibit pro-inflammatory cytokine production at the site of inflammation (Fig. 2 ). 3. TGF-b can be also involved in down-regulation of bacterial-induced MUC5AC mucin production, and that is the additional evi- Thus, TGF-b can contribute to the pathogenesis and persistence of chronic OME in many ways.\nThe allergy-associated cytokines IL-4, IL-5 and GM-CSF and their cellular and molecular networks in OME\nIL-4 was identified in the middle ear effusions of children with persistent OME 109 and in atopic children with OME undergoing myringotomy and ventilation tube placement. 110 The cytokine analysis of effusions showed a higher mean level of IL-4 in the allergy-positive group compared with the allergynegative group 109 and a higher percentage of cells expressing IL-4 in atopic patients with OME compared with that seen in non-atopic patients. 110 A higher level of IL-4 in effusions correlated with predominance of T lymphocytes, which was the sign of chronic inflammation and was also related to the atopic background of patients with OME. 110 In effusions of non-atopic patients, the level of IL-4 was lower and the predominance of neutrophils 109 demonstrated signs of an acute inflammatory response.\nInvestigation of adenoids in patients with recurrent otitis media 92,111 resulted in the following important findings: (1) expression and secretion of IL-4 was detected in adenoids; (2) adenoidal lymphocytes produced the same level of IL-4, or even slightly more, compared with the patient's peripheral blood lymphocytes; 92 and (3) the epsilon germline transcripts for IgE were detected in the adenoids and the level of IgE epsilon germline transcript expression was dependent on the level of IL-4 mRNA expression. 111 This local IL-4-induced immunoglobulin class switching to IgE in the adenoids was considered the essential molecular process contributing to chronic inflammation in the middle ear and promoting the pathogenesis and persistence of OME.\nThus, IL-4 was presented in both groups of patients with OME, allergic and non-allergic. However, local overproduction of IL-4 was associated predominantly with an allergic background of patients.\nStudies on IL-4 in experimental OME 93,112 demonstrated the involvement of IL-4 in regulation of different types of otitis media. In trans-tympanically challenged animals the expression of IL-4 was detected during acute and chronic otitis media, with the predominance of IL-4 ' cells in the acute form. 93 The trans-tympanical injection of soluble receptors for IL-4 prevented the allergic Eustachian tube dysfunction and formation of effusion in the middle ear cleft. 112 The involvement of IL-4 in regulation of MUC5AC and MUC5B mucin gene expression and secretion has been shown in human airways. 113 Á 115 The secreted mucins, MUC5AC and MUC5B, are presented in the middle ear mucosa and effusions in patients with chronic OME. 53,55,57 Á 60 Although relationships between IL-4 and production of MUC5AC and MUC5B mucins in the course of otitis media have not been investigated, the ability of a soluble receptor for IL-4 to prevent Eustachian tube dysfunction and formation of effusions in the middle ear cleft 112 demonstrated the potential involvement of IL-4 in the regulation of the middle ear mucin metabolism.\nAll these data allow us to make the following conclusions about the role of IL-4 in OME:\n1. IL-4 is involved in regulation of different types of otitis media. 2. IL-4 is one of the cytokines responsible for an allergic type of inflammation owing its ability to induce local immunoglobulin class switching to IgE (Fig 6) . 3. IL-4 can support local humoral inflammatory responses by increasing production and secretion of immunoglobulins in activated B cells (Figs 4 and 6 ). 4. IL-4 can also support cell-mediated chronic inflammation in the middle ear by promoting generation of multinucleated giant cells (Figs 5 and 6 ), although it is necessary to note that giant cells in OME have not been investigated. 5. IL-4 can also participate in the regulation of mucin metabolism and the mucociliary transport system in middle ear inflammation.\nThus, IL-4 is the important regulator of cellular and molecular processes, accompanying different types and different stages of the middle ear inflammation, and an important factor contributing to chronic condition and persistence of OME.\nIL-5 was detected in the middle ear mucosa and effusions in atopic patients with persistent OME. 110, 116, 117 The expression of IL-5 mRNA in mucosa was up-regulated and correlated with increased expression of major basic protein (cell marker for eosinophils) and CD3 (cell marker for activated T lymphocytes). 116 Examination of the cytokine profile in the middle-ear specimens showed an increased number of cells expressing IL-5 and its correlation with the percentage of eosinophils and T lymphocytes. 110 Correlation between the percentage of eosinophils and levels of IL-5 in effusions was particularly striking in OME patients with asthma, 117 suggesting that middle ear eosinophilia could be dependent not only on the local, but also on the general level of IL-5 production. The percentage of cells expressing IL-5 in effusions of atopic patients was the same or slightly higher compared with cells expressing IL-4; 110 this was evidence that both cytokines IL-5 and IL-4 contributed to development of allergic inflammation in the middle ear.\nThus, overproduction of IL-5 in OME is associated with the atopic background of patients, and IL-5 together with IL-4 is responsible for an allergic type of inflammation in the middle ear (Fig. 6) . This assumption was confirmed in an experimental model of OME, 112 where the late-phase allergic inflammatory response was prevented by pretreatment with IL-5 antibodies or soluble receptors for IL-4.\nHowever, the involvement of IL-5 in regulation of the non-allergic inflammatory response in the middle ear should not be ignored. The production of IL-5 was detected in effusions of non-atopic patients with OME, 110, 117 although it was significantly less compared with atopic patients. Analysis of immunoregulatory cytokines in experimental models of OME 93 demonstrated the absence of IL-5 in acute otitis media and significant up-regulation of IL-5 production in chronic otitis media, where IL-5 ' cells were numerous. Thus, the presence of IL-5 in OME of nonallergic patients shows the involvement of IL-5 in regulation of non-allergic chronic inflammatory reactions in the middle ear, where IL-5 can support humoral processes such as increased production and secretion of immunoglobulins, IgA in particular (Fig. 6) .\nThus, IL-5 can be considered the late mediator of middle ear inflammatory response, regulating allergic inflammation and increasing humoral inflammatory processes, which contribute to the chronic condition of OME.\nOur knowledge about GM-CSF in OME is insufficient. However, the involvement of GM-CSF in regulation of middle ear inflammation has been shown as GM-CSF was identified in effusions of acute purulent and chronic otitis media. 118 The level of GM-CSF in acute otitis media was higher compared with the chronictype otitis media.\nDespite a lack of experimental data about GM-CSF in OME, this cytokine could participate in regulation of different stages of the middle ear inflammation. GM-CSF was identified in acute otitis media, where it could activate matured neutrophils and macrophages and support the acute inflammatory response (Fig. 2) .\nGM-CSF in the presence of IL-4 can induce monocyte differentiation towards generation of mul-tinucleated giant cells, and in this way create the cellular background for chronic middle ear inflammation (Fig. 5) .\nFinally, GM-CSF being the eosinophil survivalpromoting cytokine together with IL-5 can up-regulate allergic eosinophil-mediated inflammation in the middle ear (Fig. 6) . However, in the past year of investigations GM-CSF has been shown as a potential down-regulator of allergic inflammation, because GM-CSF induced apoptosis of human eosinophils through the eosinophil receptor Siglec-8 (sialic acid binding immunoglobulin-like lectin). 119 Thus, GM-CSF can be a very important regulator of the middle ear inflammation and additional experimental work needs to be done to clarify the exact role of this cytokine in the pathogenesis of chronic OME.\nDifferent types of immunoglobulins, namely IgM, IgG, IgA, secretory IgA and IgE, have been identified in effusions and middle ear fluid of chronic OME. 120 Á 123 The effusion level of IgM in some patients with chronic otitis media was markedly elevated. 120 The mucoid type of effusions contained a high level of IgG, IgA 121 and IgE. 124 Comparison of the immunoglobulin levels measured in effusions and in sera showed that, in many cases, the effusion level of secretory IgA 122 and IgE 125 was significantly higher than the corresponding serum level, and this was the evidence of local overproduction of immunoglobulins in the middle ear.\nThe immunologic investigation of effusions detected the immune complexes of IgG (IgG Á/ICs) and FIG. 6. Cellular and molecular networks of the allergy-associated cytokines leading to the chronic condition of OME.\nIgA (IgA Á/ICs) in both acute and chronic otitis media. 126, 127 The highest level of IgG Á/ICs was found in subacute cases, whereas IgA Á/ICs were predominant in chronic OME. The immunoglobulin immune complexes provided the prolongation of inflammatory process in the middle ear.\nThe presence of immunoglobulins in chronic OME was associated mainly with the bacterial infection. 128 Á 131 IgG and IgA antibodies specific to Hemophilus influenzae and S. pneumoniae , 128 IgG, IgM, IgA and secretory IgA antibodies specific to outer membrane antigens of Moraxella catarrhalis, 129 and Staphylococcus aureus -harboured bacteria, intensely coated with secretory IgA and IgG antibodies, 130, 131 were identified in chronic effusions. Only one type of immunoglobulins, the secretory IgA, was identified in effusions infected with respiratory viruses. 132 The presence of IgE in chronic OME has not been always associated with local allergic inflammation. 124, 133 However, local overproduction of IgE was usually accompanied by local allergic reactions, such as degranulation of mast cells found in the middle ear biopsy specimens 124 and expression of IgE on mast cells detected in nasal mucosa specimens from patients with OME. 134 Thus, the activity of immunoglobulins in chronic OME is evidence of chronic humoral inflammatory processes in the middle ear, which is obviously controlled by cytokines. Correlation between the levels and types of immunoglobulins and the immunoregulatory and allergy-associated cytokines in chronic OME has not been investigated. However, the presence of the main types of immunoglobulins, IgM, IgG, IgA, secretory IgA and IgE, in effusions is indirect evidence that cytokines IL-2, IL-10, TGF-b, IL-4 and IL-5, which are involved in regulation of immunoglobulin production and secretion in general, 2, 11, 12, 16, 25 participate also in local production and secretion of immunoglobulins and regulate humoral immune reactions during the course of middle ear inflammation (Figs 3 Á/6 ).\nThe immunoregulatory cytokines and the allergyassociated cytokines can be considered the key regulators of the middle ear inflammation responsible for the molecular and cellular background of chronic OME.\nThe immunoregulatory cytokines IL-2, IL-10 and TGF-b initiate and support molecular switching of the acute phase of inflammation in the chronic stage, whereas the allergy-associated cytokines IL-4, IL-5 and GM-CSF very probably provide the molecular and cellular background for chronic humoral, cell-mediated and allergic inflammatory processes in the middle ear, which lead to the chronic condition of OME.\nHowever, it is necessary to note that additional experimental work needs to be done in order: (1) to elucidate the molecular mechanisms of cytokine regulation of the middle ear inflammation; and (2) to analyse the possibility of anti-cytokine therapy in clinical treatment of OME." }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: Since its advent more than 40 years ago, the specialty of critical care has made remarkable advances in the care of severely ill patients. Mortality rates for many commonly encountered critical illnesses such as severe sepsis [1] and acute respiratory distress syndrome (ARDS) [2] have declined sharply over the past 2 decades. As greater numbers of patients survive intensive care, it is becoming increasingly evident that quality of life after critical illness is not always optimal. For instance, nearly half of ARDS survivors manifest neurocognitive sequelae 2 years after their illness, falling to below the 6th percentile of the normal distribution of cognitive function [3] . Considering that 89% of Americans would not wish to be kept alive if they had severe, irreversible neurologic damage [4] , these findings are quite concerning.\nCognitive dysfunction (CD) is quite common in critically ill patients, not only during the acute illness but also long after the acute illness resolves [5] . Delirium, a form of acute CD that manifests as a fluctuating change in mental status, with inattention and altered level of consciousness, occurs in as many as 80% of mechanically ventilated intensive care unit (ICU) patients [6] . Most clinicians consider ICU delirium to be expected, iatrogenic, and without consequence. However, recent data associate delirium with increased duration of mechanical ventilation and ICU stay [7] , worse 6-month mortality [8] , and higher costs [9] . Chronically, critical illnessassociated CD manifests as difficulties with memory, attention, executive function, mental processing speed, spatial abilities, and general intelligence. Interestingly, patients who develop acute CD often go on to develop chronic CD after hospital discharge [10] [11] [12] [13] , suggesting that the two entities may share a common etiology.\nAlthough there are clearly defined risk factors for critical illness-associated CD, there is little understanding of the underlying pathophysiology. The precise mechanisms are unknown and there are likely to be multiple mechanisms at work in any given patient ( Figure 1 ) [5, 14, 15] . We have chosen to focus on two mechanisms that appear to have the greatest merit: neurotransmitter abnormalities and occult diffuse brain injury. In this bench-to-bedside review, we discuss the evidence supporting these mechanisms, potential markers that could be used to evaluate each mechanism in individual patients, and emerging therapies that may prevent or mitigate critical illness-associated CD. relative dopamine excess in the central nervous system (CNS). Antipsychotics such as haloperidol, which antagonize central dopamine receptors, can counteract the cognitive effects of anticholinergic medications, further supporting the anticholinergic hypothesis.\nDrugs with potent central anticholinergic effects, such as tricyclic antidepressants and antihistamines, are particularly likely to cause delirium. Many medications that are commonly used in the ICU yet not generally considered to be anticholinergic, such as H 2 blockers, opiates, furosemide, digoxin, glucocorticoids, and benzodiazepines, were recently shown to have central anticholinergic properties [16, 17] . Volatile anesthetics, such as sevoflurane, and intravenous anesthetics, such as propofol, also have anticholinergic effects and may be responsible not only for postoperative delirium but also for the more complex phenomena of postoperative cognitive dysfunction [18] . Acute illness itself may be associated with production of endogenous anticholinergic substances [19] . In one study, 8 out of 10 elderly medical inpatients had had detectable anticholinergic activity in their serum, even though no medication used by these individuals had anticholinergic activity. Characterization of such substances might improve our understanding of delirium and lead to useful intervention strategies. Considering that activation of specific cholinergic pathways can inhibit proinflammatory cytokine synthesis and protect against endotoxemia and ischemia-reperfusion injury [20] , it is tempting to speculate that inhibition of these pathways, whether exogenous or endogenous, might contribute not only to CD but also to other outcomes of critical illness.\nIn assessing the overall risk for developing CD posed by medications with central anticholinergic activity in a given patient, individual differences in drug pharmacokinetics make the dose received a poor estimate of a patient's overall anticholinergic burden [21, 22] . However, we can objectively measure anticholinergic burden in individual patients using an assay referred to as serum anticholinergic activity (SAA) [16] . First described by Tune and Coyle [23] , SAA measures the ability of a individual's serum to block central muscarinic receptors using a rat forebrain preparation. Elevated SAA levels are associated with cognitive impairment in studies of medical ward inpatients and community dwelling seniors [16, [24] [25] [26] [27] . Only a single, small study has used this assay to investigate CD in ICU patients. Golinger and colleagues [28] examined SAA levels in surgical ICU patients and found the mean SAA level drawn 4 hours after mental status change was significantly greater in delirious patients (n = 9) than in those without delirium (n = 16; 4.67 ng/ml versus 0.81 ng/ml; P = 0.007). Whether these results apply to all critically ill patients is uncertain because no study has examined SAA across a broad range of ICU admitting diagnoses or in medical ICU settings. Furthermore, because SAA measurement requires fresh rat brain preparations, its use is likely to remain limited to research settings for the foreseeable future. Pathophysiologic mechanisms and predisposing factors thought to underlie critical illness-associated cognitive dysfunction [5, 14, 15] . Apo, apolipoprotein; HIV, human immunodeficiiency virus; 5-HT, serotonin (5-hydroxytryptamine); GABA, γ-aminobutyric acid; NE, norepinephrine (noradrenaline).\nOther neurotransmitter systems such as dopamine, serotonin, γ-aminobutyric acid (GABA), norepinephrine (noradrenaline), and glutamate are also thought to contribute to critical illnessassociated CD. Dopaminergic hyperfunction is thought to underlie the cognitive symptoms of schizophrenia, and dopamine administration itself may be a risk factor for delirium [29] . Serotonin syndrome, a consequence of excess serotonergic agonism, can be seen not only with selective serotonin reuptake inhibitors but also with a variety of drugs and drug combinations [30] . Even a single therapeutic dose of an selective serotonin reuptake inhibitor can cause the syndrome, which manifests as mental status changes, autonomic hyperactivity, and neuromuscular abnormalities.\nGABA abnormalities are thought to contribute to hepatic encephalopathy, perhaps mediated by branched chain and aromatic amino acids acting as false neurotransmitters [31] . Excess GABA activity, such as that which occurs after withdrawal from chronic ethanol or benzodiazepine use, is a well known and quite dangerous cause of delirium [32] . Acutely, sedatives that stimulate GABA receptors, such as benzodiazepines and (probably) propofol, impair cognitive function and are deliriogenic [8, [33] [34] [35] . This raises the possibility that strategies to minimize sedative drug accumulation, such as daily interruption of sedative infusions [36] , which have been shown to reduce duration of mechanical ventilation, and ICU and hospital length of stay, might also reduce the incidence or duration of delirium. Whether these sedative drugs lead to neurocognitive deficits long after their use is unknown, but this has been suggested in certain high-risk groups, such as the very old (>75 years) and those with pre-existing cognitive impairment [37, 38] .\nNoradrenergic hyperfunction, as part of the 'fight or flight' response, can lead to panic attacks and delusions. Glutamate has been implicated in the 'Chinese food syndrome', in which food with high amounts of monosodium glutamate interferes with normal neurotransmission causing confusion [39] . For a more complete review of the other neurotransmitter abnormalities that may underlie delirium, the reader is referred elsewhere [40, 41] .\nIf critical illness-associated CD were solely due to acute medication effects, it would probably resolve after the exposure has ended. However, a significant percentage of individuals developing delirium in the hospital continue to demonstrate symptoms of CD after discharge [10] [11] [12] [13] . These patients manifest decreased cerebral activity and increased cognitive deterioration, and are more likely to develop dementia than patients without delirium. Also, patients who develop delirium have a greater rate of decline on cognitive tests than do nondelirious patients [10] [11] [12] [13] . Taken together, these observations raise the possibility that some degree of occult diffuse brain injury, as a consequence of the local hypoxia, hypoperfusion, cytokine-mediated inflammation and microvascular thrombosis that characterize the multisystem organ dysfunction of critical illness, might have occurred in these patients [42] . Given that every other organ system can be damaged by these forces, it seems implausible that the brain would be uniquely spared.\nMany of the data supporting occult diffuse brain injury as a cause of critical illness-associated CD come from studies of sepsis and septic encephalopathy, a form of delirium. In animal models of sepsis, oxidative damage occurs early in the hippocampus, cerebellum, and cortex [43] , and significant alterations in cerebral vascular hemodynamics and tissue acid-base balance indicate that cerebral ischemia and acidosis do occur [44] [45] [46] [47] [48] . Sharshar and colleagues completed several studies comparing brain pathology in small numbers of patients who died from septic shock with that in patients who died from other causes. Septic patients demonstrated diffuse severe ischemic and hemorrhagic CNS lesions [49] , which correlated with persistent hypotension and severe coagulation disorders. Multiple microscopic foci of necrosis involving the white matter of the pons [50] were seen, as well as ischemia and apoptosis within the cerebral autonomic centers [51] . The white matter lesions were associated with elevated levels of proinflammatory cytokines, suggesting a possible role of inflammation and microvascular thrombosis in the genesis of CNS injury [52] . Although those studies demonstrated that ischemic brain injury occurs in sepsis, they did not determine whether delirium occurred.\nTwo studies attempted to examine the relationship of ischemic brain injury to delirium. In one study of 84 patients with severe sepsis and multiple organ dysfunction [53] , severe hypotension was the only factor in multivariable analyses that was associated with delirium, suggesting that sepsis-related encephalopathy may be caused by ischemic damage rather than metabolic abnormalities. Another study examined cerebral blood flow and cerebral oxygen metabolic rates in patients with septic encephalopathy and multiple organ dysfunction [54] , and it found that both were significantly lower than those in normal awake individuals. Although these studies support the idea of occult brain injury as a cause of delirium, the authors did not use a standardized Diagnostic and Statistical Manual of Mental Disorders (DSM)-IV based tool to diagnose delirium, such as the Confusion Assessment Method for the ICU [6] .\nLending support to the hypothesis that acute inflammation leads to brain injury and subsequent development of delirium, a recent study found that delirium in postoperative hipfractured patients was significantly associated with serum levels of C-reactive protein, an acute-phase protein that is a marker of acute inflammation [55] . Importantly, patients in the study were diagnosed with delirium using the Confusion Assessment Method (the ward-based predecessor to the Confusion Assessment Method for the ICU), providing the first DSM-IV based evidence that acute inflammation may be in the causative pathway of delirium. The brain is a target for free radical damage because of its large lipid content, high rate of metabolism, and low antioxidant capacity. Free radical induced oxidative stress may play a role in the delirium seen after cardiopulmonary bypass. Karlidag and colleagues [56] noted that patients with low preoperative levels of catalase, a erythrocyte-based antioxidant enzyme, were more susceptible to delirium postoperatively. They suggested that preoperative catalase levels might some day be used to identify at-risk patients who could then be put on antioxidant treatment preoperatively. Whether this would reduce the incidence of delirium remains speculative.\nRegional cerebral blood flow appears to be reduced in delirium. Using xenon-enhanced computed tomography (CT), Yakota and colleagues [57] demonstrated significant focal and global brain hypoperfusion in 10 ICU patients with hypoactive delirium. After recovery from delirium cerebral blood flow returned to normal, implying that cerebral hypoperfusion may contribute to the development of delirium.\nStudies of ARDS survivors suggest that a combination of acute hypoxia, hypoperfusion, and hyperglycemia plays an important role in the long-term cognitive sequelae of critical illness [3, 58, 59] . However, it has been difficult to demonstrate a clear relationship, given the lengthy interval between stimulus and effect and the great number of additional contributing variables that can obscure downstream effects. Among ARDS survivors, Hopkins and colleagues showed that the degree of CD at 1 year is significantly correlated with the durations of hypoxia [58] and mean arterial blood pressure less than 50 mmHg during the ICU stay [3] . In animals, hyperglycemia markedly enhances hypoxic-ischemic brain damage due to increased brain edema and disrupted cerebral metabolism [60] . In ARDS survivors, the duration of blood glucose greater than 180 mg/dl has been shown to correlate with worse visual spatial abilities, visual memory, processing speed, and executive function at 1 year [59] . Given the recent interest in maintaining tight glucose control during critical illness as a means of reducing mortality, it will be interesting to see whether patients managed using this technique have better cognitive outcomes. Clearly, such an approach will need to balance the benefits of tight glucose control with the known risks that hypoglycemia poses to the CNS.\nOne of the perceived difficulties with looking for evidence of occult brain injury in humans is the apparent need for CNS tissue specimens to prove that brain injury actually occurred. However, studies of stroke, trauma, and cardiopulmonary bypass-associated brain injury show that serum markers of brain injury correlate well with the extent of CNS damage. S-100β, neuron-specific enolase (NSE), and myelin basic protein (MBP) are three such markers that could be used to look for evidence of occult brain injury in critical illnessassociated CD.\nS-100 is a dimeric calcium-binding protein consisting of two subunits (α and β) [61] . The β unit (S-100β) is highly brain specific, located mainly in astrocytes. Circulating levels of S-100β are elevated in patients with cerebral ischemia [62] , cardiopulmonary bypass-associated decline in explicit memory function [63, 64] , and traumatic brain injury (TBI) [65] [66] [67] . Even in mild head injury, serum levels of S-100β are correlated with clinical measures of injury severity, neuroradiologic findings, and outcomes, including postconcussion symptoms [68] . Elevated serum S-100β levels were recently demonstrated in critically ill patients with respiratory failure [69] and in porcine models of endotoxic shock [70] and acute lung injury [71] . In this latter group, elevated S-100β levels were associated with hippocampal histopathologic changes, including basophilic shrunken neurons in the pyramidal cell layer [71] . Interestingly, S-100β may have both beneficial and detrimental effects, in that lower levels may have protective neurotrophic effects, yet higher levels can lead to exacerbation of neuroinflammation and neuronal dysfunction [72] .\nWhereas S-100β is a marker of astrocyte damage, NSE and MPB are markers of neuron and white matter (myelin) damage, respectively. NSE is protein-based enzyme that is found primarily in neurons. Serum levels of NSE are elevated after TBI, exhibiting a close relationship with outcome in severe head injury [73, 74] and with volume of contusion in minor head injuries [75] . Interestingly, elevated NSE levels were recently shown to predict death in one small study (n = 29) of patients with severe sepsis [76] , even though these patients had no acute CNS disorders, such as stroke or neurotrauma. MBP is the major protein component of myelin. Serum levels of MBP are elevated in diseases in which there is myelin breakdown. Studies of patients with TBI have shown that MBP levels correlate with clinical measures of severity and may allow early prediction of outcomes [74, 77, 78] .\nNew developments in neuroimaging, such as functional magnetic resonance imaging (MRI) and positron emission tomography, have revolutionized our understanding of abnormal brain function in many disease states, including schizophrenia, Parkinson's disease, and post-traumatic stress disorder. To study further whether critical illness-associated CD is associated with occult brain injury in humans, it would be useful to have an imaging test that can detect subtle evidence of brain injury. Unfortunately, traditional CT scans and MRI do not appear to be sensitive enough to pick up the microscopic cellular changes that may underlie CD [42] . Two small studies assessed brain CT findings in critically ill patients with sepsis [79, 80] . Neither study demonstrated any CT abnormalities, although brain pathology in nonsurvivors was consistent with the previously cited findings of Sharshar and colleagues [49] [50] [51] [52] . A recent study of ARDS survivors (n = 15) [81] found that many of these individuals exhibited signs of significant brain atrophy and ventricular enlargement on head CTs obtained during their acute illness, but there were no significant correlations between these abnormalities and subsequent neurocognitive scores.\nA new MRI technique may prove useful for identifying occult brain injury in critically ill patients. Specifically, highresolution, three-dimensional MRI can be used to assess noninvasively differences in brain tissue sodium concentration, which is a highly sensitive marker of tissue viability that highlights areas that traditional MRI can miss [82] [83] [84] [85] [86] . The method is based on sodium ion homeostasis, which is tightly regulated in the body and is a major energy consuming process. Any event that perturbs the energy level of the cell enough to disrupt the sodium ion gradient, such as ischemia, has an important impact on cell viability. Although tissue sodium concentration MRI has been successfully used to evaluate the CNS, including nonhuman primate studies and clinical studies of stroke and reversible focal brain ischemia [87] [88] [89] , it has not been used to assess patients with either acute or chronic critical illnessassociated CD.\nThere are several recent developments that, although preliminary, are of interest because of their potential to prevent or mitigate critical illness-associated CD.\nHaloperidol has been used for many years to manage agitation in mechanically ventilated ICU patients, and it is the recommended drug for treatment of ICU delirium [90] . Kalisvaart and colleagues [91] compared the effect of haloperidol prophylaxis (1.5 mg/day preoperatively and up to 3 days postoperatively) with that of placebo in 430 elderly hip surgery patients at risk for delirium. Although there was no difference in the incidence of postoperative delirium between treatment and control groups, those in the haloperidol group had significantly reduced severity and duration of delirium (5.4 days versus 11.8 days; P < 0.001). Haloperiodol also appeared to reduce the length of hospital stay among those who developed delirium (17.1 days versus 22.6 days; P < 0.001). A recent retrospective cohort study examined haloperidol use in 989 patients who were mechanically ventilated for longer than 48 hours [92] . Despite similar baseline characteristics, patients treated with haloperidol had significantly lower hospital mortality than did those who never received the drug (20.5% versus 36.1%; P = 0.004), an association that persisted after adjusting for potential confounders. Because of the observational nature of the study and the potential risks associated with haloperidol use, these findings require confirmation in a randomized, controlled trial before they may be applied to routine patient care.\nLeung and colleagues [93] tested the hypothesis that using gabapentin as an add-on agent for treating postoperative pain reduces the occurrence of postoperative delirium. Patients aged 45 years or older undergoing spine surgery were randomly assigned to gabapentin 900 mg or placebo by mouth 1 to 2 hours before surgery and continued for the first 3 days postoperatively. Postoperative delirium occurred in 0% (0/9) of gabapentin-treated patients and 42% (5/12) of placebo patients (P = 0.045). Reduction in delirium appeared to be due to the opioid-sparing effect of gabapentin. Given the small size of the study, these results require confirmation.\nDonepezil, a cholinesterase inhibitor that increases synaptic availability of acetylcholine, improves cognitive function in Alzheimer's disease. Sampson and colleagues [94] randomly assigned 33 elderly patients undergoing elective total hip replacement to donepezil 5 mg or placebo immediately following surgery and every 24 hours for 3 days. Donepezil was well tolerated with no serious adverse events. Although the drug did not significantly reduce the incidence of delirium (9.5% versus 35.7%; P = 0.08) or length of hospital stay (mean ± standard error: 9.9 ± 0.73 days versus 12.1 ± 1.09 days; P = 0.09), both outcomes showed a consistent trend suggesting possible benefit. The authors project that a sample size of 95 patients would be required for a definitive trial.\nDexmedetomidine's sedative effects are due to selective stimulation of α 2 -adrenoreceptors in the locus ceruleus of the CNS. Because it does not have anticholinergic or GABA-stimulating effects, it has the potential to be a delirium-sparing sedative. In preliminary results presented in abstract form [95] , cardiac surgery patients (n = 55) randomly assigned to dexmedetomidine for postoperative sedation had a nonsignificantly lower incidence of postoperative delirium as compared with those sedated with propofol or a combination of fentanyl and midazolam (5% versus 54% versus 46%). The authors of that report plan to enroll a total of 90 patient in the study; perhaps these impressive differences will be statistically significant with a greater number of patients.\nRecombinant human erythropoietin (rHuEPO) has received considerable attention as a potential transfusion sparing strategy in the ICU. Interestingly, EPO and its receptor are both expressed by the nervous system, and systemically administered rHuEPO can reach sites within the brain. In preclinical studies, rHuEPO reduced neuronal injury produced by focal ischemia, TBI, spinal cord injury, and subarachnoid hemorrhage [96] [97] [98] . Enthusiasm regarding its use as a general neuroprotectant in the ICU has been tempered by potential risks such as thromboembolism and the considerable cost of the drug. Concerns over safety may be at least partially addressed by the recent finding of erythropoietin derivatives with tissue protective but not hematopoietic properties [99] .\nXenon is a chemically inert gas that has been used as an anesthetic agent and for contrast enhancement in CT scans.\nIn rats xenon appears to protect the brain from the neurologic damage associated with the use of cardiopulmonary bypass, an effect that is potentially related to N-methyl-D-aspartate receptor antagonism [100] . However, its tendency to expand gaseous bubbles, such as bypass-associated cerebral air emboli, could abolish any beneficial effect or even worsen cerebral outcome [101] .\nIn the setting of ischemic stroke or TBI, there are a variety of compounds with the potential to improve neurologic outcomes. For example, NXY-059, a free radical trapping agent, reduced disability at 90 days when given within 6 hours of stroke onset [102] . In a pilot randomized trial in 56 patients, simvastatin given up to 12 hours after stroke onset significantly improved neurologic functioning (National Institutes of Health Stroke Scale score) at 90 days [103] . Ethyl pyruvate, a pyruvate derivative that prevents mortality in murine sepsis models, reduced motor impairments, neurologic deficits, and infarct volume in a rat stroke model when given as late as 12 hours after middle cerebral artery occlusion [104] . In rodent models of TBI, cyclosporin A reduced acute motor deficits and improved cognitive performance, even when given after the traumatic insult [105] . A phase II dose escalation trial is currently underway in humans.\nMounting evidence suggests that mild-to-moderate hypothermia can mitigate neurologic injury. Shankaran and colleagues [106] found that whole-body hypothermia (33.5°C for 72 hours) reduced the risk for death or disability in infants with moderate or severe hypoxic-ischemic encephalopathy. In adults successfully resuscitated after cardiac arrest, moderate hypothermia (32-34°C for 12 to 24 hours) increased rates of favorable neurologic outcomes and reduced mortality [107, 108] . A practical limitation of therapeutic hypothermia is that reaching target temperatures takes at least 2 hours using the fastest currently available cooling techniques. However, Polderman and colleagues [109] demonstrated that hypothermia could be induced safely and quickly (about 60 min) by means of ice-cold intravenous fluid combined with icewater cooling blankets.\nCognitive rehabilitation involves the teaching of skills and strategies to target specific problems in perception, memory, thinking and problem solving, with the goal of improving function and compensating for deficits. The benefits of cognitive rehabilitation are well known to those that care for patients with stroke, anoxia, or TBI. Predicting who will benefit and how much has proven challenging, but even severely disabled patients sometimes make dramatic neurocognitive recoveries [110] . Although there are no studies evaluating the effectiveness of cognitive rehabilitation in patients recovering from non-neurologic critical illness, it stands to reason that such patients could benefit when they are found to be cognitively impaired. Because cognitive impairments in critically ill patients appear to be underrecognized by ICU and physical rehabilitation providers [111] , few patients are referred for cognitive rehabilitation therapy [3] . Education regarding the cognitive sequelae of critical illness is needed to enhance referrals for rehabilitation, not only for weakness and physical debilitation but also for cognitive impairments.\nCognitive function is an important and relatively understudied outcome of critical illness. Evidence suggests that neurotransmitter abnormalities and occult diffuse brain injury are important pathophysiologic mechanisms that underlie critical illness-associated CD. Markers that could be used to evaluate the influence of these mechanisms in individual patients include the following: SAA, certain brain proteins (S-100β, NSE, and MPB), and MRI tissue sodium concentration. Although recent advances in this area are exciting, they are still too immature to influence patient care. Additional research is needed if we are to understand better the relative contributions of specific mechanisms to the development of critical illness-associated cognitive dysfunction and to determine whether these mechanisms might be amenable to treatment or prevention.\nThis article is part of a thematic series on Translational research, edited by John Kellum.\nOther articles in the series can be found online at http://ccforum.com/articles/ theme-series.asp?series=CC_Trans" }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: DNA microarray technologies have proven to be extremely valuable for probing biological processes by measuring mRNA expression profiles. However, studies at the protein level have the potential to provide more direct information since most genes function through their protein products. Traditional investigations focus on individual proteins in a system and then combine such individual analyses to provide a more global perspective. Recently, technologies to analyze proteins in a high throughput and unbiased fashion have become feasible [1] . One particular powerful technology is protein microarrays, which contain a high density of proteins and allow a systematic probing of biochemical activities [2, 3] .\nThere are two types of protein microarrays [3] . A 'functional protein microarray' contains a set of proteins individually produced and positioned in an addressable format on a microarray surface. Functional protein microarrays are useful for identifying binding activities or targets of modification enzymes. The first version of a proteome microarray was reported in 2001 and contained 5,800 yeast proteins with amino-terminal glutathione S-transferase (GST) tags printed on the array [4] . A second version of yeast protein microarrays was generated recently and contained 5,600 proteins with carboxy-terminal 6His-HA-ZZ domain tags [5] . Proteins from both collections were overexpressed, purified and spotted onto the protein microarrays. Global proteome studies were performed on these chips to understand various biological mechanisms. For example, 87 yeast kinases were examined for their substrates using yeast protein microarrays and over 4,200 in vitro substrates representing 1,325 unique proteins were identified [6] . Compared with the approximately 150 known in vivo kinase-substrate interactions, this global study served as an important first step for dissecting yeast signaling networks. In addition to searching for kinase substrates, proteome chips can be probed with labeled proteins, DNA, lipids, antibodies and many other molecules to search for interacting proteins [4, 7, 8] . Large amounts of data have been generated using protein microarrays, presenting significant challenges in developing robust methods to process the raw data and building reasonable biological hypotheses from the datasets.\nThe second type of protein microarray, the 'analytical protein microarray' or 'antibody microarray', shares similarities with immunoassays and uses antibodies to detect specific probes. Studies have shown that these antibody arrays can recognize specific targets and generate dose-dependent signal intensities, indicating that they can be used to quantify levels of various targets in a crude mixture [9, 10] . Because of the crossreactivity of certain antibodies with a variety of proteins, only highly specific antibodies are suitable for this type of study. This remains a limiting factor in preparing antibody microarrays.\nBoth DNA and protein microarrays are prone to systematic errors that are usually generated from different sources, such as surface defects and spatial artifacts. Many studies have offered insight on noise subtraction in DNA microarrays [11] [12] [13] [14] , but little investigation has been done for protein microarrays. Functional protein microarrays differ in many respects from DNA microarrays. First, the goals of these two microarrays are different. DNA microarrays measure the relative DNA levels in a pool of probes, whereas functional protein arrays often aim at discovering global interactions of a single probe molecule. Second, a typical DNA microarray experiment measures signal ratios between two color channels, one for a tested mRNA sample and the other for a reference sample [15] . Signals in the second channel may serve as intrinsic controls that can help to decrease the effects of various amounts of reagent on the arrays and any local array nonuniformity. Furthermore, many current scaling methods are then based on the assumption that signal intensities should be balanced between the two color channels despite variation in slide location, intensity and other sources of systematic variation [16] [17] [18] . However, such controls are missing in onecolor-channel protein microarrays. Third, several scaling approaches in DNA microarrays are based on a set of 'housekeeping' genes that give constant signal intensities at different conditions [19, 20] . However, in protein microarrays, such a control group must be customized according to the type of activities that are assayed, and, therefore, a ubiquitous reference group does not exist. Fourth, unlike DNA microarrays, in which non-specific binding can often be addressed by signal comparison with mismatch probes [21] , cross-reactivities of protein microarrays can not be as directly corrected for. A separate slide is, therefore, often required to be probed in parallel as a negative control in protein microarray experiments. Finally, several protein-specific artifacts serve as common noise sources in protein microarrays. In the kinase assay, for example, the signal from strongly phosphorylated spots can bleed into neighboring spots, leading to incorrect background measurement. These differences are particularly applicable to functional protein microarrays in comparison to antibody arrays, and, therefore, the normalization techniques used for DNA microarrays are usually not directly applicable to functional protein microarrays.\nWe have developed a new protein chip analysis tool (ProCAT) to deal with various artifacts specific to functional protein microarrays. The work started from a careful survey and characterization of all potential sources of systematic errors in protein microarrays. Specific approaches were then designed to deal with each type of noise. A correction approach is applied to reduce measurement errors in the background signals. In addition, spatial variations can be reduced efficiently through a novel two-parameter signal normalization approach and calling positive spots locally. After generating a list of positives, negative control slides are analyzed in the same approach and spots are subtracted from the list if they appear in the control slide. Slide features with poor signal qualities are also removed. Finally, signal intensities of the positives are normalized according to their protein amounts. All modules that account for the challenges in data processing specific to protein microarrays are built into ProCAT and tested.\nProCAT contains a flexible modular design whose individual components can be adjusted according to the experimental designs and stringency level selected by the users. Six sequential modules are currently implemented in ProCAT before a final annotation report is assembled ( Figure 1 ). These modules carry out: background correction; signal normalization; positive spot identification; spot cross-reactivity filter; signal qualities inspection; and protein amount normalization. The performance of many of the steps was tested using several types of experiments as described below.\nA fundamental issue in all microarray experiments is background correction, which aims at reducing noise in background quantification. Signal intensities are generally quantified by subtracting the foreground intensities with the local background intensities, which are measured as the background signals immediately surrounding the spot of interest (termed here the 'adjacent background'; Figure 2b ). However, in protein microarrays local background regions can be easily skewed by artifacts such as small speckles. In addition, strong positive signals from on-chip kinase assays tend to produce signal smears on both film and phosphoimagers that exceed the normal feature size (Figure 2a) . In both cases, the measurement for that spot will be inaccurate. First, the background intensity will be arbitrarily high, which will diminish the real signal intensity for that spot. Second, the intensities will be affected by the alignment of the grid and extent of the smear, and, therefore, the variance of the same protein at replicate experiments will be increased.\nTwo methods can reduce the artifacts in local background. The user can manually adjust the grid size to fit the circles to each individual spot. However, the aligning process requires considerable time and effort. The size of the smear may even prevent refitting the grid without adversely affecting neighboring spots. Additionally, a larger spot size can diminish the signal of the spot because the signal density decreases with increasing spot size. The second method for background correction, which is applied in ProCAT, replaces the background intensity of the central spot with the background from its local neighborhood. A three by three surrounding window is assigned to each protein spot, and the median background of the nine spots will be used as the 'neighborhood background' value for the central spot (see Materials and methods for more details). No additional time is needed for further alignment, yet this method will significantly reduce artifacts that can produce erroneous measurements on spots background.\nIn the analysis of the phosphorylome dataset [6] , we applied the neighborhood background correction and observed a high sensitivity in identifying positive targets. To further characterize the effects of neighborhood background correction, we performed a test kinase assay with 100 nM protein kinase A (PKA) spotted at 96 locations on one slide (Figure 2a) . Each of the 48 blocks on the slide contains two PKA pairs with random yeast proteins spotted elsewhere (approximately 12,000 spots). After incubating the slide with 33 P-γ-ATP, all of the PKA spots autophosphorylated and showed strong signals, and in many cases the signal went beyond the grid circle boundaries (Figure 2b ). We then applied the neighborhood background correction to the PKA spots. As expected, the median for PKA signal intensities was enhanced by 53%. Furthermore, the PKA signals from different positions are more similar to each other; the variance within them is decreased by 41% (p value = 0.006; Fig 2d) . Therefore, the neighborhood method for accessing background provides more robust measurements than that of the adjacent background method.\nSpatial artifacts arise from uneven signal distribution across the slide, in part due to uneven probing conditions and smear artifacts [13] . Uneven probing can occur by several means, such as uneven mixing of the probe, exposure to the probe solution, or uneven washing and drying of the slides. Twocolor-channel experiments of DNA microarrays provide intrinsic controls that can be used to account for spatial artifacts. Functional protein microarrays often use only one color channel and, therefore, are especially prone to spatial artifacts. Spatial artifacts will cause inaccurate measurements of signal intensities and can hinder the identification of significant interactions. Adding more controls can help remove spatial artifacts since the signal of each spot can then be normalized according to its local controls. Due to the variable shape and size of spatial artifacts, ideally a large number of controls would be needed. However, space constraints of the protein chip and an inability to anticipate all the uses of the arrays usually prevent the necessary number of controls to fully account for spatial artifacts on the array.\nA scaling method that reduces signal variations among spots of the same proteins at different array locations decreases spatial artifacts. We developed a new normalization method to deal with the spatial artifacts specific to functional protein microarrays. By assuming that signal distribution in large windows is consistent across the slide, the foreground signal of each spot can be normalized according to signal intensities in its surrounding neighborhood. This assumption is usually valid in protein microarray experiments in which proteins are randomly printed on the array ( Figure 3 ). Two parameters, the median and the median absolute deviation (MAD), are calculated to represent the signal distribution in the local window ( Figure 4 ). To perform the normalization, the median and MAD of all sliding windows are averaged. The average values are then used to correct the signal of the central spot to To test the performance of this two-parameter scaling approach for signal normalization within one slide, we designed a test microarray containing multiple positive controls printed at different positions on the slide. The test array was organized in the same format as the commercially available protein microarrays (Invitrogen). Each protein was printed in duplicate, and the array contained 24 blocks of 16 by 16 printed proteins (Figure 5a ). Two GST-fusion proteins, Sla2p and Myo4p, were purified separately and a 1:1, 1:5, and 1:25 dilution of each protein was prepared. Sla2p and Myo4p at each concentration were printed at eight random positions on the array. Other spots were occupied with bovine serum albumin (BSA) as negative controls. In order to visualize the two fusion proteins, anti-GST antibody was used to probe the slide, and one probing with typical spatial artifacts is shown in Figure 5 . The artifact-containing slide showed different signal levels between the edges and the middle portion of the array. This produced blocks that had a variable signal distribution that ranged from high to low from one edge of the slide to the opposite edge; the variability occurred across blocks and simple block normalization methods adopted in DNA microarray normalization approaches [17] would not be suitable for dealing with this problem.\nWe applied ProCAT to normalize the slide with several different parameters ( Figure 5) . Five window sizes were tested, termed windows 1, 3, 5, 7, and 9. These numbers correspond to the window size as a function of the number of spots on one edge of a block. For example, a block of 20 by 20 spots analyzed using window 1 would have a window size of 0.1 that of the block edge, or in this case 2 spots above, below, and to either side of the central spot, whereas a window size of 9 would contain a 37 by 37 area roughly as large as 4 blocks.\nThree observations were made from the analysis of different window sizes. First, as the window size increases, the computational time used for the normalization also increases. Second, no obvious spatial artifacts were left after the normalization with any of the window sizes tested ( Figure 5b ). Third, a small window size diminishes any signal inequality that exists between positive signals and background noise. Indeed, a small scaling window tends to introduce extreme changes to the original signals and, therefore, increases the discrepancy between the duplicate spots of the same protein. The variance of the signals for the same protein after normalization with different window size was calculated. In five out of the six cases (three dilutions of two proteins) the scaling window 9 can successfully reduce the signal variance in a range from 31% to 90% (Figure 5c ). Decrease of signal variation suggests that a large scaling window will help to reduce spatial artifacts. Although larger window sizes are possible, 9 was used as the default number for ProCAT because the analysis can be done in a reasonable time and minimal improvement has been achieved after window size 7 (Additional data file 1).\nIn addition to providing accurate measurements of spot intensities, ProCAT has been developed to assign thresholds for identifying positive targets in one experiment. Traditionally, a global cutoff can be calculated from all spots and applied to the whole slide. Due to variable spatial artifacts, cutoffs were assigned locally in ProCAT. For each spot on the array the signal distribution within a nine by nine window was calculated and a cutoff defined as a number of standard deviations away from the mean; the default for ProCAT is two standard deviations. This cutoff corresponds to 5% significance level if the signal distribution within this local window is normal. When many spots with strong signals are included in the window, the cutoff will be arbitrarily high and thus decrease the sensitivity of detecting positive spots by the program. To avoid this loss in sensitivity, ProCAT has a built in function to identify possible outliers, to remove those outlier spots that have extremely strong signals, and then to calculate a cutoff for identifying positive spots using the remaining spots.\nA receiver operating characteristic (ROC) curve was used to compare the performance of local window cutoffs versus a global cutoff on the test slide [22] . Area under ROC curve (AUC) is a performance indicator that ranges from 0 to 1, with 1 for the best performing method. Using GST-Sla2p and GST-Myo4p as positive controls and BSA as negative controls, the sensitivity and specificity for both local and global cutoff methods was estimated. Five window sizes were tested and compared with the global cutoff ( Figure 6 ). Prediction performance is increased significantly when using local windows with nine or more spots on one edge. Thus, a nine by nine window is used as the default in ProCAT since a larger A representative protein microarray with high-quality data Figure 3 A representative protein microarray with high-quality data. The slide image was reconstructed from a protein microarray experiment with minimal noise in the data. Density plots of signals in local 37 by 37 windows (window size 9) for all spots were computationally combined, and they showed high similarities. window size results in increased computing time with only minimal improvement in sensitivity. The AUC value is much larger in local cutoffs (0.992) compared to global cutoffs (0.916) and the improvement is unlikely to be due to random chance (p value = 0.002) [20] . Therefore, we can conclude that the local cutoff is significantly better in identifying positive spots than a global cutoff.\nScheme for the signal scaling method Figure 4 Scheme for the signal scaling method. The signal of one spot on the array is normalized according to the distribution in its local neighborhood. For each spot, a surrounding window is chosen and all spots in this window are defined as its neighborhood. The signal of a center spot will then be normalized by comparing the local median and MAD with the average values. Norm, normalized signals; Origin, original signals.\nAverage distribution \nTwo layers of filters are implemented in ProCAT. First, all positive spots from negative control experiments are removed. For example, in on-chip kinase assays, kinase dead alleles were probed on separate arrays using the same experimental conditions as used with wild-type kinases. Spots that produce signals in the absence of active kinase were identified by ProCAT and removed from the target lists of kinase probings. When probing tagged protein to detect protein-protein interaction, testing the epitope tag in the absence of the protein of interest is also an essential control. If proper negative control experiments are available, ProCAT will analyze them in the same way as regular experiments to construct experimental positive spot lists void of proteins producing positive signals under control conditions.\nThe second filter checks the quality of each positive spot. All proteins are spotted in duplicates on protein microarrays, hence should have very similar signal intensities. ProCAT then uses the difference between duplicate signals as an indicator of the signal qualities. The difference between signals of two duplicate spots (s 1 , s 2 ) is calculated as (s 1 + s 2 )/(|s 1 | + |s 2 |) and then fitted to a normal distribution. Proteins with exceptionally large differences in their duplicate spots are more likely to be biased by certain artifacts, and thus are removed from the positive list. The default threshold for the duplicate spot difference in ProCAT is set at two standard deviations away from the mean.\nOne of the goals for protein microarray experiments is to identify the affinity of a binding interaction (in a protein-protein interaction assay) or the extent of phosphorylation (in a kinase assay) so that one can compare the relative strength of the reaction for each positive protein. Ideally, the spot intensity would directly correspond to the strength of interaction. However, a number of other factors contribute to the array signal intensities, including the systematic noise from various artifacts, as was already discussed, and the amount of protein printed on the chip. Nonetheless, semi-quantitative estimates can be obtained. After background correction and signal normalization, the raw signals can be standardized by relative protein amounts before they can be used to estimate the interaction strength.\nAlthough proteins on the microarray can have very different amounts, they do share the same epitopes for the purpose of large-scale protein purification [4, 5] . Therefore, probing with anti-epitope antibodies will provide an estimate of the relative protein amounts in each spot on the array. After the protein amount is determined for one spot at row i and column j, ProCAT divides the raw signal intensities S i,j by the protein amount signals A i,j and uses the quotient as an approximation of the strengths of interactions:\nThis approximation generally works well across the slide except for the following two situations. Less abundant proteins will be biased because the A i,j values estimated in antiepitope probings are more susceptible to background noise and slide artifacts. On the other hand, overpowering spots can also be biased if they have saturated signal intensities. A saturated S i,j value is an underestimate to the real signal. For these two reasons, only proteins with amounts more than a minimal cutoff and signal intensities lower than a saturation threshold will be normalized with protein amounts. Proteins that do not conform to these two requirements will be recorded with unnormalized signals and flagged for further inspection. An additional caveat is that the relative protein amount assessed using antibodies includes both native and denatured protein at a given spot. Therefore, the estimation of interaction strength will be an underestimate since the amount of functional protein may be an overestimate.\nProCAT was designed as a flexible tool to analyze functional protein microarray data. The program was scripted in Perl (version 5.6.1) on top of a Tomcat (version 5.0.30) web server [23] . Each module discussed above was implemented independently and can be included or excluded depending on various experimental designs. To input a dataset, the user has to characterize the data in three aspects: experimental designs, data file formats and normalization parameters. Experimental design contains parameters such as the number of test arrays and negative control arrays for one particular assay. Data file format describes the layout in the uploaded dataset so that ProCAT can recognize and extract the useful information from it. Normalization parameters allow users to try different stringency levels. These three levels supply sufficient information to uniquely characterize an experiment while still allowing ample flexibility for the individual user to customize parameters to suit many different types of experimental designs.\nAfter inputting all three descriptions and uploading the dataset, ProCAT takes five minutes on average to complete all analysis modules for each array. The time may vary depending on the selected analysis modules and the size of the protein microarrays. Each task is assigned a unique ID and results are organized into a database for future queries. Processed data including analysis parameters, a list of positive spots with protein annotations, and normalized signal intensities will be available for the users to download from the server.\nFunctional protein microarrays serve as an efficient platform for screening protein biochemical functions. Here we present ProCAT as a systematic approach to process and analyze data specific to functional protein microarrays. Calibrated by explicit test experiments, ProCAT has proven to be able to handle many types of functional protein microarray studies with three unique features. ProCAT includes novel scaling methods that provide robust and reproducible measurement for quantitative signals. This is crucial for protein microarrays as chip signal intensities often indicate strength of interactions. In addition, by calling positive candidates locally, ProCAT demonstrated excellent performance in identifying positives in comparison to global thresholds. Finally, each step has been integrated into a modular design to fit various experimental designs and stringency requirements.\nA major challenge in designing any automated data processing method is thinking of and anticipating all possible situations that may arise. ProCAT uses a local three by three window to correct background containing signal smears or dust speckles. This method assumes the artifacts are sparse enough so that the majority of the nine spots in the local window still provide correct measurements of the background signals. Since the median value of nine spots is used to correct the background, a few biased spots within the window will not severely affect the corrected background value. This assumption is usually valid since the percentage of spots that are either positive or whose signal is contaminated by artifacts in protein microarray experiments is generally quite low. In extreme cases where such spots are likely to be very close to each other, a larger window (five by five for example) can be used. Large artifacts such as bright speckles and incubation bubbles may affect many spots in a particular region. Since the shapes of these artifacts are variable, it is necessary to manually flag these spots initially and then remove them from future analysis. Many commercially available software packages for microarray experiments have a built in flagging function, and ProCAT will automatically discard flagged spots.\nA key aspect of ProCAT is the two-parameter approach for reducing spatial nonuniformity. Several factors can affect the performance of ProCAT's normalization. First, ProCAT normalizes the signal of a spot according to the signal distribution in its local neighborhood. It diminishes the signal intensity if the spot is located in a high signal neighborhood, while compensating the intensity if it is in a low signal neighborhood. This approach is based on the assumption that signal intensities across the slide share the same distribution, and it holds true if and only if the regional variations observed on the slide are due to technical artifacts and not from real biological differences. Since proteins are printed in a random order on most of the current protein microarrays, it is unlikely a particular region of the slide will gain high intensities as a result of biologically relevant reasons. Second, the size of the neighborhood window can also largely affect the performance of the normalization. Small window sizes tend to add biases to signals and diminish all local variations, whereas large window sizes increase the computational burden and tend to preserve local variations. We found that the optimal window size of ProCAT is 9 for protein-protein interactions; this figure corresponds to approximately four blocks on the chip and is used as the default. Other window sizes can also be chosen to fit various shapes of spatial artifacts.\nProCAT can be applied to many experiments using protein microarrays, such as kinase assays, protein-protein interactions and protein-DNA interactions. Thus far, the twoparameter scaling approach has only been used in single chip normalization; however, a similar strategy can be extended to rescale multiple slides by assuming signals in neighborhood windows on different slides are similarly distributed. Overall, ProCAT provides a powerful and flexible new approach for optimal processing and analysis of functional protein microarrays.\nFor the slide used for testing background correction, 100 nM PKA (Sigma, St. Louis, MO, USA) was spotted at 96 different places as positive control. The slide was incubated with 200 μl of kinase buffer (100 mM Tris pH 8.0, 100 mM NaCl, 10 mM MgCl 2 , 20 mM glutathione, 20% glycerol) plus 0.5 mg/ ml BSA, 0.1% Triton X-100, and 2 μl 33 P-γ-ATP in a humidified chamber at 30°C for 1 hour. The slide was then washed twice with 10 mM Tris pH 7.4, 0.5% SDS and once with double distilled H 2 O before being spun dry and exposed to X-ray film (Kodak, Rochester, NY, USA).\nFor the anti-GST probing, slides were printed with Sla2p and Myo4p as positive controls and 150 nM BSA as a negative control. The array surface was blocked using SuperBlock (Pierce, Rockford, IL, USA) at 4°C for 1 hour. Rabbit polyclonal IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was incubated with the slides at 1,000-fold dilution. The array was then washed with PBST (Sigma) and incubated with a 1:1,000 dilution of Cy5-conjugated anti-rabbit IgG antibody (Jackson Laboratories, Bar Harbor, ME, USA). Slides were then washed with PBST five times and scanned in an Axon\nTesting experiment for the signal scaling approach Figure 5 (see previous page) Testing experiment for the signal scaling approach. (a) The design of the test slide with positive spots shown as red spots and the five tested normalization window, indicated by red squares, for a given spot on the array, shown in blue. (b) Comparison of signal intensity before and after normalization using window size 9 on the testing experiment. The two images were computationally reconstructed from the signal files, either without or with normalization. GenePix scanner (Molecular Devices, Sunnyvale, CA, USA). Raw signals were extracted with GenePix Pro 6.0 software (Molecular Devices).\nFor one spot, let i be the row and j the column on a protein microarray. Thus, B i,j represents the adjacent background intensity and F i,j denotes the foreground intensity. The raw signal intensity S i,j is calculated as:\nIn neighborhood background correction, we use neighborhood background to replace the adjacent background. A local three by three window around B i,j is chosen and the neighborhood background is defined as:\nIn a protein slide with N rows and M columns, a local window W i,j around one spot (i, j) is defined as signals of a set of spots S i,j that satisfy:\nThe size parameter k is dependent on window size factor f win and the block size f block :\nin which f block represents the number of spots on one edge of the block, and f win is chosen by users from five options: 1, 3, 5, 7 and 9. Different windows can overlap with each other and go beyond the block edges. Let s denote signal intensities of spots within the local window; ProCAT uses two parameters to characterize the signal distribution of s: median (MED) and median absolute deviation (MAD):\nAfter calculating MED i,j and MAD i,j for all the spots on the array, they are averaged to obtain the two parameters and for the reference distribution.\nFor one spot (i, j), ProCAT normalizes its raw signal S i,j by comparing MED i,j and MAD i,j with the average values:\nFor a given spot at row i and column j, its normalized signal is compared to surrounding spots in a nine by nine window W ij (4) . Signals within this window are fit to a normal distribution. The mean μ i,j and standard deviation σ i,j will be calculated and the default threshold is set at two standard deviations above the signal mean. A spot (i, j) will be called positive only if its signal is above the threshold:\nWhen positive spots are likely to be close to each other, Pro-CAT uses box plots to examine and remove possible outliers from the surrounding window [24] . Let Q 1 be the lower quartile (25th percentile) and Q 2 be the upper quartile (75th percentile); the difference between Q 1 and Q 2 is termed interquartile range ΔQ. A spot (i', j') is then defined as an outlier if its signal:\nROC curve comparing the global cutoffs and local cutoffs in calling positive spots Figure 6 ROC curve comparing the global cutoffs and local cutoffs in calling positive spots. The test slide has six unique positive controls (Sla2p and Myo4p in three different titrations). The performance of identifying the positive controls is increased by using local cutoffs generated in relatively large surrounding windows. Five window sizes were tested and the best performance was achieved using nine by nine or larger windows. To obtain a robust threshold, the corrected mean and standard deviation are generated using the non-outlier spots.\nThe following additional data files are available with the online version of this paper. Additional data file 1 is a figure illustrating the variance reduction in positive controls using different normalization window sizes. Additional data file 2 is a table listing the raw signals generated in the autophosphorylation experiment for testing the background correction method. Additional data file 3 is a table listing the raw signals generated in the anti-GST probing experiment calibrating the signal scaling approach.\nAdditional File 1 Variance reduction in positive controls using different normaliza-tion window sizes Variance reduction in positive controls using different normaliza-tion window sizes. Click here for file Additional File 2 Raw signals generated in the autophosphorylation experiment for testing the background correction method Raw signals generated in the autophosphorylation experiment for testing the background correction method. Click here for file Additional File 3 Raw signals generated in the anti-GST probing experiment cali-brating the signal scaling approach Raw signals generated in the anti-GST probing experiment cali-brating the signal scaling approach. Click here for file" }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: Understanding transcription factor (TF) mediated control of gene expression remains a major challenge at the interface of computational and experimental biology. Computational techniques predicting TF-binding site specificity are frequently unreliable. On the other hand, comprehensive experimental validation is difficult and time consuming. We introduce a simple strategy that dramatically improves robustness and accuracy of computational binding site prediction. First, we evaluate the rate of recurrence of computational TFBS predictions by commonly used sampling procedures. We find that the vast majority of results are biologically meaningless. However clustering results based on nucleotide position improves predictive power. Additionally, we find that positional clustering increases robustness to long or imperfectly selected input sequences. Positional clustering can also be used as a mechanism to integrate results from multiple sampling approaches for improvements in accuracy over each one alone. Finally, we predict and validate regulatory sequences partially responsible for transcriptional control of the mammalian type A g-aminobutyric acid receptor (GABA A R) subunit genes. Positional clustering is useful for improving computational binding site predictions, with potential application to improving our understanding of mammalian gene expression. In particular, predicted regulatory mechanisms in the mammalian GABA A R subunit gene family may open new avenues of research towards understanding this pharmacologically important neurotransmitter receptor system.\nCo-regulation is a basic mechanism to coordinately control expression of genes in modules, biochemical pathways and protein complexes (1) (2) (3) . In eukaryotes, expression is most often mediated by transcription factors (TFs) that bind upstream of the transcription start site (TSS) and recruit the polymerase assembly (4) . TFs bind, with varying affinity, to a set of similar, short (6-20 nt) sequences (5) . Computational binding site discovery focuses on finding significantly overrepresented sequences in upstream regions of co-regulated genes (6) (7) (8) . Thus, computational TFBS prediction algorithms must begin with an input set of promoters from genes hypothetically co-regulated by a shared TF. The algorithms aim to predict the binding positions and consequently the nucleotide specificity of the TF (9) (10) (11) .\nThe first part of transcription factor binding site (TFBS) discovery, the input set, can be identified using either computational or experimental methods. Experimental techniques, such as chromatin immunoprecipitation (ChIP) (12) , have been successfully used to generate a genome scale mapping of approximate TF-binding positions (10, 13, 14) . Computational techniques, such as phylogenetic profiling (15, 16) and artificial neural networks, can also be used to identify sets of co-regulated genes. Both experimental and computational approaches, however, suffer from a significant false positive (FP) prediction rate. Inclusion of extraneous promoters in the input sets dilutes the enrichment of the shared TFBS sequences making computational TFBS discovery significantly more challenging (17) . We term such erroneously included promoters decoy sequences (DSs).\nAfter receiving a set of upstream regions co-regulated by a shared TF as input, computational methods aim to predict the binding positions of that TF (6) (7) (8) 18) . Given a set of input promoters, motif detection algorithms identify a set of short, oligonucleotide segments hypothesized to bind to the TF of interest. The predicted sequences can be used to construct a position weight matrix (PWM) representing the average nucleotide frequencies for each position in the site (19) . Ideally, computational detection will return all sequences that bind to every TF with biologically relevant function in those upstream regions. However, since the source of binding specificity for TFs is not well understood (20) , heuristic approaches and ad hoc multiple alignment based scoring schemes are used to identify locally optimal solutions (17) . Each local optimum that exists in a given set of promoters may correspond to distinctly different motifs, and may score differently relative to each other according to different scoring schemes.\nBinding site prediction algorithms are generally confounded by several factors: degeneracy in the binding site; the unknown length of the binding site; the relatively large length of promoters; and the inclusion of DSs in the input sets (17, 21, 22) . As a result as few as 10% of predicted positions correspond to biologically functional binding sites (23) . Due, in part, to the low accuracy rate, computational binding site identification has been of limited use (23) . Problems identifying binding sites are further exacerbated in mammalian genomes by larger promoter regions (24) and scarcity of reliable information on co-regulation of genes. Thus, the most demanding test of efficacy for TFBS identification approaches lies in their application to mammalian systems and subsequent validation of predictions.\nBecause of computational complexity of the problem, Gibbs sampling is often used to identify binding positions (18) . In this paper, we present a new strategy that clusters Gibbs sampling results at each input nucleotide-a technique we term positional clustering-to improve accuracy of predicted TF binding. We evaluate the efficacy of our approach using known examples of binding and regulation in yeast and experimentally testing predicted TF-binding sites upstream of the subunit genes coding for the heteromeric mammalian neurotransmitter receptor system, the type A g-aminobutyric acid receptor (GABA A R).\nThe GABA A R is the major inhibitory neurotransmitter receptor in the central nervous system (CNS) (25, 26) with important roles in development (27, 28) and disease (29) (30) (31) . The receptor is believed to be a pentamer made up of multiple subunits that come from at least four different subunit classes (a, b, g and d) (32) . At least 19 genes code for the various subunits that differentially combine to form numerous pharmacologically distinct GABA A receptor isoforms (29, 30) . Isoform utilization depends in part on the relative abundance of the subunits, which may change under various conditions (33) (34) (35) . Understanding subunit regulatory mechanisms may provide insight into GABA A receptor isoform usage and related phenotypes (36) .\nIn the current study, we test the ability of positional clustering to detect known TF-binding sites in a series of increasingly noisy sets of yeast promoters, and found marked improvement in the percentage of correct predictions over Gibbs sampling alone. We also present de novo predictions of TF-binding sites in promoter regions of GABA A receptor subunit genes (GABRs) whose expression is altered (either up-regulated or down-regulated) in an animal model of temporal lobe epilepsy (35) . Positional clustering identified a number of putative cis-regulatory sites, many of which correspond to known binding elements for TFs found in the CNS. Mobility shift assays showed several predicted GABR-binding sequences specifically bind nuclear proteins derived from primary neocortical neurons kept in culture. Furthermore, a particular non-consensus GABR putative regulatory sequence was shown to have a functional role in cultured cortical neurons demonstrating the efficacy of positional clustering in detecting functional regulatory elements in mammals.\nWe identified S.cerevisiae genes predicted at high confidence (P < 0.001) to be regulated by the TF STE12 in YPD growth media, according to whole-genome TF location data (14) . For the 51 identified genes, we collected upstream intergenic promoters. Intergenic regions were truncated at 1 kb upstream of the gene's TSS.\nWe selected for study a set of six GABRs: GABRA1, GABRA4, GABRB1, GABRB3, GABRD and GABRE. Promoters were extracted for each gene, including two alternative first exons of the GABRB3 (37), giving a set of seven promoters. The length of each promoter was: GABRA1, 3733 bp; GABRA4, 1546 bp; GABRB1, 1353 bp; GABRB3 (exon 1), 1310 bp; GABRB3 (exon 1A), 2080 bp; GABRD, 6625 bp; and GABRE, 5278 bp. We augmented the input set with orthologous promoters from rat, with the exception of GABRB3 for which an orthologous gene from mouse was used. In total, 14 promoters upstream of six GABRs were selected for analysis.\nFor a given input set of promoters, we ran the Gibbs sampler BioProspector (8) 400-550 times, evenly distributed across all motifs widths from 6-15 bp. We used a third-order background model derived from appropriate genomic promoters. We collected the best three results from each BioProspector run. We counted the number of times BioProspector identified each nucleotide in the input set. For each promoter, we identified the maximally occurring nucleotide, and extracted all positions identified by BioProspector >35% of the maximum. We clustered together neighboring positions into putative TFBS. As a dust filter, we removed all putative TFBSs <6 bp long ( Figure 1 ).\nFor sets of S.cerevisiae promoters, we used 1200 results from 400 BioProspector runs in our evaluation. For GABRps, we considered all 127 non-empty subsets of the seven promoters (orthologous sequences were always considered together). We used results from 70 000 BioProspector runs, evenly distributed across all promoter subsets, in our analysis. In addition to dust filtering, we required putative TFBSs to occur both in the human and in the orthologous rodent promoter.\nWe used positive predictive value,\nto evaluate STE12-binding site predictions. We classified predictions as true positive (TP) or false positive (FP) by comparison to the STE12-binding motif, TGAAACA, as determined by (14) . For each sequence, we calculated distance from the known STE12 PWM using a modified local ungapped sequence alignment similar to that in (38) . Alignments were scored as the sum of Pearson's correlation coefficient,\nbetween prediction X and the STE12 PWM across all aligned positions. Thus, scores ranged from zero, with no positions aligned, to seven, the length of the STE12 PWM. We observed a bimodal distribution of scores (Supplementary Figure S1) , and chose the alignment score corresponding to the minima of the distribution (alignment score ¼ 4.5) as the threshold to classify predictions as TP or FP.\nWe complemented the seed set of 51 STE12-bound promoters with 1-50 randomly chosen yeast promoters. We performed our motif detection procedure on each input set, and compared the PPV of putative TFBS with that of raw BioProspector results ( Figure 2 , solid lines).\nTo evaluate the background rate of STE12-binding site recovery, we created a seed set of 51 randomly chosen S.cerevisiae promoters. We evaluated the percentage of STE12-like binding sites identified in the random seed set, as well as in versions of the seed set augmented with 1-50 randomly chosen yeast promoters (Figure 2, dashed lines) .\nFor additional yeast evaluations (HAP4, TEC1, YAP1 and YDR026C), we substituted for BioProspector an in-house implementation of the BioProspector algorithm. Comparisons of results from each implementation show the two implementations to be approximately equivalent.\nWe ran MotifScanner (39) to search GABR promoters for all vertebrate TF-binding motifs found in TRANSFAC (40) . For each promoter analyzed, we used a prior probability of 0.1 and the corresponding organism specific third-order promoter background model from Eukaryotic Promoter Database (EPD) (41) .We considered positional overlap between Motif-Scanner predictions and putative TFBSs indicative of known binding motifs in our predictions.\nDouble-stranded oligonucleotides for EMSA contained the following sequences: Nuclear extracts were prepared (42) and used for gel shift analysis after concentration (Microcon no. 10 columns, Amicon, MA). Quantification was performed on EMSAs under conditions that yield a standard curve for band intensity.\nSingle-stranded sense and antisense phosphorothioate oligonucleotides for the predicted GGCGGCGTGCACACACACGC-CCACCGCGG binding site are annealed by boiling sense and antisense oligonucleotides for 5 min at equal molar ratios in dH 2 O. Oligos are then cooled to room temperature and placed on ice. Transfections using DOTAP (Roche)/HEPES solutions are performed with oligonucleotides corresponding to wildtype, mutant or with DOTAP (Roche)/HEPES solution lacking oligonucleotides (MOCK) as described in (29) . Effects of oligonucleotide application to neurons are assessed by real-time RT-PCR.\nSince TFBS are predicted computationally by local optimization strategies, we evaluate the extent to which one of these strategies, Gibbs sampling, identifies the same set of segments in repeated runs using the same input data. Identifying stably recurring motifs requires clustering of related results which, in turn, requires definition of 'related'. Sequence similarity based clustering is impaired by the combination of sequence variation within motifs, the short length of TF-binding sites, and aligning motifs of different lengths. Instead of using sequence based clustering, we chose to cluster results by position, counting the number of times Gibbs sampling identifies each nucleotide in the promoter (Figure 1 ). We find that Gibbs sampling predictions, generated using BioProspector (8) are power-law distributed over nucleotide position (Supplementary Figure S2) . Gibbs sampling converges on the majority of nucleotides very infrequently, and a small number of nucleotides very frequently. Thus, the most frequently recurring nucleotides appear in as few as 10% of results. Moreover, we find the power-law distribution of results is robust to Gibbs sampling algorithm and scoring scheme (data not shown). We can hypothesize that the most frequently occurring positions are the most biologically significant. Thus, discarding the least frequent Gibbs sampling results may yield higher accuracy and robust identification of biologically insignificant positions.\nAs a preliminary test of the above hypothesis, we applied repeated runs of Gibbs sampling to a set of 51 S.cerevisiae promoters enriched in STE12 binding as identified by wholegenome ChIP-chip experiments (10) . We used positional clustering of 1200 results to identify the most frequently recurring positions (see Methods). Incorporation of additional results did not significantly alter the distribution of results (data not shown). We chose STE12 because it is one of the best studied TFs, with a well known, highly conserved and experimentally well-defined binding motif (10, 43) . The most frequently recurring positions were compared with the known STE12binding motif (40) . We classified predictions into two categories: true positive (TP) if they resemble the experimentally identified STE12-binding motif, and false positive (FP) otherwise (see Methods). Finally, we calculated the positive predictive value PPV as PPV ¼ TP/(TP + FP).\nWe find that positional clustering and subsequent selection of frequently recurring nucleotides improved the PPV of the STE12 binding site by at least 37% over Gibbs sampling alone (Figure 2 ). To validate that the above results were not specific to the number of input promoters, the STE12-binding motif, or the particular Gibbs sampling implementation, we repeated the above prediction process for promoters predicted to bind to YAP1, TEC1, HAP4 and YDR026C. We also repeated the analysis replacing the original Gibbs sampling procedure with our own implementation and MotifSampler (44) . In all cases, we found positional clustering significantly improves on results over local optimization procedures alone ( Figure 3 ).\nComputational discovery of TFBS can have two types of FP predictions. One type is the identification of an incorrect motif from a set of upstream regions known to bind to a TF of interest as described above (see Methods). The second type of FP error is the background discovery rate of the correct motif using upstream regions that do not bind to the TF. To simulate this rate for STE12-like binding site recovery we repeated the analysis as described above starting with 51 randomly chosen yeast promoters. We find that positional clustering identifies STE12-like sites in <5% of results, compared with 10-15% for Gibbs sampling alone. Thus, using positional clustering, the performance of computational motif discovery is enhanced not only by improving the positive predictive value in promoters of genes co-regulated by STE12, but also by decreasing the false discovery of STE12-like sites by 10%.\nNext, we evaluated the effect of adding DSs on the performance of Gibbs sampling with and without positional clustering. Addition of DSs dilutes enrichment of the TF-binding site in the input set, making motif detection more challenging (17, 22) . Modeling DSs, we repeated our estimate of PPV of TFBS detection with the addition of 1-50 random yeast promoters (DSs) to the original set of 51 STE12-bound promoters. We found that positional clustering improves the PPV of Gibbs sampling by >20% through the addition of up to 80% noise or 40 DSs (Figure 2, Supplementary Figure S3 ). Additionally, results of Gibbs sampling both with and without positional clustering decay linearly with the addition of decoys [R 2 ¼ 0.81 and 0.95, respectively (Supplementary Figure S4) ]. Extrapolating, we predict positional clustering will maintain an improved PPV through the addition of >100% noise or 70 DSs.\nTo address issues of generality, we repeated the procedure on additional sets of S.cerevisiae promoters (YAP1, TEC1, HAP4 and YDR026C). An added benefit is that we can evaluate the effect of information content of the binding motif and number of promoters on the improvement from positional clustering (22) . Repeating the analysis, we again find that independently of the set or sampling procedure, positional clustering improves accuracy through a broad range of random DSs (Figure 3 ). Improvement appears to be limited and unreliable only when sampling alone correctly identifies the binding site in fewer than 20% of results. This result is consistent with our analysis of STE12-bound promoters (Figure 2) , and may correspond to a lower limit for the efficacy of positional clustering.\nRecently, researchers have noted that complementary motif detection approaches can be used together to predict binding sites more effectively than either method alone (23) . With this in mind, we evaluated positional clustering in terms of its ability to combine results from two different sampling implementations. For each dataset, an equal number of results from each approach were combined into a single dataset, and positional clustering was used to predict binding sites as described above ( Figure 3C ). We measured the average percent change in PPV for each TF on each dataset, and found positional clustering improved combined sampling by 94% compared with 25% and 27% improvement for BioProspector and MotifSampler, respectively. Additionally, clustering combined sampling improved 19 of the 22 datasets evaluated, whereas clustering of BioProspector and MotifSampler results improved 17 and 16 datasets, respectively. Thus, positional clustering is an effective mechanism to integrate results from multiple sampling procedures.\nIdentification of GABR cis-regulatory sequences As described above in Introduction, identifying functional TFBS in mammals is difficult due in part to inclusion of decoy sequence from long upstream regions and lack of information on co-regulation of genes. Positional clustering, as shown above, is more robust to noisy input than Gibbs sampling alone, and thus may be better suited to identify de novo cis-regulatory elements in mammalian promoters that are coordinately regulated. To test this possibility, we chose seven mammalian GABR promoters (GABRps) whose activity is potentially altered in response to status epilepticus as identified through change in mRNA levels of the gene products (10) . For each set, the initial promoters were analyzed using Gibbs sampling with positional clustering (solid triangles) and without (open triangles). Two Gibbs sampling approaches were applied to each dataset: a Gibbs sampler procedure similar to BioProspector (8) (row A), and MotifSampler (39) (row B). Row C shows the combination of both sampling procedures, along with positional clustering of the combined results. x-axis counts over addition of DSs. We evaluated the positive predictive value of each technique on each dataset, and found positional clustering generally improved the PPV through addition of 100% random DSs. (31, 35) . We also included orthologous rodent promoters in the input sets (45) . Orthologous promoters were included to provide more instances of binding sites in the input set than would be expected by random, allowing for easier detection of the sites. Inclusion of orthologous promoters has the additional effect of selectively amplifying evolutionarily conserved binding sites. Such binding sites are more likely to have major functional roles in the regulation of the GABR receptor. Thus, sensitivity to such sequences is improved at the expense of sensitivity to species-specific binding sites. With this effect in mind, we require all GABR-binding site predictions to exist in orthologous promoters. Since the mechanisms of co-regulation for the seven GABRs are unknown, hypothetical co-regulation models were evaluated by querying all 2 7 possible subsets of the seven GABRps. Clustering results on nucleotide positions and selecting the most frequently occurring positions, we predicted 13 functional TF-binding sites.\nPredictions were compared with instances of known binding motifs from TRANSFAC (40) , and 8 of the 13 predictions (61.5%) resembled known binding sites for 10 TFs (Table 1) . Of the 10 TFs, 7 have been identified in the CNS of rodents: SP-1 (46); AP-2, TST-1 (POU3F1), OCT-1 (POU2F1), OLF-1 (47); CP-2 (48); and RREB-1 (49) . Furthermore, previous analyses of GABR promoter regions agree with our predictions that assign putative regulatory roles to SP-1, OCT-1, OLF-1 in the regulation of GABRs (29) . We chose to validate novel motif predictions with EMSAs and functional studies in primary cultured neurons.\nEMSA (50) was performed with an excess of cold competitors to define specificity of protein binding in nuclear extracts derived from primary neocortical neurons and fibroblasts (FIBs) kept in culture. As shown in Figures 4-6 , out of six predicted binding sites found upstream of the (a, b, g and d) subunit genes, four (GABRA4, GABRB1, GABRB3 and GABRD) displayed specific binding. In addition to specific binding of neuronal extracts to novel GABRA4 motifs, we have evidence for specific binding using FIB extracts ( Figure 5A and B) , of especial interest given that the expression of GABRs is restricted to the nervous system and repressors such as the RE1-silencing transcription factor (REST) (51, 52) expressed in non-neuronal cells have been implicated in the neural specificity of gene expression.\nClearly, protein binding to DNA does not always necessitate regulatory function. To begin to address the functional Table 1 . Positional clustering based predictions of transcriptional regulatory sequences upstream of GABRs In total, we predict 15 orthologous pairs of regulatory sequences, representing 13 unique sequences. Comparing with known mammalian binding motifs, eight of the predictions show similarity to previously characterized TFBS, as indicated. Where no known binding motif exists, the corresponding in vitro EMSA and functional assay, if applicable, is indicated. Similar predictions are grouped together and aligned by hand. significance of our predicted regulatory motifs, we evaluated the effects of transfecting neurons with double-stranded oligonucleotides containing one of the GABRA4 novel binding motifs (dsA4O), as described above. GABRA4 is especially interesting given that it is regulated by brain derived neurotrophic factor (BDNF) after status epilepticus (31, 53) . Transfection with the dsA4O produced a significant downregulation of GABRA4 gene expression in neocortical neurons as monitored by quantitative real-time RT-PCR with no change after MOCK transfection or transfection with a dsO containing three copies of a cAMP regulatory element (CRE) (Figure 6 ).\nHow reliable are the binding site predictions returned by Gibbs sampling based TFBS identification algorithms? We began by evaluating the stability of binding site predictions via repeated runs of Gibbs sampling. To quantify the robustness of predictions, we counted the number of Gibbs sampling results at each nucleotide position in the input set ( Figure 1 ) over a large number of repeated trials. We find that the most frequently returned positions better predict TF binding sites than the maximally scoring motifs from Gibbs sampling (Figures 2 and 3 ). Since scoring functions are empirically derived and do not necessarily represent biological reality, the result is not altogether unexpected (17) . However, we find that selecting frequently recurring positions allows filtering of up to 90% of spurious sampling results caused by convergence on biologically uninformative local minima. Positional clustering allows unbiased aggregation of results from different motif widths, thus approximating the width of the binding site 'for free' (54) .\nNext we show that positional clustering improves robustness to the addition of DSs (Figures 2 and 3) . Such sequences arise from inclusion of promoter regions in input sets without direct binding to the TF either due to experimental error or computational mis-annotation (17, 22) . In the STE12 example studied, linear regression models indicate our approach will maintain an advantage over traditional Gibbs sampling through addition of up to 150% noise to the original signal (Supplementary Figure S4) . Empirical data, however, show a sharp decrease in improvement close to the addition of 45 DSs, or roughly double the input set ( Figure 2 ). Moreover, evaluations using promoters co-regulated by other TFs Figure 6 . Double-stranded oligonucleotide functional assay for GABRA4 regulation. Primary cultures of rat neocortical neurons were treated with DOTAP (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate) alone (Mock) or with DOTAP and phosphothioate oligonucleotides from either a cAMP response element (CRE Decoy) or a sequence from the GABA-A4 promoter predicted using positional clustering (GABA-A4 Decoy) (GTGCACACACACGCCCACCGCGGCTCGGG). mRNA was harvested after 24 h, and real-time RT-PCR was performed with GABA-A4 specific primers. Error bars refer to individual experiments; i.e. different platings of cells from different animals. Data was normalized to rRNA levels, and expressed as relative mRNA levels (GABA-A4/rRNA). Results are shown as mean ± SEM, N ¼ 3, asterisk indicates significantly different from control at the 95% confidence interval. Figure 5 . EMSA of three putative TF binding sites form DNA-protein complexes in neocortical and fibroblast nuclear extracts. Neocortical (NEO) and fibroblast (FIB) nuclear extracts from E18 rat embryos were incubated with three 32 P-radiolabeled probes from human A4 and D receptor subunits. Cold wild-type oligonucleotides were used to define specificity through competition. Cold oligonucleotides were added at 100-fold excess over probe. indicate positional clustering is less likely to improve predictions when Gibbs sampling identifies a correct site in <20% of repetitions ( Figure 3 ). Thus, it is possible the rather simplistic linear model overestimates improvement in robustness beyond what is practically achievable. Moreover, when multiple motifs exist in the input promoters, preliminary evidence suggests positional clustering will uniquely identify a single dominant motif (Supplementary Figure S5) . With further refinement, however, it may be possible to recover subordinate motifs, enabling identification of cis-regulatory modules. In spite of these limitations, using positional clustering of repeated runs, researchers can successfully apply sampling algorithms in identification of functional binding sites in datasets with a significant proportion of noise.\nComputational prediction of TF binding in mammalian genomes poses just such a challenge due to increased decoy sequence in large upstream regions (24) . Thus, having established increased robustness to DSs in yeast, we applied our approach to identify potentially unknown GABA A receptor subunit gene regulatory sequences that may participate in the response of the genome to seizure activity. We reasoned that GABA A receptor subunit genes either up-regulated or down-regulated in the animal model of epilepsy would share common binding motifs. Using positional clustering, we predicted 13 TF-binding sites upstream of GABA A receptor subunit genes ( Table 1) . Twelve of our predictions were verified by either comparison to known binding sites or experimental verification using in vitro binding assays. Initially positive experimental results highlight the ability of computational techniques to direct research into transcriptional regulation in mammalian models. As such, our approach may be applicable in the study of other protein complexes in the mammalian proteome.\nThe reported predictions may enable pharmacologically important downstream research. For example the predicted sites can be used as a starting point for quantifying in vivo effect on downstream transcription; for identifying the TFs bound; and even for the more complex task of understanding the role of each site in determining the relative abundance of GABA A receptor isoforms. Research along these lines may dramatically improve our understanding of GABA A receptor regulation and its role in disease and development. Additionally, a more comprehensive evaluation of the remaining GABA A receptor subunit genes may reveal additional TFbinding sites that uncover the evolutionary significance of g-a-b GABR clusters in the human genome.\nSupplementary Data are available at NAR Online.\nCharles DeLisi is partially supported by NIH grants A08 POGM66401A and J50 01-130021. Daniel S. Roberts is supported by NIH training grant 2T32 GM00854. Shelley J Russek is supported by NIH/NINDS Grant NS050393. Funding to pay the Open Access publication charges for this article was provided by the Boston University Bioinformatics Program." }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: A new highly pathogenic strain of avian influenza, H5N1, emerged in the poultry markets of Hong Kong in 1997 and subsequently re-emerged in Vietnam in 2003. From this time onwards it has rapidly spread across the globe and is likely to be endemic in poultry in many parts of the world. Although onward transmission to humans at present remains limited, the high case fatality rate in those people that are infected has raised concerns about the impact of a potential human pandemic [1, 2] . Whilst much research and planning is currently underway to contain any outbreak in humans, relatively little is known about the extent of infection in poultry and, in particular, the transmissibility of highly pathogenic avian influenzas between poultry farms. Such understanding is vital if we are to limit the potential for a human pandemic by reducing the extent of infection in poultry, either through movement restrictions, culling or vaccination.\nAvian influenza occurs naturally in wild water fowl, usually in a low-pathogenic version (LPAI) causing no symptoms or only mild disease. However, in poultry some strains also occur in a highlypathogenic form (HPAI) and result in a devastating disease which can kill up to 100% of infected birds within 48 hours, and is highly transmissible between individual birds [3, 4] . Transmission between flocks kept at different farms is thought to occur via movement of infected birds, equipment or staff, with current evidence suggesting that air-borne transmission over long distances is rare [5] . There has been an increase in HPAI outbreaks over the past ten years [4] . In addition to their implications for human health, these outbreaks also have severe economic consequences for the affected countries. Typical control measures for HPAI in poultry comprise of swift isolation and culling of flocks on infected farms, the restriction of movements between farms, increased bio-security, and the culling of flocks in the vicinity of infected farms to deplete the susceptible poultry population. Vaccination, if coupled with a strict surveillance programme, has also been demonstrated to be effective in reducing the risk of further outbreaks [5, 6] .\nThe reproductive number for infected poultry farms, defined as the average number of farms that each original infected farm infects at the start of an outbreak (i.e., when most farms are susceptible), is an important measure of the overall transmissibility of the virus in a population. It determines whether a self-sustaining epidemic will occur and, more importantly, yields a tool to assess the effectiveness of control measures. If, on average, at any point in time, each infected farm infects more than one further farm, the epidemic will continue. However, if on average, each infected farm infects less than one further farm, the epidemic will decline and the intervention measures applied at that point can be interpreted as being sufficient to control the outbreak.\nIn this paper, we analyse published data from four outbreaks of HPAI in commercial poultry in industrialised countries to estimate the farm-to-farm reproductive number of HPAI to explore the extent to which different intervention measures implemented during these outbreaks reduce the reproductive number. The results from our analyses can be used to inform current planning for an outbreak of HPAI in similar commercial poultry sectors.\nWe analyse data from three different outbreaks of HPAI that occurred in the past 8 years in industrialised countries: an outbreak of H7N1 in Italy in 1999/2000, an outbreak of H7N7 in the Netherlands in 2003, that will be treated as two distinct outbreaks due to geographic separation, and an outbreak of H7N3 in Canada in 2004. Figure 1 shows the time course of these outbreaks. Brief details of these outbreaks are given below.\n2.1.1. Outbreak of H7N1 in Italy in 1999/2000 Northern Italy has experienced a number of avian influenza outbreaks from 1997 onwards [6] [7] [8] [9] [10] . These all occurred in an extremely dense poultry production area (up to 70 000 birds/km 2 ) and involved a significant number of farms keeping turkeys, a species known from experimental studies to be highly susceptible to avian influenza [11] . Furthermore, in this region there are many wetlands and resting sites for migratory waterfowl in close proximity to the poultry industry, which likely lead to multiple introductions from the wild bird host.\nIn March 1999, H7N1 LPAI was detected in a farm keeping turkeys [6, 7, 9] . This outbreak was not controlled rigorously and so AI continued to circulate. In December, a case of H7N1 HPAI was found and strict control measures were implemented, including culling of affected flocks, movement restrictions and pre-emptive slaughter of flocks deemed at high risk. However, due to LPAI circulating at the time, the confirmation of HPAI was delayed, and so the disease had already infected a number of farms by the time control measures were enforced. This resulted in an HPAI epidemic affecting a total of 413 flocks. The LPAI/HPAI epidemic lasted until April 2000, and involved a total of over 13 million birds.\nThe H7N7 epidemic in the Netherlands in 2003 affected a total of 255 commercial flocks in two distinct geographical and temporal clusters. The outbreak was situated in the Gelderse Vallei, the densest poultry production area in the Netherlands, in which over 10 million birds are kept in 984 flocks with a density of 4 flocks/ km 2 [12, 13] . Two months into the outbreak the infection passed to Limburg, another very dense poultry production area, where it continued to spread.\nIn the Gelderse Vallei, HPAI was confirmed on 28 th February, 6 days after clinical signs appeared in the first infected farm, and between March and early April, a total of 212 farms were infected. In Limburg, a further 43 farms were infected between April and early May.\nA number of control policies were enforced in several stages. From 1 st March all movement of poultry and poultry products was banned, the tracing of dangerous contacts was initiated and reinforcement of strict bio-security measures was implemented. Two days later, from 3 rd March, culling of infected farms was initiated. On 5 th March the additional pre-emptive culling of farms within a 1 km radius of any infected farms was put in place. This was further extended to a 10 km radius for turkey flocks and 3 km radius for all other flocks on 7 th April [14] . However, these control measures were insufficient and it is hypothesized that the epidemics in both areas finally came to a halt due to depletion of susceptible flocks, after the culling of 30 million birds in 1,255 commercial and 17,421 hobby flocks [15] . During this epidemic, 89 human infections were also reported, most of whom presented with conjunctivitis or mild influenza-like illness. One person died from their infection. There was also evidence of limited human-to-human transmission [16, 17] . Following detection of the index case, a broiler breeder farm, a surveillance program was initiated, which led to the detection of the second case on 11 th March. The Fraser Valley south of the River Fraser was declared a Control Area, restricting movements of birds, bird products and equipment. Furthermore, active surveillance was undertaken in a High Risk Region (HRR, 5 km around the index case) and in flocks deemed dangerous contacts in a Surveillance Region (SR, 10km around index case).\nAfter the identification of 7 infected farms, all birds within the HRR were slaughtered from 24 th March onwards, but as this failed to stop transmission, on 5 th April it was decided to depopulate the whole Control Area, containing approximately 19 million birds.\nInfected farms were located mainly in three distinct local clusters within the Control Area. It is hypothesized that long distance spread between these clusters was due to bird, equipment or people movement, whereas once a farm in a densely populated area became infected, where sheds are sometimes within a few hundred metres from each other, the virus spread via dust or feather debris.\nAssuming homogeneous mixing, that all farms are equally infectious, and that the time-dependence of infectiousness from the point of infection is identical, we can estimate both the distribution of generation time intervals and the reproductive numbers of individual farms from the time-course of an epidemic using the following method [19] .\nSuppose there are N infected farms, labelled i = 1,…,N, and ordered so that the first k farms are those that contracted their infection from outside sources. The infection times of these farms are t = (t 1 ,…,t N ) such that t 1 = … = t k = 0. Under the simplest model that neglects any differences between farms, spatial locations, etc., the probability that farm j[fkz1,:::,Ng was infected by farm i[f1,:::Ng is\nwhere the generation time distribution has density w(T;h), which is defined to be 0 if T,0 and indexed by unknown parameter vector h. Under the above assumptions, the number of farms infected by farm i (i.e. the reproductive number of farm i) in the outbreak can be represented as an outcome from a random variable\nthat is, a sum of Bernoulli random variables, which has expected value\nNow denote the 'infection tree' by v = (v k+1 ,…,v N ), defined such that v j = i if farm j was infected by farm i. Under (1), the likelihood for h when v and t are observed is\nBut as v is unobserved we sum over all possible infection trees to obtain the 'integrated likelihood'\nwhere S j = {1,…,N}\\{j} is the set of all indices other than j. The integrated likelihood is a genuine likelihood (up to a multiplicative constant) permitting valid inferences about h conditional on outbreak size N.\ntime The maximum likelihood (ML) estimateĥ is obtained by minimizing twice the negative log-likelihood\nMore details on how ML estimation is performed are given in Appendix S1. We assume the generation times T = t j 2t i are Weibull distributed, with density\nand so h = (k, g). Minimization was performed using the Downhill Simplex method [20] , the code used for these calculations is given in Code S1 and Code S2; to ensure the global minimum is reached, the procedure was run from 10000 different starting points. We further investigated whether the generation time distribution changed after control measures were introduced. To do this, we extended the above model to allow for distinct parameters for the generation times before and after controls, h pre and h post , see Code S3 and Code S4. The improvement in fit compared to the original model was assessed using a likelihood ratio test.\n2.2.2. Estimation of the reproductive number Givenĥ we can estimate the mean and variance of the generation time distribution. Moreover, we can estimate the reproductive number for each infected farm via equation (3), and the mean reproduction number for any subset of infected farms.\nTo calculate confidence intervals for the reproductive number we use an approximation of the parametric bootstrap percentile interval method [21] . To obtain proper parametric bootstrap intervals would involve generating infection times and trees according to the underlying epidemic model, which we do not wish to specify completely. Instead, the following two-step approximation is used, which we propose will be a good approximation for large N . These two steps approximate generating realisations from the underlying epidemic process. First, we take bootstrap samples of parameter values from the conventional approximation to the sampling distribution of the ML estimator,\nthat is, from a bivariate normal distribution with mean (k k,ĝ g) and\nvariance-covariance matrix V based on the inverse of the observed information matrix (see Appendix S1 for further details, the code used to generate the bootstrap sample is given in Code S5 and Code S6). This first stage can be loosely thought of as sampling the mean behaviour for a subgroup of possible outbreaks. To allow for variability within each subgroup, stage two involves fixing (k * ,g * )and independently generating reproductive numbers for each farm according to model (2) . Steps one and two together give R * = {R * i :i21,…,N}, an approximate bootstrap sample of the reproductive numbers for each farm. Here, 1000 samples of (k,g)pairs were drawn, and 500 sets of reproductive numbers generated for each. Finally, the approximate 95% CI for each R i is given by the 2.5 th and 97.5 th percentiles of the bootstrap distribution. The second stage of the calculation of the approximate CIs was done using Code S7 and Code S8.\nThe generation time is defined as the time between the infection of a farm and the time at which the farm passes on infection to another farm. We have assumed the generation time distribution is Weibull. While this is a biologically plausible choice, we cannot verify it empirically. As such, we assessed robustness to this choice using other plausible choices such as the gamma distribution (results not shown). However, the following results under these alternatives did not differ substantively from those shown below. Figure 2 shows the estimated generation time distribution for the four different outbreaks; the parameter estimates are detailed in Table 1 . The estimates, and hence the distribution, differs substantially between the outbreaks. It could be hypothesised that the generation time would shorten after measures were put in place to isolate the infected farms. However, allowing for different generation time distributions for the pre-and post-control time periods did not significantly improve the model fit. Table 2 ) with upper 95% bounds in the range 1.5-3.6.\nThe impact of control measures on the effective reproductive number can be clearly seen in all four outbreaks. For the outbreak in Italy (Figure 3a) , their introduction rapidly reduced the reproductive number, hovering around the threshold of 1 for the next few months before finally dying out. In British Columbia (Figure 3b ) controls were put in place after detection of the first IP. However our estimates of the reproductive number remain high until 24 th March when the decision was taken to cull the whole high risk region. Our estimates show that the control activities following this decision were effective in reducing the reproductive number to below one.\nOur results show that the situation in the Gelderse Vallei, The Netherlands (Figure 3c ) differed in that the initial control measures failed to bring the reproductive number reliably below 1, and the epidemic only died out at the end of March after the depletion of susceptible flocks in the affected area [15] . The same controls were applied to the Limburg epidemic but our estimates show in this case the reproductive number was reduced to just below 1 (Figure 3d) , and so potentially effective in controlling the outbreak. However, the end of the epidemic in late April coincided here too with the depletion of susceptible flocks and therefore it is possible that the epidemic would have taken substantially longer to control had there been a larger pool of susceptible flocks in the area.\nOur estimates of the farm-to-farm reproductive number prior to interventions for HPAI are in the range 1.1 to 2.4 and were remarkably consistent across the four datasets. However, these estimates are substantially lower than those previously reported for the Dutch epidemic. Prior to the implementation of control measures we obtained estimates of 1.1 (95% CI 0.9-1.5) in the Gelderse Vallei and 1.9, (95% CI 1.0-3.0) in Limburg which are significantly lower than those previously reported for the same outbreak prior to notification (6.5 (95% CI 3.1-9.9) for the epidemic in the Gelderse Vallei). However, as demonstrated in Figure 3c , there was substantial variation in our estimates of individual reproductive numbers prior to interventions. In addition, in the previous study, the generation time was not estimated directly from the data but based on observational and experimental data on the course of infection in the farms. Our estimate of the generation time for this region is of the order of 2 days, whereas the values previously assumed for the infectious period were defined per flock as the time between detection and culling plus an additional 4 days to cover the time before the infection was detected but during which birds were infectious. The previously published estimates therefore assumed a much longer mean generation time and this could also lead to a higher estimated reproductive number. Our results showed substantial differences between the estimated generation time distributions for the different outbreaks. Whilst much is known from experimental studies on the course of infection in individual birds [11, [22] [23] [24] , estimates of the generation time at the farm-level are more difficult to obtain. Although it is perhaps surprising that the generation time differs between the outbreaks, it is plausible that such differences could arise because of variation in farming practices or in the contact patterns between farms. In addition, the latent and infectious periods determining the generation time may differ by the strain of HPAI. Alternatively, the estimates may be biased because of assumptions made in the method. In particular, we assumed that the datasets were complete (and thus that all infected farms were detected) and that only the first farm in each outbreak was infected from an outside source. If, however, further undetected farms had played a role in transmission, this would substantially alter the estimates of the generation time and the reproductive number, particularly if these infections occurred towards the beginning or end of the epidemics where overall cases are sparser.\nAll of the outbreaks investigated here occurred within dense poultry farming areas and hence were difficult to control. The control policies implemented in the different outbreaks were similar, comprising strict bio-security measures for movement of poultry and poultry products, swift culling of infected flocks, and if these failed to control the epidemics, additional pre-emptive culling of flocks in the neighbourhood of any infected farms. Our results demonstrate that the bio-security measures, movement restrictions and culling of infected farms, all of which were initiated early on in the outbreaks, did have an effect but for all four outbreaks only reduced the reproductive number to close to the threshold value of 1. The additional pre-emptive culling of flocks and de-population of the areas was needed to fully control the outbreaks. Current contingency plans for HPAI outbreaks in Europe focus on the former set of control measures to contain any outbreak [25] . Whilst differences in farming practices between countries mean that it is difficult to predict whether these measures will be sufficient for a new outbreak, our analyses suggest that additional interventions may well be required. Close monitoring of outbreaks, coupled with quantitative estimation of the reproductive number, is therefore needed to ensure that such additional measures, if required, are promptly implemented.\nThe method used here to estimate the reproductive number and generation time parameters is an extension of that developed by Wallinga and Teunis [19] for the SARS-epidemic. This method requires only time-series data for an outbreak, and is therefore easily applied even in real-time. Technically, appropriate censoring terms should be added to the likelihood to account for infection times yet to occur, but a straightforward application of the method as described here will give estimates unbiased in an asymptotic sense. If data on the spatial location of infected farms are also available, this information can easily be incorporated to estimate the spatial transmission kernel and improve the estimation of the reproductive numbers. Such an approach was successfully applied to the Foot-and-Mouth epidemic in the UK in 2001 [26] . Further work is required to explicitly incorporate missing data, as this is likely to have a strong influence on the estimates of both the generation time and the reproductive number. Such methods are of particular importance to estimating the reproductive number for outbreaks of HPAI in Asia in which, with high general levels of poultry mortality, cases are likely to be less well documented.\nAppendix S1 Code S1 Source code for the maximum likelihood estimation of the parameters of the generation time distribution, shown in Figure 2 and Table 1 Code S7 Source code for estimating R0 and the confidence intervals based on the uncertainties inherent in the estimation procedure and the uncertainty of the generation time distribution, shown in Figure 3 . " }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: Preparedness against pandemic influenza has become a high priority public health issue and many countries that have pandemic preparedness plans [1] . For the design of such plans, mathematical models and computer simulations play an essential role because they allow to predict and compare the effects of different intervention strategies [2] . The outstanding significance of the tools for purposes of intervention optimization is limited by the fact that they cannot maximize realism, generality and precision at the same time [3] . Public health planners, on the other hand, wish to have an optimal combination of these properties, because they need to formulate intervention strategies which can be generalized into recommendations, but are sufficiently realistic and precise to satisfy public health requirements.\nPublished influenza models which came into application, are represented by two extremes: generalized but oversimplified models without dynamic structure which are publicly available (e.g. [4] ), and complex computer simulations which are specifically adjusted to real conditions and/or are not publicly available (e.g. [5, 6] ). The complexity of the latter simulations, however, is not necessary for a reliable description of infection dynamics in large populations [7] . A minimum requirement for a pandemic influenza planning tool is a dynamic modelling structure which allows investigation of time-dependent variables like incidence, height of the epidemic peak, antiviral availability etc. The tool should, on the other hand, be adjustable to local conditions to adequately support the pandemic preparedness plans of different countries which involve considerably different assumptions (Table 1) .\nHere we describe a publicly available influenza pandemic preparedness planning tool [8] which is designed to meet the requirements in preparedness planning. It is based on an explicitly formulated dynamic system which allows addressing time-dependent factors. It is sufficiently flexible to evaluate the impact of most candidate interventions and to consider local conditions like demographic and economic factors, contact patterns or constraints within the public health system. In subsequent papers we will also provide examples and applications of this model for various interventions, like antiviral treatment and social distancing measures.\nThe model is based on a system of 1,081 differential equations which extend the classic SEIR model. Demographic parameters reflect the situation in Germany in 2005, but can be adjusted to other countries. Epidemiologic and clinic values were taken from the literature (see Tables 1, 2 , 3, 4, 5, 6 and the sources quoted there). Pre-set values can be varied by sliders and input fields to make different assumptions on the transmissibility and clinical severity of a new pandemic strain, to change the costs connected to medical treatment or work loss, or to simply apply the simulation to different demographic settings. Model properties can be summarized as follows. The mathematical formulation of this model is presented in detail in the online supporting material. The corresponding source code, programmed in Java, and further information can be downloaded from [8] .\nAccording to the German National Pandemic Preparedness Plan [9] , the total population is divided in age classes, each of which is subdivided into individuals of low and high risk ( Table 2) . Transmission between these age classes is based on a contact matrix (Table 3) which is scaled such that the model with standard parameter values yields a given basic reproduction number R0. Values for the R0 associated with an influenza strain with pandemic potential are suggested to lie between 2 and 3 [10] . This value is higher than the effective reproduction number which has been estimated to be slightly lower than 2 [11, 12] . As a standard parameter, we use R0 = 2.5 which means that cases infect on average 2.5 individuals if everybody is susceptible and if no interventions are performed.\nSusceptible individuals who become infected, incubate the infection, then become fully contagious and finally develop protective immunity (Table 4) . A fraction of cases remains asymptomatic; others become moderately sick or clinically ill (i.e. they need medical help). Depending on the combination of age and risk group, a fraction of the clinically ill cases needs to be hospitalized, and an agedependent fraction of hospitalized cases may die from the disease ( Table 5 ). This partitioning of the cases into four categories allows combining the realistic description of the transmission dynamics with an easy calculation of the resources consumed during an outbreak. The degree and duration of contagiousness of a patient depend on the course of the disease; the latter furthermore depends on the age of the patient (Table 5) . Passing through the incubation and contagious period is modelled in several stages which allows for realistic distributions of the sojourn times ( Table 4 ). The last two stages of the incubation period are used as early infectious period during which the patient can already spread the disease. Infectiousness is highest after onset of symptoms and thereafter declines geometrically (Table 6 ). Clinically ill patients seek medical help on average one day after onset of symptoms. Very sick patients are advised to withdraw to their home until their disease is over, whereas extremely sick patients need to be hospitalized and may die from the disease (Table 4) . After the end of their contagious period, clinically ill patients go through a convalescent period before they can resume their ordinary life and go back to work (Table 4) .\nWe provide some examples of model output of InfluSim [8] , version 2.0, by means of four sensitivity analyses; further investigations will be presented elsewhere. Figure 1 shows the graphical user interface of the software which is divided into input and output windows. The user may set new values in the input fields or move sliders to almost simultaneously obtain new results for the course of an epidemic in a given population. Figures 2A and 2B show pandemic waves which result from varying the basic reproduction number from 1.5 to 4.0. Using the standard parameter values as given in Tables 2, 3 , 4, 5, 6 and omitting all interventions in a town of 100,000 inhabitants results in a pandemic wave which lasts for about ten weeks (Figure 2A , with R 0 = 2.5). The peak of the pandemic wave is reached after six to seven weeks, with a daily incidence of up to 2,340 influenza patients seeking medical help, with up to 280 hospital beds occupied by influenza cases and with up to 14,000 out of 60,000 working adults unable to go to work because of illness or convalescence. These results depend on the assumptions concerning the yet unknown contagiousness and pathogenicity of the virus. Figures 2C and 2D show how the shape of the curves depends on the course of contagiousness: the pandemic wave proceeds relative slowly if the contagiousness does not change during the infectious period (x 50 = 50%), but proceeds quickly if the contagiousness is highest after onset of symptoms and decreases thereafter (x 50 > 50%).\nThe influenza pandemic preparedness planning tool InfluSim stands between simple spreadsheet models and sophisticated stochastic computer simulations. It describes a pandemic wave within a homogeneously mixing population like a town or city, but surprisingly produces the same dynamics as individual-based simulations which explicitly consider geographic spread through the US (cf. [6] and [5] with Figure 2 using R 0 = 2). Similar observations were made with a simple deterministic compartmental model [7] . Stochastic models are known to behave quasi-deterministically when the simulated population becomes very large.\nA further reason for the congruence of complex stochastic and simple deterministic models must lie in the incredi-bly quick way in which pandemic influenza spreads geographically. Unless being controlled at the place of origin [12, 13] , a pandemic starting in a far-off country will lead to multiple introductions [14] into the large industrialized nations where it can be expected to quickly spread to neighbouring towns and to rural areas. The large populations which have to be considered susceptible to a pandemic virus and the quick geographic spread tend to diminish the differences between the results of sophisticated individual-based and simple deterministic models.\nHowever, a deterministic model like InfluSim cannot reliably represent effects originating from stochasticity, from effects in small populations, or from heterogeneities. Examples are: (i) a geographically limited spread and fairly effective control measures can imply that the epidemic affects only a small population and thus, may be strongly influenced by stochastic events [15] [16] [17] ; (ii) transmission which predominantly occurs in households or hospitals, or which is driven by other substantial features of the contact network is not in agreement with the assumption of homogeneous mixing in the deterministic model cannot reliably predict the spread of infection [18] [19] [20] [21] [22] [23] . In particular, (iii) super-spreading events can substantially change the course of an epidemic compared to the deterministic prediction [24] [25] [26] [27] . Apart from such factors, the predictability of intervention success is generally subject to uncertainties in the choice of parameter values, Assumed scenarios and outcomes of pandemic preparedness plans. * Gross attack rate (i.e. clinically ill and moderately ill cases). A population of N = 100,000 inhabitants of Germany is subdivided according to age a and risk category r. We assume that all age groups are fully susceptible at begin of the outbreak. A fraction of F a = 6% of all children (age < 20 years) are regarded as being under high risk (r = r 1 ) after an influenza infection whereby the remaining 94% are under low risk (r = r 2 ). The high risk fractions of working adults (ages 20-59) and elderly (ages 60+) are F a = 14% and F a = 47%, respectively. Source: [9] demanding additional efforts like Bayesian approaches [28] to evaluate the reliability of predictions [29] .\nPandemic preparedness plans must consider constraints and capacities of locally operating public health systems. The time-dependent solutions of InfluSim allow assessing peak values of the relevant variables, such as outpatients, hospitalizations and deaths. Various interventions may be combined to find optimal ways to reduce the total number of cases, to lower the peak values or to delay the peak, hoping that at least part of the population may benefit from a newly developed vaccine.\nSpecial care was taken when implementing a variety of pharmaceutical and non-pharmaceutical interventions which will be discussed in subsequent papers. Despite its comprehensible structure, the model does not suffer from over-simplifications common to usual compartment models. Instead of implicitly using exponentially distributed sojourn times, we have implemented realistically distributed delays. For example, the model considers that individuals may transmit infection before onset of symptoms, and that some cases may remain asymptomatic, but still infecting others. Such features have serious implications for the success of targeted control measures.\nInfluSim is freely accessible, runs on a regular desktop computer and produces results within a second after changing parameter values. The user-friendly interface and the ease at which results can be generated make this program a useful public health planning tool. Although we have taken care of providing a bug-free program, including the source code, the user is encouraged to treat results with due caution, to test it, and to participate in bug-reports and discussions on the open-source platform [30] which also provides regular updates of InfluSim. \nThe author(s) declare that they have no competing interests.\nME developed the model, MS designed the software, HPD wrote the manuscript and SOB formulated the public The who-acquires-infection-from-whom matrix shows the frequency of contacts (per week per person) between different age classes.\nSource: [38] . Distribution of sojourn times (the last two stages of the latent period are used as early infectious period with an average duration of D L = 0.5 days). Sources: A [11] , B [39, 40] , C assumed, D [41] health requirements of the software. All authors read and approved the final manuscript.\nSusceptible individuals S a, r are infected at a rate λ a (t) which depends on their age a and on time t. Infected individuals, E a, r , incubate the infection for a mean duration D E . To obtain a realistic distribution of this duration, the incubation period is modelled in n stages so that progression from one stage to the next one occurs at rate δ = n/D E .\nThe last l incubation stages are regarded as early infectious period during which patients may already spread the infection (this accounts for an average time of lD E /n for the \"early infectious period\" which is about half a day for the standard set of parameters). After passing through the last incubation stage, infected individuals become fully contagious and a fraction of them develops clinical symptoms. The course of disease depends on the age a of the infected individual and on the risk category r to which he or she belongs: a fraction c a, r (A) becomes asymptomatic (A a ), a fraction c a, r (M) becomes moderately sick (M a ), a fraction c a, r (V) becomes very sick (V a ) and the remaining fraction c a, r (X) becomes extremely sick (X a ) and need hospitalization (i.e., c a, r (A) + c a, r (M) + c a, r (V) + c a, r (X) = 1 for each combination of a and r). ) . A fraction f V (t) of all severe and a fraction f X (t) of all extremely severe cases who visit the doctor within D T days after onset of symptoms are offered antiviral treatment, given that its supply has not yet been exhausted. As our model does not explicitly consider the age of the disease (which would demand partial differential equations), we use the contagious stages to measure time since onset and allow for treatment up to stage m a, T Sources: Contagiousness of asymptomatic cases: [11] ; degree of contagiousness during the early infectious period and equality of the contagiousness of moderately and severely sick cases: assumed. Independent of age a and risk group r, a fraction c a, r (A) = 33% of infections result in asymptomatic cases, a fraction c a, r (M) = 33.5% become moderately sick and the remaining fraction develops severe disease. An age-and risk-dependent fraction h a, r of untreated patients with severe disease needs hospitalization. An age-dependent fraction d a of hospitalized cases dies. Sources: fraction of asymptomatic cases: [11] ; 50% of symptomatic cases see a doctor: [9] ; hospitalizations per severe case: [9] ; case fatality of hospitalized, but untreated patients calculated from [4] .\n(see below for details). This imposes some variability to the maximum time until which treatment can be given, which may even improve the realism of the model with respect to real-life scenarios. Antiviral treatment reduces the patients' contagiousness by f I percent and it reduces hospitalization and death by f H percent. Extremely sick patients, whose hospitalization is prevented by treatment, are sent home and join the group of treated very sick patients(W a, T ). The remaining duration of disease and contagiousness of treated cases is reduced by f D percent so that their rate of progressing from one stage to the next has To obtain a realistic distribution of this sojourn time, convalescence is modelled in j stages so that progression from one stage to the next occurs at rate ρ = j/D C . Fully recovered patients who have passed through their last stage of convalescence join the group of healthy immunes I; working adults will go back to work. Further interventions, describing the reduction of contacts, will be discussed after the presentation of the differential equations.\nInfluSim user interface Figure 1 InfluSim user interface. x 50 = 95% means that 95% of the cumulative contagiousness is concentrated during the first half of the contagious period, see Table 6 ). D: Cumulative number of deaths for values of x 50 as in C. All other parameters as listed in Tables 2-6 . Hospitalized, but untreated cases\nContact matrix For the mixing of the age classes, we employ a whoacquires-infection-from whom matrix which gives the relative frequency of contacts of infective individuals of age a i with other people of age a s . In this paper, we assume bi-directional contacts (e.g. children have the same total number of contacts with adults as adults with children). Multiplication of this matrix with an appropriate constant scaling factor κ (see below)\nresults in the matrix of crude contact rates .\nIn the absence of interventions, we have to multiply these contact rates with the contagiousness factors b L , b A , b M and b V to obtain the effective contact rates:\nduring the early infectious period, of asymptomatic cases, of moderately sick cases, of (untreated) very sick cases.\nTo assess the effect of day care centre and school closing on the transmission of an infectious disease, we have to first make an assumption on what fraction r sch of the contacts among healthy children who are in the same age class occurs in day care centres and schools. The contact rates between very sick or hospitalized children (who do not attend day care centre or school) and other children need, therefore, be reduced to (contact rate between healthy and very sick children in the same age class, i.e. a i = a s ).\nAs very sick children have to be taken care of by adults at home or in hospital, their contact rate to adults increases by a factor F HC (contact rate between very sick children of age a i and adults of age a s ).\nContacts between very sick children and other children in a higher or lower age class remain unchanged: (contact rate between healthy children of age a s and very sick children of a different age a i ).\nClosing day care centres and schools at time t will not necessarily prevent all the contacts that would have happened with other children. During the closing of schools and day care centres, the contact rates between susceptible children of age a s and infected children of age a i who are in their late incubation period ( ), who are asymptomatic ( ), or who are moderately sick ( ) are reduced by the factor r sch if the children are in the same age class:\nwhere 1 sch (t) is a function which indicates when schools and day care centres are opened or closed: \n,..., While day care centres and schools are closed, children (age a i ) need adult supervision at home. Their contact with susceptible adults (age a s ) increases by the \"child care factor\" F CC :\nChild care at home also increases the exposure of healthy children (age a s ) to contagious adults (age a i ):\nCancelling mass gathering events effects only the contacts of adults who are healthy enough to attend such events. Assuming that such an intervention at time t reduces contacts by a fraction r mass , we get for all contacts between susceptible adults of age a s and infectious adults of age a i the following contact rates:\nwhere 1 mass (t) is a function which indicates when mass gathering events are possible or when they are closed:\nAs contacts with adults who are too sick to attend such mass gathering events cannot be prevented by this measure it is .\nDuring some time in the epidemic, the general population may effectively reduce contacts which can be a result of wearing facial masks, increasing \"social distance\", adopting improved measures of \"respiratory hygiene\" or simply of a general change in behaviour. This will be implemented in the program by reducing the contacts of susceptible individuals at that time t by factor r gen (t \nwhile mass gathering events are forbidden while m mass gathering events are allowed. while the population reduces their contacts while the population behaves as usual.\nThe contact rates of cases in the late incubation period and that of asymptomatic cases remain unchanged:\nfor infected individuals in the late incubation period, for asymptomatic cases.\nTo allow for a contagiousness which changes over the course of disease, we multiply each contact rate with a weighting factor whereby k is the stage of contagiousness. This leads to the following contact rates:\nfor asymptomatic cases in For x = 1, contagiousness is equally high in all stages; for x = 0, only the first stage is contagious; for 0 <x < 1, the contagiousness decreases in a geometric procession. We make the simplifying assumption that contagiousness does not change during the late incubation period for cases in stage k = n -l,..,n of the incubation period.\nAt time t = 0 and in the absence of interventions, the next generation matrix has the following elements where is the fraction of untreated extremely severe cases who die from the disease (see below for details). The dominant eigenvalue of this matrix is called the basic reproduction number R 0 . If κ (which determines the value of the contact rates ) is given, the eigenvectors of this matrix can numerically be calculated. The user-specified value of R 0 is now used to determine numerically the scaling factor κ. Let be the eigenvector which has the largest eigenvalue R 0 . \n) ) − \nUsing the user-specified numbers of people N a in the age classes and the fractions F a of people under high risk within each age class (Table 2) , we obtain the initial population sizes according to age and risk class: Using these initial values, the set of differential equations is solved numerically with a Runge-Kutta method with step-size control. \nif and in treatment window otherwise 0 ⎩ ⎩" }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: While evident in social organisms ranging from humans to birds (Brown & Brown, 2004; Masuda et al ., 2004) , the impacts of sociality on disease are especially vivid within social insect colonies. Here, typically thousands of individuals interact in close quarters, at densities far exceeding those of even the most crowded vertebrate social groups (Wilson, 1971) . This density, coupled with a relatively homeostatic nest environment and the presence of stored resources, makes social insects attractive targets for disease agents (Schmid-Hempel, 1998) . As expected based on their parasite and pathogen pressures, social insects have evolved both individual and group strategies to combat disease. Grooming, nest hygiene and other behavioural traits found throughout the social insects can reduce the impacts of pathogenic bacteria, fungi and parasitic mites. For example, 'hygienic behaviour' first described for honey bees (Rothenbuhler, 1964) is now a classical example of a social defence, whereby workers identify and remove infected larvae from among the healthy brood (Spivak & Reuter, 2001) . Other defences enabled by sociality include the construction of nests from antimicrobial materials (Christe et al ., 2003) , the raising of offspring in sterile nurseries (Burgett, 1997) , social 'fever' in response to disease (Starks et al ., 2000) , transference of immune traits (Traniello et al ., 2002; Sadd et al ., 2005) , and heightened risk-taking by infected individuals (Schmid-Hempel, 2005) . Like most eukaryotes, colony members also possess individual defences, including immune responses toward disease agents (Casteels-Josson et al ., 1994; Evans, 2004) . The recent sequencing of the honey bee genome (Honey Bee Genome Sequencing Consortium, 2006) allows the first global analysis of immune components in honey bees, and the second opportunity (after humans) to use genomic insights to better understand disease resistance in a highly social organism.\nInsects have diverse mechanisms to combat infection by pathogens. Many insects are protected by a layer of antimicrobial secretions on their exterior, and by a gut environment that is hostile to pathogens. When pathogens move beyond these defences, the epithelium is often sufficient to stop further progress. Should pathogens defeat the morphological defences of insects, they are often met by efficient cellular and humoral immune defences. Insect immunity shows many parallels to the innate immune responses of humans and other vertebrates, involving a diverse set of actions including the secretion of antimicrobial peptides, phagocytosis, melanization and the enzymatic degradation of pathogens (Hoffmann, 2003; Hultmark, 2003) . Further, insect immune pathways share both an overall architecture and specific orthologous components with the innate immune system of vertebrates (Beutler, 2004) . This suggests both a shared root for these immune pathways and selection to conserve many components over hundreds of millions of years.\nIn the first part of this paper, we propose honey bee models for four non-autonomous pathways implicated in inducible host defence, Toll, Imd, Janus kinase (JAK)/STAT and JNK (Boutros et al ., 2002) , based primarily on extensive searches for orthologues to well-studied fruit fly, mosquito and moth species. While these pathways engage in cross-talk and can direct some of the same immune effectors, they have well-defined structures and interaction sets, and are best tackled as individual entities. Most honey bee components for these pathways remain to be validated by functional tests, yet we feel that the presented models serve two important purposes. First, they point toward the most likely orthologues involved with all stages of the immune response, thus setting the stage for postgenomic functional work on honey bee immunity. Second, the models themselves show intriguing differences between species for these canonical immune pathways with respect to gene losses and duplications.\nNext, we show that many immune-gene families in bees appear to be reduced in number, when compared to Drosophila and Anopheles . While genome-wide analyses of the honey bee have identified many gene families with reduced diversity (Honey Bee Genome Sequencing Consortium, 2006) , such reductions appear to be especially pervasive in the immune system. These reductions hold for each stage of immunity, from recognition and signalling to immune effectors. We couple gene-family data with data on specific orthologues to test five hypotheses:\n(1) missing genes are not represented in the current draft honey bee genome assembly but are present in the genome; (2) immune-related genes in the bee have diverged especially quickly at the sequence level and, as such, have escaped annotation based on sequence similarity to other species; (3) honey bees enact immune responses using pathways and/or components not currently identified as immune players in other insects; (4) honey bees are targeted by a small set of coevolved pathogens and their immune systems are thereby tuned to these pathogens at the expense of being responsive to a wider range of threats; and (5) 'social' defences and barriers in honey bee colonies are effective in reducing pathogen pressure and, as such, bees are not as reliant as other insects on individual immune responses.\nHoney bees possess apparent orthologues for the core members of each of the four pathways implicated in immunity (Figs 1 and 2, Supplementary Material Table S1 ) and precise 1 : 1 : 1 orthology between honey bees and the flies Drosophila melanogaster and Anopheles gambiae is evident for most pathway members, especially for the intracellular components. Of the dozens of described actors in four signalling pathways predicted to play a role in insect immunity, only one protein appears to be completely absent in the bee genome: the ligand unpaired from the JAK/STAT pathway. The presence of the JAK/STAT cytokine receptor domeless and all other members of this pathway (Fig. 2) suggest that JAK/STAT remains functional in honey bees and is triggered by a currently unrecognized ligand.\nInsect Toll and the Toll-like receptors (TLRs) are transmembrane signal transducing proteins that play critical roles in both immunity and development. They are orthologous to mammalian TLRs, all of which have been implicated in immunity (Beutler, 2004) . In Drosophila , the Toll signalling pathway is enacted when the cytokine-like molecule Spaetzle binds to the extracellular domain of the transmembrane receptor Toll. The Drosophila genome encodes a family of six Spaetzle-related molecules, that are believed to function as ligands for the nine Drosophila Toll receptors (Parker et al ., 2001) . Two plausible Spaetzle orthologues are evident in the bee genome (GB15688 and GB13503; Fig. 1 ), and functional tests will be needed to determine which act as Toll-binding cytokines. Following conformational changes of the activated receptor, several intracellular death-domain (DD) containing proteins are recruited to form a receptor complex. Activation of this complex leads to the degradation of the NF kappa B inhibitor (I κ B) Cactus and subsequent nuclear translocation of the NF-κ B transcription factor Dorsal (or the Dorsal-related immune factor, Dif, in Drosophila (Royet et al ., 2005) . Two homologues of Dorsal were found in the honey bee genome (Fig. 1) , neither of which was orthologous with Dif. This lends support to the view that Dif is a highly derived branch found in brachyceran flies but absent from other insects. In mosquitoes (Shin et al ., 2005) , and arguably honey bees, Dorsal (called REL1 in mosquitoes) is a functional alternate for Dif. Functional tests can help determine which of the two dorsal paralogues is the key transcription factor for this pathway. The intracellular components Tollip, Pellino, Cactin and TNF receptor associated factor-2 (TRAF-2) are believed to aid the main players of this pathway, and all appear to be present in both fly species as well as the honey bee. Candidate effectors for the immune-related Toll pathways in honey bees include a compliment of antimicrobial peptides, the melanizing agent phenoloxidase and three lysozymes. While it has not been confirmed that these effectors are triggered by the Toll pathway as opposed to other pathways described below, it is evident that some of the bee effectors are responsive to pathogens and/or mechanical wounding of bees (Fig. 3) . Candidate honey bee members for the Toll pathway. Names are given for the Drosophila pathway components, along with vertebrate orthologues (in parentheses). Honey bee matches given as named during the genome project. Honey bee names in italics refer to genes with close paralogues which cannot readily be distinguished with respect to pathway components from Drosophila. Underlining indicates genes shown to be transcriptionally up-regulated after immune challenge.\nCandidate honey bee members for the Imd, JNK and JAK/STAT pathways, below names for Drosophila pathway components along with vertebrate orthologues (in parentheses). Honey bee matches presented as named during the genome project. Honey bee names in italics refer to genes with close paralogues which cannot readily be distinguished with respect to pathway components from Drosophila. Underlining indicates genes shown to be transcriptionally up-regulated after immune challenge.\nWhile Toll signalling in flies serves a dual purpose in development and immunity, the signalling process activated by peptidoglycan recognition protein (PGRP)-LC and Imd is specific for antimicrobial defence and is dispensable for normal development (Hultmark, 2003) . Via the NF-κ B-like transcription factor Relish, this signalling induces transcription of all major antimicrobial effector peptides in Drosophila . In Drosophila , Imd signalling is often said to be specific for Gram-negative bacteria, although Gram-positive bacteria with diaminopimelic acid-type peptidoglycans are at least as strong as elicitors. A weaker response is also seen to other types of peptidoglycan and even to fungi (Hultmark, 2003; Werner et al ., 2003; Stenbak et al ., 2004) . This broad specificity is caused by the three alternative splice forms of Drosophila PGRP-LC, which carry different peptidoglycan recognition domains (Werner et al ., 2003; Mellroth et al ., 2005 . Chang et al ., 2006 . Interestingly, the Imd signalling pathway is highly conserved in the honey bee, with plausible orthologues for all components (Fig. 2) . While this strongly suggests that Imd signalling is similar in flies and bees, it does not necessary imply similar biological roles.\nBesides the activation of Relish, Imd signalling also leads to activation of components of the JNK signalling pathway (Boutros et al ., 2002) , and recent evidence indicates that this pathway can provide both positive and negative feedback for the expression of the antimicrobial peptides (Wojda et al ., 2004) . Plausible orthologues for each of the major components of the JNK signalling were also identified in the honey bee genome (Fig. 2) .\nThe JAK/STAT signalling pathway may also contribute to innate immunity by induction of complement-like factors and the overproliferation of haemocytes. JAK/STAT appears to be initiated via cytokine-like molecules in blood cells (Agaisse & Perrimon, 2004) . In flies, the extracellular glycosylated protein Upd acts as a ligand that activates the JAK/STAT pathway, which in turn promotes phagocytic activity of haemocytes. The JAK/STAT pathway has also recently been shown to participate in an antiviral response in Drosophila (Dostert et al ., 2005) . Honey bee homologues for the Drosophila JAK/STAT signalling pathway (Fig. 2 ) comprise the cytokine receptor domeless (Dom), JAK tyrosine kinase (Hopscotch), the STAT92E transcription factor 3. Transcript abundances for immune candidate genes in adult workers 24 h after injections of Escherichia coli (Ec), saline buffer, or the bee pathogen Paenibacillus larvae , and controls (left four columns). Two columns on right show transcript abundances in 2nd-instar larvae challenged orally with an infective dose of P. larvae or unchallenged controls. Cluster A = genes strongly up-regulated by adult injection or wounding, Cluster B = genes up-regulated in infected larvae, Cluster C = genes down-regulated or minimally changed in challenged bees. and two negative pathway regulators SOCS (suppressor of cytokine signalling) and PIAS (protein inhibitor of activated STAT). Orthologues of two recently identified components of this pathway (Baeg et al ., 2005; Muller et al ., 2005) , the tyrosine phosphatase Ptp61F (XP392429) and the WD40and bromo-domain-containing protein BRWD3 (XP395263), are also present in the honey bee. Although the key ligand (Upd) for the JAK/STAT pathway was not found in the honey bee genome, the presence of the gp130 cytokine receptor homologue Domeless and all other members of the signalling pathway indicates that this mechanism may be common across insects and is intact in honey bees as well as in flies. In addition to up-regulating the complement-like thiolestercontaining proteins (TEPs; Lagueux et al. , 2000; Boutros et al ., 2002) , the JAK/STAT pathway in Drosophila regulates expression of the Turandot (Tot) genes that encode humoral factors induced by severe stress Agaisse et al ., 2003) . None of the Tot factors (Tot A-Z) are apparent in honey bees.\nWhile honey bees appear to have maintained each of the known insect immune-related pathways, they appear to do so with a reduced number of paralogous members. When comparing a set of 17 gene families and functional groups implicated in immune responsiveness (Christophides et al ., 2002) , honey bees have substantially lower paralogue counts than either Drosophila or Anopheles (Table 1 ). The 71 genes placed into these groups for honey bees are in sharp contrast to the 196 and 209 found in Drosophila or Anopheles , respectively. Bees have the lowest gene counts for 12 of the 17 families and are tied for the lowest count two more times. Drosophila and Anopheles were lowest for only one family each (defensins and dorsal, respectively). In contrast, Drosophila and Anopheles show the highest paralogue counts for this triad seven and eight times, respectively, versus once in bees (for the Toll-pathway candidate cactus, with three copies). These rankings are significantly different under an ordinal contingency-table analysis ( P < 1.0 × 10 -4 ), and reflect differences for genes involved with pathogen recognition and signalling, as well as effectors.\nPGRPs, major players in pathogen recognition (Hultmark, 2003; Steiner, 2004; Royet et al ., 2005) are less diverse in honey bees versus flies and other insects for which genomic data exist (e.g. the moth Bombyx mori ). There are only four PGRPs in the honey bee genome, compared to 13 and seven in Drosophila and Anopheles , respectively (Fig. 4A , Table 1 ). Further, bees show no capacity for the splice variation that contributions to diversify peptidoglycan recognition specificity in flies and mosquitoes. Specifically, the single membrane-bound PGRP in bees (PGRP-LC, GB17188) is similar to fly and mosquito PGRP-LC but lacks the potential to insert alternative peptidoglycan recognition domains by alternative splicing, a factor in recognition breadth for flies. PGRP-LC and PGRP-S2 are both up-regulated in honey bees after disease challenge (as in flies), suggesting that the products of these genes indeed play a defensive role (Fig. 3) . As in Anopheles , there is only one Class C Scavenger Receptor (SR-C) in the honey bee. This group has diversified into four members in Drosophila , three of which show selective signs suggestive of an immune role (Lazzaro, 2005) . There are 10 Class B scavenger receptors in the bee, a number roughly similar to that in the fly and mosquito (Fig. 4B , Table 1 ). Several other recognition classes also seem to be reduced in honey bees, including β -glucan recognition proteins ( β GRPs), galectins and fibrinogen-related proteins (Table 1) . Of these, the fibrinogen-domain genes are especially striking, due to the absence in bees of high lineage-specific diversification found in mosquitoes and Drosophila [resulting in 57 and 13 domain-family members, respectively (Christophides et al ., 2004) ].\nAlong with PGRP-LC and SR-C , two other receptors were recently found to participate in the phagocytosis of infectious non-self in Drosophila , DSCAM and Eater. DSCAM, long implicated in neuronal development, was shown to have a likely role in the binding of bacteria by Drosophila haemocytes (Watson et al ., 2005) . This gene, which has > 12 000 potential splice variants in honey bees thanks to three sets of highly interchangeable exons (Graveley et al ., 2004) , is a very interesting candidate for determining the extent to which bees might better tune their immune response to specific pathogens. Another Table 1 . Gene counts for a subset of gene families implicated in insect immunity. Anopheles gambiae and Drosophila melanogaster counts based on Christophides et al. (2002 Christophides et al. ( , 2004 recently identified protein involved in Drosophila cellular immunity is Eater, a phagocytic receptor characterized by several repeats of an EGF motif in its extracellular domain (Kocks et al., 2005) . Many genes with EGF motifs are present in the bee genome (e.g. GB14654, Supplementary Material Table S1 ), as in flies, although their orthology with Eater is unclear. Bees are also likely to engage in the encapsulation of endoparasites and pathogens, and show typical integrins (Honey Bee Genome Sequencing Consortium, 2006) implicated in lamellocyte encapsulation . None of these cellular immunity components appear to be more diverse in bees than in fly species (Supplementary Material Table S1 ).\nIn addition to their function in digestion of food, serine proteases (SPs) in insects participate in regulatory cascade pathways in embryonic development and in immune responses (Kanost & Clarke, 2005) . Many haemolymph SPs and serine-protease homologues (SPHs) implicated in immunity contain one or more clip domains at their amino terminus, which may regulate or localize the immune responses stimulated by protease cascades (Jiang & Kanost, 2000) . Among the 57 SP-related proteins in the honey bee genome, 12 SPs and six SPHs contain at least one clip domain, significantly fewer than in Drosophila (24 SPs and 13 SPHs) (Ross et al., 2003) or Anopheles (26 SPs and 15 SPHs), and smaller than the number of clip domain SPs identified to date (n = 14) from expression data in Manduca sexta (Jiang et al., 2005) . Additional phylogenetic and functional relationships among insect SPs and SPHs are discussed in Zou et al. (2006) . Serine protease inhibitors from the serpin superfamily regulate protease cascades in mammals and in arthropods (Reichhart, 2005) . In insect haemolymph, serpins inhibit activated proteases to maintain homeostasis and prevent unregulated activation of immune responses such as melanization or Toll-mediated antimicrobial protein synthesis (Kanost & Clarke, 2005) . In the honey bee genome, there are seven annotated genes encoding five serpins and two proteins with serpin-like regions (GB10078 and GB15070). The number of serpin genes in the honey bee is much lower than in Drosophila (28) or Anopheles (14), mirroring the reduced size of the protease gene family in bees.\nNine Toll-related receptor genes are known from the Drosophila genome and 10 from Anopheles (Tauszig et al., 2000; Christophides et al., 2004) . In Drosophila, Toll is the primary family member implicated in immune-related function (Lemaitre et al., 1996) , although it appears that Drosophila Toll-5 and Toll-9 (the paralogue that is structurally most similar to mammalian TLRs), are also involved in immune-related signalling (Ooi et al., 2002; Bilak et al., 2003) . We have identified only five Toll-related genes in the honey bee: Toll1, Toll2/18w, Toll6, Toll8/Trex/Tollo and Toll10.\nAdditional Toll members in Drosophila (Toll-3, -4, -5, -7 and -9) apparently reflect gene duplication events in the fly lineage ( Fig. 4) or, in the case of Toll-9, arguably a loss in the honey bee and lepidopteran lineages. For instance, the ancestral Toll appears to have diverged into two different groups, Toll-1 and Toll-5, in flies. In contrast, Toll-1 remains the only member of this clade in honey bees. Similarly, the Toll-7/2 clade is represented only by a single honey bee homologue Am18w (Aronstein & Saldivar, 2005) . Presumably, the ancestral Toll 7/2 gene was duplicated in flies following their divergence 300 Mya from the lineage leading to honey bees. A protein named Apis mellifera toll7 (Kanzok et al., 2004) , seems more likely to be an orthologue of Toll-10. The five Toll receptors present in the A. mellifera genome (Toll-1, -6, -2/7, -8, -10) are also present in the sequenced genomes of other insects that belong to the orders Diptera, Lepidoptera and Coleoptera, with few exceptions. For example, whereas orthologues of Toll-6, -7 and -8 are found in D. melanogaster and A. gambiae (Diptera), Bombyx mori (Lepidoptera), and Tribolium castaneum (Coleoptera), Toll-1 appears to be absent from the genome of the lepidopteran insect B. mori, whereas Toll-10 appears to have been lost in the fruit fly. This suggests that these five genes encode the basic set of Toll receptors that was present in the common ancestor of these insects. These five receptors are highly expressed, in a dynamic and tissue-specific manner, during Drosophila embryogenesis (Kambris et al., 2002) . Of note, Toll-6 and Toll-8, are adjacent in both the D. melanogaster and A. mellifera genomes.\nAmong the immune effectors, the total of six honey bee antimicrobial peptides contrasts with the 20 and nine found in Drosophila and Anopheles, respectively (Table 1) . Two of these (abaecin, and apidaecin) are in the class of prolinerich antimicrobial peptides, two are conventional defensins and two (apisimin and hymenoptaecin) are distinct from all other recognized antimicrobial peptides. Genomic analysis reveals that the gene encoding apidaecin consists of a conserved N-terminus followed by several exons, each of which encodes a complete 28-amino acid peptide. Peptide and cDNA evidence for this gene was used to predict a mechanism for ratcheting up expression of apidaecin in response to bacterial challenge (Casteels-Josson et al., 1993) . The genomic structure of apidaecin raises the possibility of a mechanism for generating specific responses to pathogens by splice variation. As each exon is a functional and distinct antimicrobial peptide, it is conceivable that splice variation at this locus can further refine this gene as an immune effector. Apidaecin exons differ greatly across individual honey bees in both number and in their encoded amino acid sequences. The two bee haplotypes sequenced in this project differed in sequence and exon number and also differed from three previously described apidaecin cDNAs (Casteels et al., 1993) . Collectively, the two haplotypes sequenced here, and the three described by Casteels et al. encode a range of 4-11 secreted peptides each. While some peptides are shared between the various haplotypes for this gene, there is a surprisingly high level of sequence variation, such that the 35 peptides expressed by these five haplotypes reflect 23 different amino acid variants.\nWith the exception of defensin-2 (identified from genomic sequences generated during this project; Klaudiny et al., 2005) all of the honey bee antimicrobial peptides were first characterized by protein sequencing (Casteels-Josson et al., 1994) , a fact that belies the difficulty in discovering such genes by sequence similarity across the millions of years separating insect species. Still, there is no evidence for close paralogues for any of the honey bee antimicrobial peptides, in contrast to such patterns in other insects (e.g. the gene-rich cecropin family). Five of the six bee antimicrobial peptides are up-regulated across diverse immune challenges ( Fig. 3; Evans, 2004) . Honey bees possess only one prophenoloxidase (proPO) gene, versus three and nine in Drosophila and Anopheles, respectively. Like most proPO, the honey bee proPO lacks a signal peptide and has the consensus sequence of NRFG around the activation site. The gene encoding proPO is expressed more strongly in older honey bee larvae and pupae (Lourenço et al., 2005) . This gene was not up-regulated in our challenge experiments, but a gene identified as a proPO activator was up-regulated during natural infection (Fig. 3) . There are only three lysozymes in the honey bee genome, two c-(chicken) type and one i-(invertebrate) type. One of the c-class lysozymes is up-regulated by challenged honey bees (Fig. 3) .\nThere were fewer thiolester-containing proteins (TEPs) in the bee genome (four) than expected based on flies ( Table 1 ). The Anopheles genome encodes 15 TEPs, most of them originating from species-specific expansion (Christophides et al., 2004) versus six members of this gene family in Drosophila (Agaisse & Perrimon, 2004) . TEPs are induced after septic injury and promote phagocytosis in mosquitoes (Blandin & Levashina, 2004; Moita et al., 2005) . They also play a central role in vertebrate innate immunity as the complement factors. In Anopheles, TEP1 was found to promote phagocytosis of Gram-negative bacteria and is also a major player in the host response to plasmodium infection. Members of this group are implicated as both recognition proteins and effectors (opsonins) in insects (Levashina et al., 2001; Moita et al., 2005; Stroschein-Stevenson et al., 2006) .\nAs social animals, honey bees are at considerable risk from parasites and pathogens. Specifically, increased genetic relatedness and the high population densities that typify honey bee societies can strongly favour pathogen spread and epizootic outbreak. However, such social costs to honey bee immunity might be offset by social defences including mating strategy (e.g. multiple mating by queens: Tarpy, 2003) , mutual grooming, and the maintenance of a sheltered environment for colony members. Given their well-studied natural pathogens, immune pathway models from the current annotated draft genome and unique genetic traits, honey bees can join with several fly and moth species as important systems with which to understand the genetic causes of immunity and disease. They also join humans as organisms for which there is great interest in understanding the social drivers of disease, and in using this information to improve host survival.\nOur analyses indicate that the basic set of molecules defining the insect host-defence system is present in honey bees, including intact pathways for the key processes implicated in immunity and development. Single orthologues can be assigned for many pathway members, while others show several potential bee genes for which further work is needed to confirm roles. Interestingly however, whereas in Drosophila and Anopheles the host appears to have diversified its molecular arsenal through species-biased gene duplication (Table 1) , we have not found examples of such gene expansions in honey bees. Thus, despite having wideranging parasites and pathogens, and tremendous losses to these pests at least in domesticated settings (Morse & Flottum, 1997) , bees appear to have relatively diminished capacities to respond to and defend against pathogens.\nOur first hypothesis to help explain this observation, that bee gene families are systematically smaller than in other insects, is not supported because there is no evidence for a systematic downward bias in paralogue counts across the bee genome at the level seen for immune-gene families (Honey Bee Genome Sequencing Consortium, 2006) . Hypothesis two, that bee genes were simply missed due to sequence divergence, should apply particularly for immune genes that are short and subject only to limited sequence constraints, such as those encoding antimicrobial peptides. Indeed, few such peptides have been identified from bees (n = 6) and the means with which these few were discovered (directly at the protein level in all but one case) support the idea that sequence-level searches might have missed additional family members. Nevertheless, members of the remaining gene-poor families do not comprise especially short genes, nor genes divergent enough to be missed completely by comparisons at the level of insect orders. In fact, at least one significant bee:Drosophila:Anopheles orthologue is present in each of the remaining discussed families, indicating sufficient sequence-level conservation for identification of bee counterparts. Our third hypothesis, that bees simply have an undescribed mechanism for broadening their immune efficacy, is not testable at this point but would be very surprising given similarities in immune actors across the diverse insect orders studied to date. Two of our initial hypotheses, that bees face a less diverse set of successful parasites and pathogens, and that societal defences by bees lessen pathogen pressures, therefore seem best supported by the data in hand and can now be compared. On one level, bee pathogens are diverse, ranging from Gram-positive and Gram-negative bacteria to fungi, RNA viruses, microsporidia and amoebae (Morse & Flottum, 1997) . Bees are also parasitized by mites and other arthropods, raising risks of both pathogen infection (Kanbar & Engels, 2003; Chen et al., 2004) and lower abilities to combat disease (Gregory et al., 2005; Yang & Cox-Foster, 2005) . Still, despite having pests that range over several kingdoms, common disease agents in bees are in fact restricted to several pathogens, two of which (the bacterium Paenibacillus larvae and the fungus Ascosphaera apis) are predominant. While functional data on the specificity of responses toward these and other pathogens are not yet available, gene-expression changes after challenge do not appear to be especially precise with respect to honey bee pathogens vs. exotics (e.g. Escherichia coli) or stress generally (Fig. 3) . Thus, there is no compelling evidence at this point that the bee immune response is channelled just toward a small set of 'true' pathogens.\nWith respect to the final hypothesis, bees, like many social insects, are relentlessly hygienic, removing alien organisms from their nests, and secreting antimicrobial substances that can reduce the viability and growth of pathogens in the colonies. Bees also raise their young in individual cells using, as a food source, substances with strongly antimicrobial properties (e.g. royal jelly; Albert & Klaudiny, 2004) . A testament to this hygiene is the fact that, even when facing severe colony-level infections by bacterial pathogens such as Paenibacillus larvae (for which < 10 spores are normally fatal to young larvae (Brodsgaard et al., 1998) , the vast majority of larvae show no signs of exposure (Evans & Pettis, 2005) . 'Social' barriers might also reduce exposure to minor, opportunistic, pathogens or saprophytes that have been proposed as generalized targets of insect immune defences (Hultmark, 2003) . While bees do carry an assemblage of microbes, and bacteria in particular (Gilliam, 1997) , exposure to these microbes is arguably lower than in free-living Drosophila (decaying plant material as larvae and adults) or Anopheles (septic aqueous environments as larvae). Further, bacteria found in bee colonies have only rarely been associated with disease pathologies, despite extensive study. In fact, some resident bacteria in colonies appear to add to external defences through their inhibition of bee pathogens (Evans & Armstrong, 2006) .\nFuture genomic work can help reveal whether other species of highly social insects, including ants, wasps, bees and termites, also appear to have more simplistic innate defence systems. More generally, social and solitary insects with more 'exposed' life histories are predicted to have a greater number and higher functional diversity of immunepathway genes and end products, when compared to sister taxa that are more sheltered. Data on parasite and pathogen abundance across social (e.g. Boomsma et al., 2005) and solitary insects could be used as a surrogate for disease loads in different taxa, although field and epidemiological data are most needed to assess the relative fitness impacts of disease and the efficacies of different lines of defence.\nThrough this analysis, we present the first plausible models for immune pathways in a social insect, the honey bee. We show nearly complete conservation of candidate genes for these pathways yet show that bees have consistently undercut numbers of genes that embellish these pathways in other insects. Genome-wide expression studies newly available for bees, and the proven success of gene knockdown techniques such as RNA inactivation (Lourenço et al., 2005) , allow for more refinement of the roles played by pathway members as well as the discovery completely novel players in honey bee immunity. The latter discoveries, combined with analyses across more species of social and solitary bees, will help determine whether the observations described here are unique to the highly social honey bees. Longstanding agricultural interest has helped generate a wealth of data on honey bee pathologies (Morse & Flottum, 1997) , and it is now possible to connect these data with immune traits that help limit pathogen efficacy. Through these connections, honey bees will provide a valuable and tractable model for disease transmission, immunity, 'socialized medicine' and pathology.\nImmune-gene candidates from other insects were used in several ways to query the honey bee genome, primarily using the BLAST family of search functions (www.ncbi.nlm.nih.gov). Most searches were initiated by BLASTP queries against the consensus protein list (GLEAN3, derived from HBGP assembly 2.0) using BLASTP and algorithms (BLOSUM and PAM variants) appropriate to gene size and structure. Honey bee orthologues were also identified by searching honey bee genome assemblies 2.0 and 3.0 directly using TBLASTN and either local databases or the BeeBase BLAST server (http://racerx00.tamu.edu/blast/blast.html). Searches for missing genes were also carried out a on smaller coverage set of honey bee contigs that were too short to be included in the assembly, as well as the unassembled reads from the project (http://www.hgsc.bcm.tmc.edu/projects/honeybee/). Given honey bee candidates, searches were repeated in the hope of identifying paralogues missed by interspecific comparisons. PSI-BLAST was used to identify honey bee genes on the basis of conserved domains, followed by RPS-BLAST to confirm the significance of these domain matches. Putative matches were aligned and, in the case of serine proteases, scavenger receptors and C-type lectins, screened for additional motifs using pfam categories (http://sanger.ac.uk/software/pfam). Tentative matches were aligned and checked for gene-prediction errors (in the case of genes from the official protein list) as part of the annotation of immune candidate genes for the Honey Bee Genome Project (Honey Bee Genome Sequencing Consortium, 2006). All protein matches were ported to Apis mellifera assembly 3.0 using the alignment program BLAT (Jim Kent, University California, Santa Cruz) to establish scaffold locations (Supplementary Material Table S1 ).\nBest-match honey bee sequences were then aligned with counterparts from D. melanogaster and A. gambiae, along with other insects where sufficient genome-level data were available. Amino acid sequence alignments were carried out using GONNET series weight matrices, with the program CLUSTAL_X (Chenna et al., 2003) . Alignments were used to propose phylogenetic relationships using maximum-parsimony and neighbour-joining algorithms, with the programs PAUP* (Sinauer, Sunderland, MA) or PHYLIP (http:// evolution.genetics. washington.edu/phylip.html). The PGRP tree is based on the conserved domain region only. Other alignments were edited manually to reduce or remove ambiguous regions. For scavenger receptor class B proteins, human (NP_005497) and mouse (NP_031669 CD36) proteins were added to alignments. Honey bee scavenger receptor AmelSCRB8 was not included because its relatively short predicted length (apparently the result of an intercontig gap) precluded an unambiguous alignment. All alignments are available on request.\nTwo experiments were carried out to screen for immune-related transcript changes. In the first, adult worker bees from a single local A. mellifera ligustica colony were removed, then injected abdominally with either dilute phosphate-buffered saline or saline solution containing 103 live cells of E. coli or 103 vegetative spores of the honey bee bacterial pathogen Paenibacillus larvae. These bees, along with uninjected controls, were maintained for 24 h at high humidity and 34 °C and then were immediately frozen at −70 °C prior to RNA extraction. To assess immune responses following natural infection, eight 1st-instar larval bees from the same stock were given per os challenges of P. larvae in their food [(50 spores/µl as described in Evans (2004) ], then maintained 24 h at 34 °C and high humidity. Parallel control larvae were given the same food without bacterial spores. All samples were frozen at −80 °C following incubation.\nRNA was extracted from whole abdomens of the adult bees using a standard TRIzol (Invitrogen, Carlsbad, CA, USA) procedure while RNA was extracted from individual larvae using the RNAqueous kit (Ambion, Austin, TX). RNAs were pooled by sample duration for the eight larvae challenged with the bacterial pathogen P. larvae, and the eight controls prior to cDNA synthesis, giving six RNA pools. DNA was removed from all extracts, then first-strand cDNA was synthesized as described by Evans (2004) . Transcript abundances for these cDNAs were assayed by quantitative real-time PCR with an Icycler real-time PCR machine (Bio-Rad, Hercules, CA, USA). Primer pairs were designed to amplify 120-300 bp sections of 39 honey bee immune-related genes derived from Supplementary Material Table S1 and ribosomal protein S5 as a control gene (primers in Supplementary Material Table S2 ). Primer sequences were modified, where necessary, to run in duplicate on 96-well plates using a fixed thermal protocol consisting of 5 min at 95 °C, then 40 cycles of a four-step protocol consisting of 94 °C for 20 s, 60 °C for 30 s, 72 °C for 1 min, and 78 °C for 20 s was used (Evans, 2006) . Reactions were carried out on 0.5-2 µg cDNA along with 1 U Taq, the provided PCR buffer (Roche Applied Sciences, Indianapolis, IN, USA), 1 mM dNTP mix, 2 mM added MgCl 2 , 0.2 µM each primer, 1× concentration SYBR-Green I dye (Applied Biosystems, Foster City, CA, USA), and 10 nM fluorescein in a 25 µl reaction volume. Amplification was followed by a meltcurve dissociation program in order to confirm expected product size. Thresholds were calculated individually for each target gene on the 96-well plate. For adult bee samples, data were pooled for the three replicates in each single-bee injection treatment (or controls). Results were screened for the appropriate dissociation (melt-curve) values, and by 1% agarose gels, in order to ensure against primer artefacts and the presence of DNA contamination (which would have been evident for numerous primers spanning two exons). Immune-gene transcripts were normalized relative to expression levels for the gene encoding ribosomal protein S5, a gene with consistent expression across honey bee life stages and disease status (Evans & Wheeler, 2000; Evans, 2004) . For display purposes, transcript abundance values (CTcontrol-CTtarget) for each gene were median-normalized across each panel of genes and clustered by average linkage clustering (using Cluster 3.0, M. Eisen, www.rana.lbl.gov/EisenSoftware.htm) and presented as relative grey-scale values (using Treeview, M. Eisen)." }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: Ubiquitin, as well as Ubls such as Nedd8 and SUMO, are proteins (almost) exclusively expressed by eukaryotes [1] . Ubiquitination controls many cellular processes, including degradation of proteins by the proteasome and intracellular trafficking. Nedd8 is a ubiquitin-like modifier that regulates the rate or extent of ubiquitination, whereas SUMO1 is involved mostly in regulation of transcription factors and in nuclear import. The attachment of ubiquitin or ubiquitin-like modifiers to substrate proteins is covalent, yet reversible [2] . A large family of eukaryotic cysteine proteases is involved not only in generation of ubiquitin(-like) proteins from their precursors, but also in their removal from modified substrates [3, 4] . Pathogens can tamper with the ubiquitin-proteasome system to cripple the cell's defenses [5] . For instance, ubiquitination and proteasomal degradation of p53, initiated by a Human Papillomavirus protein [6] , or stabilization of Ikb-a by Yersinia deubiquitinases [7] have been described. The continuing discovery of new deubiquitinating proteases in viruses broadly hints at how important it is for these pathogens to seize control of posttranslational modifications in host cells [8] [9] [10] . Here, we focus on cysteine proteases of the CE clan [11] , as defined by the MEROPS database [12] . CE peptidases are expressed by viruses, bacteria and eukaryotes. In eukaryotes, this clan represents the family of Ubl-specific proteases (ULP/SENP), which remove SUMO or Nedd8 from substrate proteins [13, 14] . Viral homologs of ULP/SENPs can act as deubiquitinases, but they also cleave unrelated proteins, as long as a glycine motif is present at the C-terminus of the substrate [15, 16] . Examples of bacterial deubiquitinases include YopJ and ChlaDUBs. First, YopJ is a protease that is secreted into host cells by Y. pestis [17] , and homologs to this peptidase can be found in other bacteria, too [18, 19] . Injection of YopJ eventually suppresses the inflammatory response in affected cells. The precise molecular mechanism of YopJ family peptidases has not yet been solved, as they lack a hallmark tryptophan following the active-site histidine [20] . Yet, in vitro and in vivo data strongly suggest that these proteins are indeed proteases with specificity for ubiquitin or SUMO [7, 18, 19] , although their effectiveness as virulence factors might depend on additional functions such as acetylation [21, 22] . A second example of bacterial CE peptidases can be found in pathogenic Chlamydiae. We have shown that pathogenic Chlamydiae, but not a nonpathogenic environmental strain, express proteases that specifically recognize ubiquitin and Nedd8. They do so presumably to remove both modifiers from target proteins of the host cell, as Chlamydiaelike most other bacteria-possess neither a ubiquitin nor a Nedd8 homolog [23] .\nIntrigued by the finding of CE peptidases in bacteria and considering their functional similarity to eukaryotic ULP/SENP proteases, we explored whether additional bacterial homologs with deubiquitinating activity might exist.\nTo search the sequenced genomes of bacteria for new members of the CE protease clan, we employed PHI-BLAST [24] to find proteins with the typical catalytic triad of histidine, aspartate (or glutamate or asparagine), and the active-site cysteine [25] . Candidate proteins were subjected to a second round of analysis, in which we excluded proteins without the hallmark oxyanionstabilizing group, consisting of at least one glutamine (or asparagine) close to the active-site cysteine. Lastly, the predicted secondary structure of candidate proteases was compared to the solved structure of eukaryotic CE clan homologs [26, 27] . Apart from YopJ and the ULP/SENP homologs in Chlamydiae, we found potential CE peptidases in Alpha-, Beta-, and Gammaproteobacteria ( Figure 1 ), but not in bacteria from other branches. We also found a peptidase homolog in the giant Acanthamoeba Polyphaga Mimivirus [28] and in African Swine Fever Virus, the first virus in which a ubiquitin conjugating enzyme was discovered and for which deubiquitinating activity has been suggested [29] [30] [31] [32] . The bacteria we identified share a close (symbiotic or pathogenic) relationship with eukaryotes, and all viruses with CE proteases possess a dsDNA genome. The sequence variations among these peptidases are too extensive to allow for significant bootstrap values and it is therefore not possible to faithfully infer phylogeny from this dataset. Yet, this is also true for the eukaryotic ULP/SENPs, which fall into three functional classes, specific for either SUMO (ULP1 and ULP2 group) or Nedd8 (SENP8 group). While best reciprocal BLAST hits and consensus phylogram trees tend to correctly predict to which functional class a particular ULP/SENP homolog belongs, bootstrap support is generally not significant [33] .\nWe were especially interested in the protein elaD (belonging to ''group II'', see Figure 1 ), expressed by E. coli and with orthologs in Legionella pneumophila and in all currently sequenced strains of Salmonella ( Figure 2 ). E. coli is an abundant commensal in the human gut and also a relevant pathogen. Nonetheless, little is known about genes that define pathogenicity of various E. coli strains, apart from those that encode obvious toxins [34] [35] [36] . We furthermore chose elaD, because it represents one of the more distantly related hits in our bioinformatics screen and we aimed to test the robustness of our prediction by examining this protein's function. Figure 1 . Phylogram representation of CE clan proteases in viruses, bacteria, and eukaryotes. Eukaryotic peptidases (in blue) belong to the C48 subfamily and can be separated into three groups: ULP1 (including the mammalian proteases SENP1, 2, 3, and 5), ULP2 (including SENP6 and 7), and the SENP8 group with proposed specificity for SUMO (ULP1 and ULP2 group) and Nedd8 (SENP8 group), respectively. Bacterial proteins are indicated with a preceding ''B'', viral proteins with a ''V''. We have further divided microbial protease homologs by color: green for biochemically tested proteases, red indicating the absence of published data on the function of these putative proteases, and yellow for the group representing elaD and its orthologs. The C5 family contains Adenovirus proteases with deubiquitinating activity, C55 comprises the bacterial YopJ homologs, and C57 the Vacciniavirus I7 peptidases. Based on sequence similarity, two bacterial C48 family groups can be distinguished: a group of Proteobacteria (located at one o'clock) which appear to be closely related to fungal SENP8 homologs (common node indicated with a circle, bootstrap support.60%), and Chlamydiae, for which we had previously shown the presence of deubiquitinating and deneddylating activity. Three additional groups have not yet been assigned to specific CE clan subfamilies in the MEROPS database, including Mimivirus (''group I''), Gammaproteobacteria (''group II''), and Rickettsiae (''group III''). The African Swine Fever Virus protease and the I7 Vacciniavirus protease have not been tested for deubiquitinating or Ublspecific activity, but they both require a glycine-based motif at the C-terminus of the substrate, as found in ubiquitin or Ubls. The unrelated CD clan peptidase Clostripain is used as outgroup in this phylogram. For clarity, this tree does not contain all orthologs and paralogs of the different groups or families. Sequence information is provided in Table 2 . doi:10.1371/journal.pone.0000381.g001\nThe goal of our first experiment was to confirm protease activity and to determine the substrate specificity of elaD. Because of its relationship to ULP/SENPs, we hypothesized that elaD might recognize Ubls or ubiquitin. We expressed elaD by in vitro transcription/translation in rabbit reticulocyte lysate and incubated the metabolically labeled polypeptide with electrophilic probes, in which a Michael acceptor was added to the C-terminus of ubiquitin or the Ubls SUMO1, Nedd8, and ISG15 [14] . As shown in Figure 3 , elaD readily forms a covalent adduct with the ubiquitin probe and to a much lesser extent also with the Nedd8 probe, but not detectably with SUMO1 or ISG15. Moreover, when mutating the putative active-site cysteine at position 313 to serine, covalent binding to the electrophile is abolished. This indicates that the cysteine residue in elaD is essential for catalytic activity, as has been observed for YopJ [7] , for the Chlamydia protease CT868 [23] and for the eukaryotic ULP/SENPs [2] . To date, specific labeling of putative proteases with activity-based probes has shown excellent correlation with enzymatic activity [14, 37, 38] .\nNext, we assessed enzyme kinetics by measuring hydrolysis of fluorogenic substrates derived from ubiquitin, SUMO1 and Nedd8. For these experiments, we expressed elaD in E. coli. The growth rate of the bacteria was unaffected when overexpressing elaD, but we only recovered about 50% of the wildtype protein when compared to the amounts of C313S mutant ( Figure 4A ). As demonstrated by release of the fluorophore 7-amino-4-methylcoumarin (AMC), elaD cleaves ubiquitin-AMC, but not SUMO1-AMC or Nedd8-AMC, and the C313S mutant of elaD fails to cleave either substrate ( Figure 4B and data not shown). The initial rate of hydrolysis with 100 nM ubiquitin-AMC and 50 nM elaD is in the order of 0.3-0.6 per minute, defining elaD as a moderately active deubiquitinase, compared to the rapid Isopeptidase T with a rate of ca. 8 per minute [39] or to the much slower ubiquitinprotease USP14 with a rate of,0.01 per minute (data not shown). It should be noted that we could not assess V max , because the enzymatic rate increased linearly with substrate concentration (we tested up to 20 mM ubiquitin-AMC; 50 nM elaD then hydrolyzed ubiquitin-AMC at an initial rate of 5 per minute). Similar observations have been made with the SARS virus deubiquitinase [8] . Overall, our functional analyses confirm the prediction of our bioinformatics screen and define elaD as a deubiquitinating protease.\nWhy do eukaryotic CE peptidases show specificity distinct from their prokaryotic counterparts? Could this indicate a profound discrepancy, hinting towards a separate origin of these two protease groups? We set out to challenge the presently held notion that ULP/ SENP proteases do not exhibit ubiquitin-specific activity [13] , while most tested bacterial homologs apparently do. To this end, we chose to biochemically define CE clan members of a more deeply branched class of eukaryotes. Pezizomycotina, a subgroup of fungi that includes A. fumigatus, A. nidulans, M. grisea, N. crassa, and G. zeae encode putative SENP8 proteases that are related in amino acid sequence to a group of yet uncharacterized bacterial C48 homologs ( Figure 1 ). In our phylogram, the common node between the respective prokaryote C48 group and SENP8 homologs of Pezizomycotina replicates with a bootstrap support of.60%. We cloned, expressed and tested the putative SENP8 protease of G. zeae, as a representative of Pezizomycotina. Unlike mammalian SENP8, the G. zeae ortholog displays dual activity towards Nedd8 and ubiquitin, similar to the previously defined CE clan protease CT868 Figure 2 . Sequence comparison between SENP8 and its homologs in human pathogenic bacteria. Multiple sequence alignment of the catalytic core region of human SENP8 (NCBI protein sequence identifier GI: 33942066, shown are residues 100-165) with the homologs in C. trachomatis (CT868, GI: 76789615, residues 199-284) [23] , E. coli (elaD, GI: 15832411, residues 228-319), L. pneumophila (GI: 52843101, residues 189-265), and S. typhi (sseL, GI: 29141091, residues 197-264) [42] . The arrows indicate active-site histidine, aspartate (or asparagine), the catalytic cysteine, and the oxyanion-stabilizing group. Predicted secondary structures are shown at the bottom and have been confirmed with the solved structure of SENP8 [27] . doi:10.1371/journal.pone.0000381.g002 Figure 3 . Biochemical assay for substrate specificity of elaD. 35 S-methionine-labeled in vitro translated wildtype elaD forms covalent adducts with suicide inhibitors based on ubiquitin (ubiquitin-vinylmethylester, VME) and Nedd8 (Nedd8-vinylsulfone, VS), but not with probes based on SUMO1 and ISG15. All probes were tested for activity with bona fide substrates (not shown) [14] . Mutation of the active-site cysteine at position 313 to serine abolishes adduct formation of elaD to electrophilic probes. Samples were resolved by reducing SDS-PAGE and visualized by fluorography. Indicated at the right is the molecular mass in kDa. doi:10.1371/journal.pone.0000381.g003 of the prokaryote C. trachomatis ( Figure 5 ) [23] . This result indicates that ubiquitin-specificity is not restricted to bacterial or viral CE peptidases, but also exists in some ancient eukaryotic members of this protease clan.\nWe have found previously undescribed CE peptidase homologs in several bacterial and in one viral species (Figure 1) . We have furthermore proven that one of the more distantly related homologs-the protein elaD in E. coli-can act as a deubiquitinating enzyme in vitro. Earlier, we had shown that related Chlamydia proteases also have deubiquitinating activity and similar observations had been made regarding the YopJ protease family. This suggests that the yet undefined bacterial CE peptidases could also display ubiquitin-or Ubl-specificity, especially considering their even closer sequence relationship to eukaryotic ULP/SENP proteases. These findings raise two important questions. First, are these proteases really specific for ubiquitin in vivo, or are we simply measuring an offtarget artifact, with the true substrates merely resembling ubiquitin? Second, why are these proteases-which are present in every eukaryote-so widely distributed in bacteria and viruses as well, and what can we learn about their genetic origin?\nOne might argue that the bacterial CE clan peptidases have distinct specificity for bacterial substrates. For instance, it has been proposed that the origin of the ubiquitin system predates the split between eukaryotes and prokaryotes [40, 41] . Factors that distantly resemble Ubls and their conjugating and deconjugating enzymes can be found in bacteria. In this manner, the cleavage of ubiquitin by elaD could just be an artificial byproduct of our assays, while the true substrate is of bacterial origin. Similarly, the Adenovirus CE protease has relaxed specificity for a consensus site that is present in ubiquitin, but also in certain viral substrates [15, 16] . However, we have extended our analysis of elaD's specificity to the ubiquitin homologs Nedd8 and ISG15. Both share significant sequence similarity to ubiquitin, and ISG15 is even identical at the critical C-terminal region. We observed no reactivity between elaD and ISG15-vinylsulfone, and the binding of elaD to Nedd8vinylsulfone was significantly weaker than to the ubiquitin probe. Furthermore, we detected hydrolysis of the C-terminal peptide bond in ubiquitin-AMC, but not in Nedd8-AMC. These features clearly distinguish elaD from the more promiscuous viral CE peptidases. Given the similarity of primary, secondary and tertiary structure among these Ubls, we conclude that hydrolysis of ubiquitin by elaD reflects a highly specific interaction. With the exception of A. avenae, no bacterial strain in our dataset encodes a homolog of ubiquitin, making it likely that the substrate of elaD is indeed eukaryotic ubiquitin. Moreover, the ortholog of elaD in Salmonella-sseL-has recently been shown to be a virulence factor and to display deubiquitinating activity in vitro and in vivo [42] . This enzyme is encoded by all currently sequenced Salmonellae, but only present as a pseudogene in Shigellae [43] . Likewise, elaD is not essential for E. coli under laboratory conditions [44] . A comparison of the genomes of all 16 sequenced E. coli strains reveals that elaD is present in the commensal E. coli strain K12, and in all intestinal pathogenic strains (EAEC, EHEC, EIEC, EPEC, ETEC), but absent from all ExPEC strains (APEC, NMEC, UPEC) ( Table 1 ).\nTo answer the second question raised above, the fact that all CE proteases show the same signature motifs at the catalytic domain and that they have substrate specificity for ubiquitin, Ubls, or related products, suggests a common genetic and functional origin of these proteases. The viral and bacterial organisms that express these CE proteases all share an intimate relationship with eukaryotes, either as commensals, symbionts or as pathogens. Additionally, lateral gene transfer has been proposed between eukaryotes and dsDNA viruses [28] , as well as between eukaryotes and bacteria such as Chlamydiae, Rickettsiae, and L. pneumophila [45, 46] . The notion of a dynamic horizontal gene transfer involving deubiquitinases is further underscored by the distribution of ubiquitin-specific proteases in Chlamydiae: all pathogenic strains express CE peptidases, except for C. pneumoniae, which instead encodes a homolog of the eukaryotic otubain-type deubiquitinating enzymes [47] . Also, one group of bacterial C48 peptidases in particular clusters close to the SENP8 homologs of Pezizomycotina ( Figure 1 ). The sequence relationship and the shared habitat of these organisms-plant symbionts and phytopathogens vs. environmental fungi-raises the possibility of gene transfer between them [48] . From a functional perspective, we could show that a homolog of SENP8 in Pezizomycotina does exert ubiquitin-specific hydrolase activity, like previously characterized bacterial CE proteases ( Figure 5 ). The dual specificity of SENP8 from the fungus G. zeae towards ubiquitin and Nedd8 is not trivial. Although Nedd8 is arguably the closest relative to ubiquitin, there are sequence differences that require distinct conjugating machineries and deconjugating proteases [27, 49] . In particular position 72-an arginine in ubiquitin, and an alanine in Nedd8-can act as a compatibility switch and a single replacement at this side chain can cause ubiquitin to mimic Nedd8 and vice versa [50, 51] . In this respect, the recognition of both ubiquitin and Nedd8 by the SENP8 homolog of G. zeae is in contrast to what has been observed in mammalian SENP8 [27] .\nOne possible explanation for these observations is that CE proteases originally derived from a deubiquitinase, a protease specific for this most conserved eukaryotic protein. As CE peptidases in eukaryotes structurally diversified to accommodate the evolving family of Ubls, protease counterparts in bacteria, viruses, and some deeply branched eukaryotes retained their specificity for the ''ur-substrate'' ubiquitin.\nTogether with the published literature, our data supports the notion that the clan of CE proteases was acquired by bacteria and viruses via horizontal gene transfer from eukaryotes. Why this family of enzymes forms such a particularly attractive substrate for genetic exchange is an intriguing question. The distribution of CE proteases in symbiotic and pathogenic prokaryotes and viruses is suggestive of a general role in host-microbe interactions, as exemplified by the Salmonella protease sseL [42] .\nProtein and DNA sequence data were obtained from the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov), the Institute for Genomic Research (www.tigr.org), and the University of Wisconsin E. coli Genome Project (www.genome.wisc.edu). Protein sequence identifiers are listed in Table 2 . Sequences containing and surrounding the catalytic core of the proteases were aligned with the ClustalX algorithm (default parameters) (bips.u-strasbg.fr/fr/Documentation/ClustalX) [52] , manually edited with Genedoc (www.psc. [53] . Secondary structures were predicted with JPred (www.compbio.dundee.ac.uk/,www-jpred/) [54] . Phylograms were constructed with the MEGA software package [55] , using the Neighbor-Joining Method with Poisson correction (all substitutions, homogeneous pattern, c-distribution 2.0) and pairwise deletion of gaps. Figure 1 shows a consensus tree based on 100 bootstrap replications.\nCloning, expression, and biochemical analysis of elaD and G. zeae SENP8\nThe full-length elaD gene (NCBI protein sequence identifier GI: 16130204) was amplified by PCR from the K12 strain BL21 (Novagen) and cloned into pET28a (Novagen The putative proteases are separated by groups (as shown in Figure 1 ), by species, and by subdivision of Proteobacteria. This list is not complete in terms of orthologs/paralogs or bacterial species. " }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: The bacterial pathogens Neisseria meningitidis (Nm) and Haemophilus influenzae (Hi) are frequently found in the nasopharynx of a substantial proportion of the healthy population but are capable of causing serious infections in susceptible individuals (Turk, 1984; Foxwell et al., 1998) . Nm and typable Hi (THi) can invade the nasopharyngeal epithelial barrier to cause septicaemia and meningitis, which in the case of Nm, may rapidly become life threatening (van Deuren et al., 2000) . Non-typable Hi (NTHi), which lack a polysaccharide capsule, are associated with localized respiratory tract and conjunctival infections (Foxwell et al., 1998) . Strains belonging to Hi-biogroup aegyptius (Hi-aeg) are also associated with Brazilian purpuric fever (Foxwell et al., 1998) . The factors that determine susceptibility to infection by these frequent colonizers are not entirely clear.\nFor both colonization and pathogenesis, the first essential step is adherence to mucosal epithelial cells. Many investigations have shown bacterial targeting of specific human signalling molecules such as integrins, sialic acid binding Ig like lectins (Siglecs) and carcinoembryonic antigen (CEA)-related cell adhesion molecules (CEACAMs) can lead to cellular invasion (Virji et al., 1995; 1999; Hauck and Meyer, 2003; Jones et al., 2003) . Of these, CEACAMs have emerged as common targets of several respiratory mucosal pathogens and include Nm, Hi, Moraxella catarrhalis, as well as the urogenital pathogen Neisseria gonorrhoeae and enteric pathogens Escherichia coli and Salmonella (Leusch et al., 1991; Virji et al., 1996a; Chen et al., 1997; Gray-Owen et al., 1997; Hill and Virji, 2003) .\nCarcinoembryonic antigen-related cell adhesion molecules belong to the immunoglobulin (Ig) superfamily. Several members of the CEACAM subgroup are expressed on human epithelial cells and include the widely expressed transmembrane CEACAM1 as well as GPI-anchored CEA and CEACAM6. All CEACAMs have an N-terminal IgV-like domain and variable numbers of IgC2-like A and B domains. CEACAM1 comprises up to four extracellular domains: N, A1, B and A2 and either a long or a short cytoplasmic tail (Tsutsumi et al., 1990; Prall et al., 1996; Hammarstrom, 1999) . Various functions have been attributed to CEACAM1 including cell-cell adhesion, insulin regulation and angiogenesis (Obrink, 1997; Hammarstrom, 1999; Wagener and Ergun, 2000; Najjar, 2002) . Targeting of CEACAMs by N. gonorrhoeae, as well as Nm and Hi leads to cellular invasion and passage across polarized monolayers (Virji et al., 1999; Gray-Owen, 2003; M. Soriani, K. Setchfield, D.J. Hill, and M. Virji, unpublished data) .\nA wide range of bacterial adhesins are involved in targeting the CEACAM N-terminal domains and includes Opa proteins, a major adhesin family of pathogenic Neisseria and P5 proteins of Hi (Chen and Gotschlich, 1996; Virji et al., 1996a; Hill et al., 2001) . Nm and N. gonorrhoeae contain multiple copies of Opa genes that encode conserved domains which form b-barrel structures in bacterial membranes and variable domains that form surface exposed loops. In spite of the surface diversity afforded by the hyper-variable domains of the loops, the majority of the Opa proteins are capable of targeting CEACAMs (Virji et al., 1996a; Hauck and Meyer, 2003) . The P5 proteins of Hi are similar b-barrel forming proteins, also with surface variable loops (Webb and Cripps, 1998; Vandeputte-Rutten et al., 2003) . The interactions between these bacterial ligands and CEACAMs are complex and the binding domain appears to involve more than one variable loop of the bacterial adhesins (Virji et al., 1999; Bos et al., 2002; de Jonge et al., 2003) . Interestingly, antibody inhibition studies have shown that the diverse ligands of neisseria and haemophilus bind to an overlapping site on the N-domain. In addition, mutational analysis of the N-domain of CEACAM1 has identified several critical residues particularly Ile-91. Alanine substitutions at these sites abrogated binding of most Opa-and P5-expressing bacteria to CEACAM1. Additional residues such as Tyr-34, Ser-32, Val-39, Gln-44 and Gln-89, most of which located in the vicinity of Ile-91, appear to determine the efficiency of interactions of various Opa and P5 molecules (Virji et al., 1999; . The bacterial binding surface on the CEACAM1 N-domain is the protein face composed of the beta strands C′′, C′, C, F and G (CFG for brevity). Despite the extensive investigations in a number of laboratories particularly on neisserial Opa proteins, it remains unclear as to precisely how CEACAM-binding ligands engage with the receptors.\nA three dimensional structural model of the human CEACAM1 N-domain has been previously generated based on other Ig family molecules (Virji et al., 1999) . In the current investigation, we have refined our previous model based on murine CEACAM1 crystal structure (Tan et al., 2002) . Examination of this model showed a better confluence of the above residues into a continuous binding site and suggested that substitutions at positions 34 and 89 at the core of the binding region may affect bacterial access to the implicated key binding residue Ile-91. By introducing conservative and non-conservative substitutions at positions 34, 89 and 91, we have examined the binding of a variety of bacterial strains to the receptor constructs. The data suggest that single nucleotide polymorphisms (SNPs) in individuals or populations that may introduce substitutions in CEACAM sequence particularly at the bacterial binding site, could not only decrease but also significantly increase the functional affinity of pathogen interactions. Increased binding affinity may result in increased cellular invasion and thus may lead to increased host susceptibility to infection by CEACAM-targeting bacteria.\nA three-dimensional model of CEACAM1 N-domain was previously produced based on the Ig family molecules REI, CD2 and CD4 (Fig. 1A ) (Virji et al., 1999) . In this model, whilst most mutations affecting binding of Nm and Hi were located centrally on the CFG face of the protein, Val-39 involved in Opa binding, was located at a distance towards the bottom of the CFG face (Fig. 1A) . Subsequently, a crystal structure of murine soluble CEACAM1a domains 1 and 4 was reported (Tan et al., 2002) . Based on this, we have remodelled the human CEACAM1 Ndomain. The CC′ loop that contains Val-39 folds back against the CFG face of this model such that it lies in close proximity to the other critical residues involved in bacterial adhesion (Fig. 1B) . Further examination of this model suggested that the bacterial binding pocket forms a rather flat surface in the centre of the CFG face. It also appears that substituting neighbouring residues Tyr-34 and Gln-89 by Phe and Asn, respectively, could result in a further flattening of the bacterial binding surface (Fig. 1C-E) , providing increased access to the key Ile-91. These residue changes also increase the size of the hydrophobic patch centred on I91 (Fig. 1C-E) . Both of these effects might facilitate binding of some bacterial ligands to the target receptor.\nIn order to assess the importance of these residues in interactions with mucosal pathogens, substitutions were introduced by site-directed mutagenesis at three sites (91, 34 and 89) on the N-domain. The substitutions introduced in CEACAM1 [NA1B]-Fc are shown in Fig. 2 . Chimeric receptors proteins with the native sequence or with sub- (Virji et al., 1999) . (B) New model based on the murine CEACAM1a N-domain crystal structure (Tan et al., 2002) . The models are presented as Van der Waals surface representation. In the latter case V39 locates more centrally with the other critical residues for bacterial binding. (C-E, left) Stereo pairs presented are ribbon diagrams for CEACAM1 N-domain in the native form (C), with Y34F (D) or Q89N (E) substitutions show a flattening of the bacterial binding region on CEACAM1 and improving accessibility of the primary binding residue I91. The side-chain atom colouring (C-green, O-red, N-blue) show the increased hydrophobic nature of the binding site around I91 in the mutant structures. (C-E, right) Surface presentations of the binding site (views corresponding to a 90°rotation of the stereo images in the horizontal axis) coloured according to hydrophobicity. stitutions were produced by transient transfection of COS cells for functional studies described below.\nThe N-domain specific monoclonal antibody (mAb) YTH71.3 has been shown to require Gln-89 and Ile-91 for receptor recognition and substitution of these amino acids had no effect on the binding of the polyclonal anti-CEACAM antibody A0115 (Virji et al., 1999) . The novel receptor constructs produced in the current studies were first examined for their ability to bind to YTH71.3, A0115 as well as Kat4c. The latter mAb recognizes the A and B domains of the receptor (Jones et al., 1995) . AO115 and Kat4c bound to various receptor constructs and to the native NA1B-Fc molecule with equal efficiency. For YTH71.3, Leu but not Thr at position 91 supported binding to the same extent as Ile of the native molecule. Similarly at position 34, only Phe could be effectively substituted for Tyr. Finally Q89A and Q89N completely abrogated the antibody binding.\nOpa-expressing phenotypes of two Nm strains (C751 and MC58) were used to investigate their binding to the modified NA1B-Fc receptors. Three C751 derivatives express-ing distinct Opa proteins (OpaA, OpaB and OpaD) and the MC58 derivative expressing an Opa protein designated OpaX (Virji et al., 1999) were assessed by receptor overlay experiments (Fig. 3) .\nAll the Nm isolates showed reduced binding to the soluble receptor with Y34A and I91A substitutions whilst Q89A affected OpaD binding most significantly confirming previous studies (Virji et al., 1999) . Introduction of a Leu or Thr residue at position 91 was generally less disruptive to Opa interactions. I91T substitution either did not affect binding (OpaB and OpaX) or reduced binding by 50-60% (OpaD and OpaA). I91L substitution had no effect on OpaD or OpaB whilst having opposite effects on OpaA (~50% reduction) and OpaX (twofold increase) binding.\nSubstitutions at position 34 other than Ala also supported binding of some but not all Opa proteins. Y34S substitution was unsuitable for the three Opa proteins of strain C751 but was tolerated by OpaX of MC58. Interestingly, Phe proved to be a more favoured residue in at least two cases with OpaX as well as OpaB binding to Y34F construct at threefold higher levels than the native receptor. Substitutions of Asn at Gln-89 revealed distinct patterns of interactions and binding reduced in the order OpaA > OpaX > OpaB/D (Fig. 3) .\nOverall, the data suggest that Opa binding to the receptor requires an extended aliphatic chain at position 91 and certain arrangements of the chain may facilitate binding of most Opa proteins (Ile = leu > Thr). At position Α,Β D, E β−strand: (Virji et al., 1999) . Side chains of the residues introduced at positions 34, 89 and 91 in this study are shown in B for comparison 34, the removal of the hydroxyl residue (Y43F) is tolerated or preferred whereas the absence of aromatic ring (Y34S) has an overall deleterious effect on Opa binding. Finally, reduction of the side chain extension (Q89N) reduces binding of three of the four Opa proteins.\nComparison of C751 Opa-A, -B and -D in which the differences exist only between their surface exposed loop structures (HV1 and HV2, shown in Fig. 9 ), suggests further that a combination of the two loop structures must be involved in presenting the appropriate binding partners for the distinct residues of the receptor.\nOne non-typable (A950002) and two THi strains (Rd and Eagan) were used in receptor overlay experiments as above (Fig. 4) . Alanine substitutions at Ile-91 confirmed previous results (Virji et al., 2000) . Taken individually, substitutions of Ile-91 for Ala or Thr abrogated binding of Fig. 3 . Relative binding of CEACAM1-Fc constructs to N. meningitidis isolates expressing distinct Opa proteins. Bacterial lysates were dotted on to nitrocellulose and overlaid with NA1B-Fc constructs as indicated. Binding relative to the native receptor was determined by densitometric analysis of immno-blots using NIH Scion Image programme. One hundred per cent binding level is indicated by the horizontal line allowing comparison to native receptor binding. Mean values and SE of > 3 replicates are shown in each case, except Q89A (n = 1) for OpaA, B and X. However, alanine substitution at all three positions confirm previous observations (Virji et al., 1999) . Black inserts in C show the levels of binding of the various receptor constructs to Opaisolate of strain C751; experiments were conducted simultaneously with C751OpaD in the presence of the receptors shown. all three strains. However, substitution I91L reduced binding only of the THi strain Eagan, suggesting a requirement for the extended hydrophobic arm of Ile in Eagan binding. Alanine substitution of Tyr-34 abrogated the binding of Rd and Eagan, whereas no effect on NTHi A950002 was observed. In contrast, Y34F substitution led to an increased binding of all three strains varying from 45 to 80% above native receptor binding levels, whereas Y34S had a differential effect on the three strains tested, ranging from no binding of Rd to a slight increase in the binding of A950002. These data broadly reflect results with neisserial Opa binding. Finally substitutions at Gln-89 show that reduction or abrogation of the side chain (Q89N and Q89A) has no deleterious effect, rather its effect is often that of enhanced binding (Fig. 4) .\nIn summary, Hi strains primarily require Ile-91 to enable receptor targeting. Tyr-34 influences binding of THi and as for Nm, removal of the OH (Y34F) provides a more favourable environment. Gln-89 side chain also limits bacterial interaction and its substitution to shorter side chains (Q89A and Q89N) is more favourable especially for Rd-CEACAM binding. \nIn previous studies, interactions of Hi-aeg strains were shown to be more analogous to Nm (Virji et al., 2000) . The current studies accordingly demonstrated a requirement both for Ile-91 and Tyr-34 for all Hi-aeg (Fig. 5) . In addition, as with some Nm strains, the requirement for hydrophobic and aliphatic chains was partly fulfilled by Leu and to a lesser extent by Thr. Substitutions of residue Tyr-34 with Phe or Ser produced a complex profile. Phe generally created a suitable or better environment but the loss of the aromatic ring was also tolerated (Y34S). At position 89, Q > N substitution enhanced binding dramatically for Ha3.\nOverall, the three strains tested exhibited similar binding to all receptor constructs that had substitutions at residue\nIle-91 as well as those with alanine substitution at residue Tyr-34 (Fig. 5) . However, strains exhibited differences in binding to the receptors with substitutions Y34F and Q89N demonstrating the structural diversity of the CEACAM1-binding ligands of distinct Hi-aeg strains also.\nFrom the above data, it is clear that receptor constructs with Q89N substitution have the capacity to increase the binding of some Hi over that of Nm. In previous investigations, we have shown that certain isolates of the two bacteria are capable of competing with the native receptor and that Nm C751 derivatives can displace Ha3 as well as Eagan from the receptor (Virji et al., 2000) . Here we investigated how changes in receptor structure at position 89 may influence this phenomenon. We used receptor dot blot overlay in the presence of varying amounts of the soluble receptor constructs to estimate the ability of C751OpaB and Ha3 to selectively adsorb the receptor. Consistent with the lower affinity of Ha3 compared with C751OpaB/D for CEACAM1 (Virji et al., 2000) , the proportion of the native CEACAM1 that interacted with Ha3 compared with C751OpaB was lower and declined further at limiting concentrations of the receptor (Fig. 6A ). In contrast, Ha3 and C751OpaB had similar affinities for the receptor when the Q89N construct was present in excess (> 0.13 mg ml -1 ; Fig. 6B ). Further, at limiting concentrations of Q89N, the greater affinity of Ha3 was apparent and bound this construct threefold more than the Nm derivative (Fig. 6B ). The data demonstrate the potential influence of structural modulations at the bacterial binding site in changing the colonization profile of the target tissue.\nTo assess whether CEACAM mutations that either significantly increase or decrease bacterial binding to the soluble constructs also affect bacterial binding to cellexpressed receptor constructs, we analysed COS cells transiently transfected with CEACAM1-4L containing Q89A, Q89N and I91A substitutions. Initially, binding of the anti-CEACAM antibodies to transfected CHO cells was examined and their binding was as observed for the soluble receptors (Fig. 7) . However, bacterially expressed ligands may overcome decreased affinity for specific mutant proteins due to multiple ligand-receptor engagement at the target cell surface. Examination of adherent bacteria and levels of receptor expression by microscopy revealed that bacteria bound to all transfectants other than sham-transfected cells (Fig. 8) . However, receptor expression levels had to be considerably high in the case of I91A for significant bacterial numbers to bind the transfectants. Whereas for Q89A and Q89N, bacteria could bind to cells with barely detectable levels of receptors (Fig. 8) .\nUsing CHO cells expressing full length CC1 or CC1(Q89N), the ability of the soluble native (CC1-Fc) or CC1(Q89N)-Fc to inhibit bacterial binding was assessed using C751 OpaB bacteria (Fig. 9 ). Data show the following: (i) Binding to CHO cells as reported previously (Virji et al., 1996b) was observed only with Opa+ and not Opa-bacteria and no binding was seen with untransfected cells (not shown), (ii) Despite the low levels of binding of the isolate to the soluble CC1 construct carrying the Q89N mutation in the dot immunoblot analysis (Fig. 3B) , bacterial binding to the receptor expressed on the cells was clearly visible (Fig. 9D) , (iii) As in the case of THi Rd described above, association was only significantly high with cells expressing high levels of the receptor (assessed in parallel experiments, not shown), (iv) CC1-Fc could compete with bacterial binding to the homologous native structure expressed on CHO cells (Fig. 9B) , confirming previous results (Virji et al., 1996b) , (v) In comparison, CC1-Fc could more efficiently compete out C751 OpaB binding to the CC1(Q89N) receptor expressed on CHO cells (Fig. 9E) , consistent with its higher affinity for the isolate than CC1(Q89N)-Fc (Fig. 3B) , (vi) CC1(Q89N)-Fc construct was largely ineffective in competing with either cell-expressed receptor ( Fig. 9C and F) ent bacterial numbers are thus also low (cf. Fig. 9D and F: lack of low level binding in F compared with D). The data emphasize the importance of cell presentation of receptor in addition to receptor structure in bacterial interactions.\nThe final outcome of this depends on the balance between the effects exerted by residue substitutions and receptor density. Both can affect functional affinity of bacteria-host interactions. \nTo assess the relative importance and the degree of influence of each substitution on bacterial interactions, degrees of receptor recognition were assigned several categories as shown in Fig. 10A . The data show that substitution I91A has a profound effect on the interactions of all strains and whilst the side chain of Ile-91 is best suited to all, Leu can effectively substitute for Ile for several strains, whilst the polar Thr is less well tolerated. However, some Nm Opa proteins can also tolerate I91T substitution. Tyr-34 is also required in most cases. Interestingly, Phe-34 is preferred in general at this position over the native Tyr. Substitutions at Gln-89 have a delete- For the native receptor, expression levels and bacterial binding correlated more frequently than for the mutated molecules. In the case of substitutions at position 89, bacterial binding could be seen at very low levels of receptor expression (arrows in Q89A and Q89N panels). The reverse was the case with I91A, where even at high receptor levels (arrowhead, left panel), bacterial binding (arrowhead middle panel) was relatively low. In sampling of 100 cells, c. 50% exhibited this phenomenon. Note that the receptor recognition by Kat4C was not affected by mutations in the N-domain as determined by immunoblotting or by immunofluorescence microscopy (Fig. 7) .\nrious effect on binding of some Nm Opa derivatives and the smaller Q89N apparently is better suited overall for binding by Hi and especially Hi-aeg strains. The data emphasize the extensive inter-and intraspecies ligand variations in this receptor targeting, perhaps with greater interstrain differences in Nm. Structural aspects of bacterial ligands that affect the receptor recognition can be considered for strain C751 Opa proteins whose structures are known (Fig. 9B ) (Hobbs et al., 1994) . The hypervariable loops HV1 and HV2 of Opa proteins have been implicated in the binding of CEACAMs (Virji et al., 1999; Bos et al., 2002; de Jonge et al., 2003) . Given that the combinations of HV regions of OpaA, B and D of strain C751 provide three distinct combinations (Fig. 10C) , this is consistent with the distinct patterns of targeting of the variant receptor molecules observed in the study. However, the proteins must all contain sufficient similarity to bind the hydrophobic region around I91.\nThe N-terminal domains of the cell-expressed CEA family of molecules are highly homologous and the majority of CEACAMs are targeted by one or more pathogenic neisserial adhesins belonging to the Opa family of proteins (Virji et al., 1996a; Chen et al., 1997; Gray-Owen et al., 1997) . Despite the structural variability, all Opa proteins target a common site on the receptors whose centre of binding appears to be Ile-91 on the CFG face of the N-domains. Ile-91 is conserved throughout the CEA members. Most of the other important residues on CEACAM1 identified by alanine scanning mutagenesis located in a close proximity of Ile-91 (Virji et al., 1999) . Precisely how variant Opa proteins can bind to a common receptor site is not entirely clear but a complementary set of sequences of more than one variable domains of Opa proteins may be involved. This variability of the ligands may determine the binding preference for distinct members of the CEA family which represents one mechanism that may determine tissue tropism (Virji et al., 1999; Bos et al., 2002; Gray-Owen, 2003; Hauck and Meyer, 2003; de Jonge et al., 2003) .\nDiverse strains of typable and NTHi lineages including the biogroup aegyptius also bind to the CFG face of the N-domain of CEACAM1 (Virji et al., 2000) . In our previous analysis of receptor binding, THi isolates were shown to behave similarly: each strain tested having a primary requirement for Ile-91 and in addition, was affected by Y34A and Q44A substitutions. In the current study also, the THi strain Rd and Eagan demonstrate similar overall binding patterns. However, in all cases, intraspecies dif- Fig. 9 . Binding of N. meningitidis isolate C751OpaB to cell-expressed receptors: competition between cell-expressed and soluble receptors. Bacterial binding to cell-expressed CC1 or Q89N construct was detected using anti-Nm antisera and TRITC-conjugated secondary antibodies. The interactions of bacteria with the cell-expressed receptors were investigated in the absence (A, D) or presence of competing soluble receptors. Although binding to the soluble receptor Q89N is much lower than the native CC1 in dot-blots (Fig. 3B) , it is relatively high on certain cells (presumably, on those expressing high levels of the receptor) (D). In competitive experiments, the native CC1-Fc (B, E) and the CC1(Q89N)-Fc (C, F) were preincubated at 10 ug ml -1 with bacteria for 15 min, prior to infection of target cells. CC1-Fc inhibited bacterial binding to the homologous receptor significantly (B), and almost abrogated binding to cell-expressed Q89N (E); whereas the soluble Q89N was inefficient at inhibiting bacterial binding to CHO-CC1 (C). However, a level of homologous inhibition was apparent when examining the adhesion of bacteria to cells expressing low levels of the receptor (e.g. 'peppered' areas shown in D are less evident in F).\nCHO-CC1(Q89N)\nferences in response to different amino acid substitutions were apparent. One consistency between all species and strains tested was the dramatic loss of binding following I91A substitution which, as observed previously, appears to be central in an overlapping bacterial binding footprint on CEACAM1 (Virji et al., 1999; . A similar situation occurs on the mouse CEACAM1a (MHVR1a) N-terminal domain, in which Ile-41 appears to engage with the mouse hepatitis virus spike protein. Replacement of Ile-41 by Thr in the MHVR1b allele reduces virus binding significantly (Tan et al., 2002) . Cell surface receptor interactions with their ligands often involve hydrophobic contact points which provides the major binding energy. Hydrophobic residues surrounding these contribute to the specificity of binding (Clackson and Wells, 1995; Kwong et al., 1998; Kim et al., 2001) . Thus in murine CEACAM1, the protruding Ile-41, which is surrounded by a number of surface exposed charged residues, e.g. Asp-42, Glu-44, Arg-47, Asp89, Glu-93 and Arg-97, might form such a binding area (Tan et al., 2002) . Accordingly from current studies also, mutations introduced at position 91 in the human receptor support the requirement for a hydrophobic pocket at this site. In addition, an extended aliphatic chain is preferred as Ala disrupted binding of all bacteria and the polar Thr reduced binding of all Hi and several Nm strains. Only I91L was more frequently tolerated. This binding pocket is flanked by several polar residues (Fig. 1 ) whose contribution to bacterial ligand binding is apparent and variable (Virji et al., 1999; . The importance of the Ile-91 and the surrounding residues on human CEACAM1 in bacterial binding is also supported by the observation that the mAb YTH71.3 directed against the N-domain also requires several residues in common with bacteria and blocks binding of all CEACAM1-binding bacteria we have investigated (Virji et al., 1999; Hill and Virji, 2003) .\nThe murine N-domain strand arrangement derived form crystal structure depicts the CC′ loop to assume a convoluted conformation. The previous model of human CEACAM1 contained a flat CC′ loop like the Ig-folds on Fig. 10 . Binding of bacterial ligands to CEACAM1-Fc constructs as determined in the current study. A. Receptor recognition categories are colour coded according to percent binding relative to the native molecule*. B and C. Diagrams showing CEACAM-binding meningococcal ligand structures. As only the Opa protein structure has been analysed in details so far with respect to CEACAM binding (de Jonge et al., 2003) , for clarity and ease of discussion, a 2D Opa protein structure (B) and relationship of the three variable domains of strain C751 Opa proteins are shown (C). SV, semivariable domains of strain C751 Opa-A, -B and -D are identical as are the hypervariable structures HV1 of OpaA and OpaB and the HV2 of OpaB and OpaD. which it was based. This resulted in Val-39 and Gly-41 being located at a distance from the binding focus Ile-91. Both Val-39 and Gly-41 have been implicated in bacterial binding from alanine scanning and homologue scanning mutagenesis (Bos et al., 1999; Virji et al., 1999) . The remodelling shown here of CEACAM1 produces the convoluted structure of the CC′ loop relocating Val-39 close to Ile-91. Further, the aromatic ring of Tyr-34 suggested to be required to maintain the convoluted structure of the CC′ loop (Tan et al., 2002) when substituted with Phe almost always supported bacterial binding. Indeed, Y34F provides a better environment for most bacterial strains tested. On the other hand, Y34A frequently abrogated receptor recognition. However and surprisingly, Y34S is tolerated by a substantial number of bacterial ligands. Whether this is due to the flexible variable loop domains of the bacterial ligands which may produce an induced fit around the receptor needs consideration. Interestingly, Tyr-34 is conserved in the majority of the human CEACAMs with the exception of CEACAM4 which contains His at this site. However, the importance of Tyr-34 in human CEACAM1 maintaining the three dimensional structure requires human receptor crystallographic data.\nSubstitutions Y34F and Q89N also produced interesting data from the point of view of pathogenesis. Whilst Q89A and Q89N appear to affect some Opa proteins by reducing receptor recognition, suggesting its potential contribution in determining tissue tropism (Virji et al., 1999) , Q89N substitution occasionally caused dramatic increase in bacterial adhesion, especially of Hi isolates. Y34F, as observed above, increased binding of strains within all species examined. As it is possible that multiple receptors presented on the target cells may overcome the reduced binding affinity of mutated receptors, we examined strain Rd binding to I91A and Q89A/N-substituted receptors expressed in transiently transfected COS cells. Whilst the latter receptors were targeted on cells with low levels of receptor expression, only a proportion (c. 50%) of cells with very high levels of I91A receptors had significant numbers of bacteria attached. Thus point mutations of CEACAMs can both decrease and increase bacterial load and additional factors that dictate bacterial binding include receptor levels on the target cells.\nThe ligands of Neisseria (i.e. Opa proteins) and of Hi so far identified (i.e. P5 proteins) share similar beta-barrel structure with surface-exposed variable loops. The regions of P5 that may engage with the receptor have not been identified but those of Opa proteins were studied by mutagenesis of strain H44/76 (de Jonge et al., 2003) . This strain is related to strain MC58, one of the strains used in the current study. The studies of de Jonge et al. implicated G ¥ (I/V/l) ¥ (S/E/Q) as the key motifs of HV2 regions (Fig. 9 ) of meningococcal Opa proteins in receptor targeting. Together with this, an 99 ELK motif of the Opa HV1 region might be involved in the three dimensional presentation of the receptor-engaging residues of the bacterial ligand (de Jonge et al., 2003) . Within the strain C751, OpaB and OpaD proteins contain the motif GxLxS at positions 172-176 and 167-171, respectively, whereas Opa A contains a 168 PxIxN motif. In the HV1 region, OpaA and B contain 99 DLK whereas OpaD contains EDK. Studies in our laboratories are in progress to assess the precise variant C751 Opa and receptor residue pairs involved in mutual recognition.\nPolymorphisms that affect host susceptibility may be found at various sites in the genes encoding host receptors targeted by pathogens and may result in loss or gain of receptor-associated functions. Some SNPs may lead to multiple and diverse downstream effects, e.g. altered transcriptional response and manifestation of disease (Sakuntabhai et al., 2005) . Extracellular domain polymorphisms may have a more direct effect via altered binding of pathogen ligands to their receptors. SNPs of the innate immune system especially those affecting pathogen associated pattern recognition receptors and cytokines have been studied extensively. Changes such as Asp299Gly and Thr399Ile in the extracellular domains of LPS-binding Toll-like receptor 4 (TLR4) have been implicated in increased risk to bacterial infections (Schroder and Schumann, 2005) . These SNPs have also been associated with severe respiratory syncytial virus (RSV) bronchiolitis in infants. In this case, altered interaction of the viral fusion (F) protein, implicated as a ligand for TLR4, is regarded as the primary mechanism (Tal et al., 2004; Schroder and Schumann, 2005) . However, these TLR4 SNPs could not be correlated with meningococcal disease (Read et al., 2001) . In contrast, rare SNPs were found more commonly in the TLR4 genes of patients with meningococcal disease (Smirnova et al., 2003) . This supports the notion that rare rather than common variants of TLR4 may be associated with infectious disease susceptibility.\nStudies presented here suggest some possible polymorphisms that can increase bacterial load. Several SNPs in CEACAMs have been identified and are listed in the NCBI SNP database and three have been identified in the N-domain of CEACAM1 including one at Gln-89. In this case, a Gln-89 to His substitution is observed. Such a residue difference occurs within the members of CEACAM family. For example, CEA contains H at position 89 (Fig. 2) . This is the only major difference between CC1 and CEA that could affect bacterial binding (Fig. 2) . As such, the mutation Q89H in CC1 would be expected to produce CEA-like binding pattern and could affect tropism of the bacteria as observed for CEA (Virji et al., 1999; de Jonge et al., 2003) . Further studies are required to assess whether other CEACAM polymorphisms, for example, substitutions such as Y34F and Q89N occur in human Molecular analysis of bacterial ligand-CEACAM1 interactions 341 populations and their frequencies in susceptible populations. SNP substitutions may also change the colonization profile of the nasopharynx, because in our competition studies we could demonstrate that increased binding afforded by Q89N to Hi-aeg isolate Ha3 increases the binding of Ha3 such that it out-competes Nm isolate C751OpaB in an in vitro competition assay for this receptor. The situation with the native receptor was the reverse. As shown in Figs 8 and 9, additional factors that affect bacterial binding to cell-expressed receptor include receptor density. Whilst certain residue substitutions, e.g. I91A, reduce functional affinity of bacterial interactions, high receptor densities increase such affinity. The final outcome must depend on the interplay between these two parameters. In recent studies, the role of receptor density on enhancement of bacterial attachment and invasion have been evaluated in detail (Bradley et al., 2005; Rowe et al., 2006) . It would be interesting to analyse bacterial invasion in cell lines expressing variant CEACAM1 carrying the above mutations by employing cell lines in which the receptor expression levels can be controlled, which are under development.\nBesides receptor polymorphisms, several other scenarios may lead to increased bacterial ligand binding to CEACAMs. Both Nm and Hi CEACAM-binding ligands (Opa and P5) are known to undergo antigenic variation. Thus, in any population, antigenic/structural variants are present and may be selected for during the course of host colonization and subsequent pathogenesis (Virji et al., 1996a; Duim et al., 1997; Meyers et al., 2003) . The receptor repertoire and subtypes may select bacteria capable of binding with high affinity. As mentioned above, upregulation of receptor expression on target cells may also increase bacterial binding affinity. In such cases, high affinity interactions result in cellular invasion, whereas lower affinity or load of bacteria may not proceed beyond surface adhesion (Tran Van Nhieu and Isberg, 1993; Bradley et al., 2005) . The expression of CEACAMs on normal epithelia of the respiratory tract has been reported, which would allow bacterial attachment and possible subsequent penetration into these tissues (Tsutsumi et al., 1990; Virji, 2001) . Following exposure to cytokines such as IFN-g or TNF-a, CEACAM expression by colonic carcinoma cells has been shown to increase (Fahlgren et al., 2003) . In addition, certain viral infections have also been shown to upregulate CEACAM1 expression in several epithelial cell lines (Avadhanula et al., 2006) . Increased cytokine levels following viral infection could lead to increased CEACAM expression and bacterial association with respiratory epithelia and subsequent invasion of deeper tissue by these organisms. Such a situation may explain the epidemiological association of increased incidence of Nm and Hi infections following certain viral infections (Cartwright et al., 1991; Takala et al., 1993) .\nIn summary, little is known about why certain people are more susceptible to infection by some of the frequent colonizers of the human nasopharynx. Interestingly, opportunistic pathogens such as Nm and Hi as well as M. catarrhalis (not investigated here) target CEACAM1. As specific substitutions such as Y > F and Q > N produce more favourable targets for distinct mucosal isolates, it is possible that occurrence of such receptor polymorphisms in the human population could lead to greater bacterial binding thus increasing the chances of cellular invasion. Given the colonization rate of these organisms (generally > 10% of the population) and the frequency of invasive infection (up to 3:100 000 population), a combination of events may be required to increase host susceptibility. Inflammatory conditions that increase receptor density in populations carrying specific polymorphisms could provide the worst scenario.\nThe sequence of mature human CEACAM1 N-domain was aligned with the corresponding domain of murine CEACAM1, giving a gapless alignment for residues 1-109 with a residue identity of 42%. The crystal structure co-ordinates of mouse CEACAM1 (residues 1-109) were taken from the structure file containing domains 1 and 2 (PDB code 1L6Z) and a homology model of human CEACAM1 N-domain was built using standard methods. Final refinement of the model was performed by soaking it with a 5 Å thick layer of water and energy minimizing while constraining the backbone atoms to their original positions in the template structure. The final round of minimization was for 2000 conjugate-gradient steps, constraining the backbone heavy atoms with a force constant of 0.5 kcal/Å. A stereochemical analysis of the structure was performed using Procheck and found to be of similar quality to the template crystal structure.\nProduction of mutants was carried out by site-directed mutagenesis of the pIG construct containing the DNA encoding the CEACAM1 NA1B domains described previously (Watt et al., 1994) . The oligonucleotide primers used to create amino acid substitutions at positions 91, 34 and 89 of the N-domain are shown in Table 1 . Some primer sequences have been published previously (Watt et al., 1994) . For introducing mutations, CEACAM1 was amplified by polymerase chain reaction from the pIG-NA1B construct using either the common forward primer and a reverse primer containing the desired mutation, or a complementary forward primer containing the mutation and a common reverse primer. CEACAM1 with the appropriate mutation was amplified using the common forward and common reverse primers. The gene was then cloned into pIG using the restriction sites HindIII and EcoRI.\nChimeric soluble receptor proteins containing the appropriate amino acid substitutions were prepared as previously described by transient transfection of COS cells (Teixeira et al., 1994; Virji et al., 1999) . The CEACAM1-Q89A-Fc used in overlay experiments was kindly donated by Dr S. Watt (Virji et al., 1999; Watt et al., 2001) .\nThe strains used in this study have been described previously (Virji et al., 1999; . Nm strain C751 (serogroup A) variants used were C751OpaA, C751OpaB and C751OpaD. The strain MC58 (serogroup B) variant used expressed an Opa previously designated OpaX, which is encoded by the opaB locus. Opa -C751 isolate, which has been shown not to bind to CHO-CC1 (Virji, 1999) and RdCC-, a derivative of THi Rd, known not to bind to CHO-CC1 (M. Virji, unpublished) were used as controls. THi strain Rd is an acapsulate serotype d isolate, Eagan is serotype b isolate and A950002 is a NTHi strain. Hi-aeg strains Ha3, Ha30 and F2087 are all conjunctiva isolates. Nm was grown on brainheart infusion (BHI) agar supplemented with 10% heated horse blood (HBHI). Hi strains were grown on HBHI agar further supplemented with Levinthal base (10 mg ml -1 each of NAD and haemin). All strains were cultured at 37°C in 5% CO2.\nCOS-1 cells (African green monkey kidney cells) used for transient transfection were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 2-10% heat-inactivated Foetal Calf Serum (FCS, Gibco™), 2 mM glutamine, 50 mg ml -1 penicillin and 50 mg ml -1 streptomycin in a humidified atmosphere of 5% CO2 at 37°C.\nAntibody binding to CEACAM1 constructs. NA1B-Fc proteins were dotted at 0.2 mg ml -1 on to nitrocellulose and non-specific binding sites blocked using 3% (w/v) BSA in Dulbecco's PBS containing 0.05% Tween-20 (PBST) for 1 h at room temperature. Receptor was detected using the following antibodies, rabbit polyclonal AO115, rat monoclonal YTH71.3 both directed against the N-domain and mouse monoclonal Kat4c which recognizes the A and B domains (Jones et al., 1995) . Bound antibody was subsequently detected using an appropriate secondary antibody conjugated to alkaline phosphatase and developed using nitroblue tetrazolium and 5-bromo-4-chloro-3-indolylphosphate.\nBacterial interactions with receptor constructs. Bacterial lysates (c. 2 ¥ 10 7 bacteria) were applied to nitrocellulose strips, air-dried and non-specific binding sites blocked using 3% BSA in PBST for 1 h at room temperature. Strips were overlaid with either native or mutated soluble NA1B-Fc diluted in 1% BSA in PBST at required concentrations for 1 h at room temperature. In most experiments, excess (1-3 mg ml -1 ) of the receptor was used. In competition studies, a range of concentrations (0.008-0.5 mg ml -1 ) was employed. Following washing to remove unbound NA1B-Fc, receptor binding was detected using anti-human-Fc alkaline phosphatase conjugate and substrates as described above. For quantification, densitometric analyses of the developed immunoblots were carried out using NIH Scion Image software. In most cases, multiple estimations were carried out and means and SE of each determination have been reported.\nSite directed mutagenesis of CEACAM1-4L receptor gene was performed using the QuickChange® Site Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA) according to the manufacturer's instructions. Primers (Table 1) were used to introduce the desired mutations into the pRc/CMV-CEACAM1-4L construct (kindly provided by Professor Wolfgang Zimmermann). Following sequencing to ensure the desired substitution had been obtained, the pRc/CMV-CEACAM1-4L construct was transiently transfected into COS-1 cells for functional analysis using DEAE dextran method described previously (Teixeira et al., 1994; Virji et al., 1996a) . " }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: Since its introduction, ART has led to significant declines in AIDS-associated morbidity and mortality [1] . These benefits are, in part, a result of partial recovery of the immune system, manifested by increases in CD4 + T-lymphocyte counts and decreases in plasma HIV-1 viral loads [2] . After initiation of ART, opportunistic infections (OI) and other HIV-related events still occur secondary to a delayed recovery of adequate immunity [3] .\nSome patients initiating ART experience unique symptoms during immune system recovery. In these patients, clinical deterioration occurs despite increased CD4 + Tlymphocyte counts and decreased plasma HIV-1 viral loads [4] . This clinical deterioration is a result of an inflammatory response or \"dysregulation\" of the immune system to both intact subclinical pathogens and residual antigens [5] [6] [7] [8] [9] . Resulting clinical manifestations of this syndrome are diverse and depend on the infectious or noninfectious agent involved. These manifestations include mycobacterial-induced lymphadenitis [5] , paradoxical tuberculosis reactions [6, 7, 10, 11] , worsening of progressive multifocal leukoencephalopathy (PML) [12] , recurrence of cryptococcosis and Pneumocystis jirovecii pneumonia (PCP) [8, [13] [14] [15] [16] , Cytomegalovirus (CMV) retinitis [17] , shingles [18] , and viral hepatitis [19] , as well as noninfectious phenomena [20] .\nBecause clinical deterioration occurs during immune recovery, this phenomenon has been described as immune restoration disease (IRD), immune reconstitution syndrome (IRS), and paradoxical reactions. Given the role of the host inflammatory response in this syndrome, the term immune reconstitution inflammatory syndrome (IRIS) has been proposed [21] and has become the most widely used and accepted term to describe the clinical entity. Possible infectious and noninfectious etiologies of IRIS are summarized in Table 1 .\nTo date, no prospective therapeutic trials concerning the management of IRIS have been conducted. All evidence regarding the management of IRIS in the literature relates to case reports and small case series reporting on management practice. This does not provide reliable evidence regarding either the safety or efficacy of these approaches, but merely guidance regarding the practice of others in managing this difficult condition. In severe cases where the discontinuation of ART is a possibility, the potential disadvantages of therapy cessation, such as the development of viral resistance or AIDS progression, should be considered.\nDespite numerous descriptions of the manifestations of IRIS, its pathogenesis remains largely speculative. Current theories concerning the pathogenesis of the syndrome involve a combination of underlying antigenic burden, the degree of immune restoration following HAART, and host genetic susceptibility. These pathogenic mechanisms may interact and likely depend on the underlying burden of infectious or noninfectious agent.\nWhether elicited by an infectious or noninfectious agent, the presence of an antigenic stimulus for development of the syndrome appears necessary. This antigenic stimulus can be intact, \"clinically silent\" organisms or dead or dying organisms and their residual antigens. IRIS that occurs as a result of \"unmasking\" of clinically silent infection is characterized by atypical exuberant inflammation and/or an accelerated clinical presentation suggesting a restoration of antigen-specific immunity. These characteristics differentiate IRIS from incident opportunistic infections that occur on ART as a result of delayed adequate immunity.\nExamples of IRIS in response to intact organisms include, but are not limited to, the unmasking of latent cryptococcal infection [22] and infection with Mycobacterium avium complex (MAC) [4, 5, 23, 24] . The most frequently reported IRIS symptoms in response to previously treated or partially treated infections include reports of clinical worsening and recurrence of clinical manifestations of Mycobacterium tuberculosis (TB) and cryptococcal meningitis following initiation of ART [6, 7, 10, 13, 16, [25] [26] [27] [28] . In noninfectious causes of IRIS, autoimmunity to innate antigens plays a likely role in the syndrome. Examples include exacerbation of rheumatoid arthritis and other autoimmune diseases [29] . Given the role of this antigenic stimulus, the frequency and manifestations of IRIS in a given population may be determined by the prevalence of opportunistic and non-opportunistic infections to initiation of ART.\nThe mechanism receiving the most attention involves the theory that the syndrome is precipitated by the degree of immune restoration following ART. In assessing this theory, investigators have examined the association between CD4 cell counts and viral loads and the risk of IRIS. Some studies suggest differences in the baseline CD4 profiles or quantitative viral load at ART initiation or their rate of change during HAART between IRIS and non-IRIS patients [4, [30] [31] [32] [33] [34] , while other studies demonstrate only trends or no significant difference between IRIS and non-IRIS patients [7, 35] . These immunological differences between groups have been difficult to verify due to small numbers of IRIS cases and lack of control groups. An alternative immunological mechanism may involve qualitative changes in lymphocyte function or lymphocyte phenotypic expression. For instance, following ART an increase in memory CD4 cell types is observed [36] possibly as a result of redistribution from peripheral lymphoid tissue [37] . This CD4 phenotype is primed to recognize previous antigenic stimuli, and thus may be responsible for manifestations of IRIS seen soon after ART initiation. After this redistribution, naïve T cells increase and are thought to be responsible for the later quantitative increase in CD4 cell counts [38] . These data suggest IRIS may be due to a combination of both quantitative restoration of immunity as well as qualitative function and phenotypic expression observed soon after the initiation of ART.\nThe third purported pathogenic mechanism for IRIS involves host genetic susceptibility to an exuberant immune response to the infectious or noninfectious anti-genic stimulus upon immune restoration. Although evidence is limited, carriage of specific HLA alleles suggest associations with the development of IRIS and specific pathogens [39] . Increased levels of interleukin-6 (IL-6) in IRIS patients may explain the exuberant Th1 response to mycobacterial antigens in subjects with clinical IRIS [9, 40] . Such genetic predispositions may partially explain why manifestations of IRIS differ in patients with similar antigenic burden and immunological responses to ART.\nDespite numerous descriptions of the infectious and noninfectious causes of IRIS, the overall incidence of the syndrome itself remains largely unknown. Studies to date are often retrospective and focus on specific manifestations of IRIS, such as tuberculosis-associated IRIS (TB-IRIS). In a large retrospective analysis examining all forms of IRIS, 33/132 (25%) of patients exhibited one or more disease episodes after initiation of ART [4] . Other cohort analyses examining all manifestations of IRIS estimate that 17-23% of patients initiating ART will develop the syndrome [32] [33] [34] . Another large retrospective study reported 32% of patients with M. tuberculosis, M. avium complex, or Cryptococcus neoformans coinfection developed IRIS after initiating ART.\nRisk factors identified for the development of IRIS in one cohort included male sex, a shorter interval between initiating treatment for OI and starting ART, a rapid fall in HIV-1 RNA after ART, and being ART-naïve at the time of OI diagnosis [31] . Other significant predictors have also included younger age, a lower baseline CD4 cell percentage, a lower CD4 cell count at ART initiation, and a lower CD4 to CD8 cell ratio at baseline [4, 32] . It should be noted cohorts differ substantially in study populations and the type of IRIS (i.e. TB-IRIS only) examined, making conclusions regarding risk factors for IRIS difficult. Clinical factors associated with the development of IRIS are presented in Table 2 .\nCase reports describing different clinical manifestations of IRIS continue to appear, expanding the clinical spectrum of the syndrome. Because the definition of IRIS is one of clinical suspicion and disease-specific criteria have yet to be developed, determining the true incidence will be difficult. Taken together, these studies suggest IRIS may affect a substantial proportion of HIV patients initiating ART. Future epidemiologic and genetic studies conducted within diverse cohorts will be important in determining the importance of host susceptibility and underlying opportunistic infections on the risk of developing IRIS.\nIn order to aid clinicians in the management of IRIS, we review the epidemiology, clinical features, and treatment options for the common infectious manifestations of IRIS. Additionally, manifestations associated with significant morbidity and mortality, such as CMV-associated immune recovery vitritis (IRV) or immune recovery uveitis (IRU), are also reviewed. Treatment options and their evidence are presented. Until disease specific guidelines are developed for IRIS, therapy should be based on exist- [4, 6, 7, 10, 11, 26, 30-32, 41, 43, 45] Rheumatoid arthritis [29] Systemic lupus erythematosus (SLE) [91] Graves disease [92] , Autoimmune thyroid disease [93] Mycobacterium avium complex [4, 5, 23, 31, [94] [95] [96] Sarcoidosis & granulomatous reactions [20, 97] Other mycobacteria [4, 56, 57, 98, 99] Tattoo ink [100] Cytomegalovirus [4, 33, 61, 63] AIDS-related lymphoma [ [112] Molluscum contagiosum & genital warts [32] Sinusitis [113] Folliculitis [114, 115] ing evidence and individualized according to the severity of presentation.\nMycobacterium tuberculosis (TB) is among the most frequently reported pathogen associated with IRIS. Narita et al performed the first prospective study to evaluate the incidence of paradoxical responses in patients on TB therapy and subsequently initiated on ART. Of 33 HIV/TB coinfected patients undergoing dual therapy, 12 (36%) developed paradoxical symptoms [7] . The frequency of symptoms in this group were greater than those observed in HIV-infected controls receiving TB therapy alone, supporting the role of an exaggerated immune system response in the pathogenesis of the syndrome. Retrospective studies corroborate the finding that a significant proportion of HIV/TB coinfected patients undergoing HAART have symptoms consistent with IRIS, with estimates ranging from 7-45% [10,26, 30, 35, [41] [42] [43] .\nThe association between a shorter delay between TB treatment initiation and ART initiation is an area of debate. While some investigators have found no difference in time from TB therapy to initiation of ART between IRIS and non-IRIS subjects [30] , others have reported a significant differences between groups [31, 35] . In general, IRIS occurred in subjects initiated on ART within two months of TB therapy initiation [35] . Based on these and other data, a decision analysis on ART initiation timing in TB patients found the highest rates of IRIS occurred in patients initiated on ART within two months of TB therapy initiation [44] . However, withholding or deferring ART until two to six months of TB therapy was associated with higher mortality in scenarios where IRIS-related mortality was less than 4.6%. Future reports from large, prospective observational cohorts may aid in resolving this difficult issue.\nAlthough consisting primarily of case reports [45, 46] , TB-IRIS affecting the central nervous system (CNS) poses a unique problem. As the availability of ART increases in endemic countries, the incidence of CNS TB-IRIS may increase. Thus, clinicians should be vigilant in its diagnosis.\nThe commonest clinical manifestations of TB-IRIS are fever, lymphadenopathy and worsening respiratory symptoms [47] . Pulmonary disorders, such as new pulmonary infiltrates, mediastinal lymphadenopathy, and pleural effusions are also common [7] . Extrapulmonary presentations are also possible, including disseminated tuberculosis with associated acute renal failure [6] , systemic inflammatory responses (SIRS) [48] , and intracranial tuberculomas [45] . Pulmonary TB-IRIS can be diagnosed by transient worsening of chest radiographs, especially if old radiographs are available for comparison. Other symptoms are nonspecific, and include persistent fever, weight loss, and worsening respiratory symptoms. Abdominal TB-IRIS can present with nonspecific abdominal pain and obstructive jaundice.\nIn most studies, TB-IRIS occurs within two months of ART initiation [6, 7, 10, 11, 25, 35, 45, 48] . Among 43 cases of MTB-associated IRIS, the median onset of IRIS was 12-15 days (range 2-114 days), with only four of these cases occurring more than four weeks after the initiation of antiretroviral therapy [7, 10, 25, 26, 30] . These studies suggest the onset of mycobacterial-associated IRIS is relatively soon after initiation of ART, and clinicians should maintain a high level of vigilance during this period.\nParadoxical CNS TB reactions are well described in HIVnegative patients, and include expanding intracranial tuberculomas, tuberculous meningitis, and spinal cord lesions [49] [50] [51] . TB-associated CNS IRIS has also been reported in HIV-positive patients [45, 46, 52] . Compared to non-CNS TB-IRIS, symptoms tend to occur later, usually 5-10 months after ART initiation [45, 50, 52] . Crump et al [45] described an HIV-seropositive patient in who developed cervical lymphadenopathy after five weeks of ART. Five months later, CNS symptoms associated with an expanding intracranial tuberculoma appeared after initiation of antituberculous therapy. The significant morbidity in this case illustrates the importance of maintaining a high clinical suspicion for the disease, particularly in endemic areas.\nTreatment for mycobacterial-associated IRIS depends on the presentation and disease severity. Most patients present with non-life threatening presentations which respond to the institution of appropriate antituberculous therapy. However a range of life threatening presentations, such as acute renal failure [6] and acute respiratory distress syndrome (ARDS) [11] , are described and have significant morbidity and mortality. Morbidity and mortality might also be greater in resource-limited settings where limited management options exist. Since the pathogenesis of the syndrome is an inflammatory one, systemic corticosteroids or nonsteroidal anti-inflammatory drugs (NSAIDS) may alleviate symptoms. In studies where therapy for IRIS was mentioned, the use of corticosteroids was variable [7, 24, 25, 31, 41, 43] and anecdotally effective. Therapies ranged from intravenous methylprednisolone 40 mg every 12 hours to prednisone 20-70 mg/day for 5-12 weeks. These practices reflect the lack of evidence from controlled trials for the use of anti-inflammatory agents in IRIS. A randomized, placebo controlled trial examining doses of prednisone 1.5 mg/kg/day for two weeks followed by 0.75 mg/kg/day for two weeks in mild to moderate TB-IRIS is currently underway in South Africa. Until data become available, it is reasonable to administer corticosteroids for severe cases of IRIS such as tracheal compression due to lymphadenopathy, refractory or debilitating lymphadenitis, or severe respiratory symptoms, such as stridor and ARDS. Interruption of ART is rarely necessary but could be considered in life-threatening situations.\nIn HIV-negative patients, adjuvant corticosteroid use in tuberculous meningitis provides evidence of improved survival and decreased neurologic sequelae over standard therapy alone [53, 54] . Once other infectious etiologies, have been excluded, standard antituberculous therapy should be initiated or continued as the clinical situation dictates, and a course of corticosteroid therapy should be considered for CNS TB-IRIS. Continuation of ART is desirable, although its discontinuation may be necessary in unresponsive cases or in those presenting with advanced neurological symptoms.\nIn addition to TB, atypical mycobacteria are also frequently reported as causative pathogens in IRIS. Early observations involving atypical presentations of Mycobacterium avium-intracellulare (MAC) were first noted with zidovudine therapy [55] . Reports of atypical presentations of both Mycobacterium tuberculosis (MTB) and MAC increased in frequency with the introduction of protease inhibitors and ART. In larger cohorts, MAC remains the most frequently reported atypical mycobacterium [4, 5, 24] . Other atypical mycobacteria rarely associated with IRIS are referenced in Table 1 .\nIn general, MAC-associated IRIS typically presents with lymphadenitis, with or without abscess formation and suppuration [5] . Other less common presentations include respiratory failure secondary to acute respiratory distress syndrome (ARDS) [56] , leprosy [57] , pyomyositis with cutaneous abscesses [23], intra-abdominal disease [58] , and involvement of joints, skin, soft tissues, and spine [58, 59] .\nSeveral studies have characterized the time of onset of Mycobacterium-associated IRIS. In one study of MAC lymphadenitis, the onset of a febrile illness was the first sign of IRIS and occurred between 6 and 20 days after initiation of antiretroviral therapy [5] . In another study, the median time interval from the start of antiretroviral therapy to the development of mycobacterial lymphadenitis was 17 days (range 7-85 days) [24].\nAs with TB-IRIS, evidence for treatment of IRIS due to atypical mycobacteria are scarce. Occasionally, surgical excision of profoundly enlarged nodes or debridement of necrotic areas is anecdotally reported [23, 59] . However, healing is often poor leaving large, persistent sinuses. Needle aspiration is another option for enlarged, fluctuant and symptomatic nodes. Otherwise, treatment is similar to TB-IRIS (see Mycobacterium tuberculosis IRIS -Treatment).\nIn the pre-ART era, CMV retinitis, a vision-threatening disease, carried a high annual incidence and was one of the most significant AIDS-associated morbidities [60] . After the introduction of HAART, Jacobson et al described five patients diagnosed with CMV retinitis 4-7 weeks after ART initiation. They speculated that an HAART-induced inflammatory response may be responsible for unmasking a subclinical infection [17] . In addition to classical CMV retinitis, ART led to new clinical manifestations of the infection, termed immune recovery vitritis (IRV) or immune recovery uveitis (IRU), in patients previously diagnosed with inactive AIDS-related CMV retinitis [61] . Distinct from the minimal intraocular inflammation of classic CMV retinitis, these manifestations exhibit significant posterior segment ocular inflammation thought to be due to the presence of residual CMV antigens or proteins which serve as the antigenic stimulus for the syndrome [62] . Clinical manifestations include vision impairment and floaters.\nIn a retrospective cohort, CMV-related IRIS was common (6/33 of IRIS cases, or 18%) [4] . In prospective cohorts, symptomatic vitritis occurred in 63% (incidence rate 83 per 100 p-yr) of ART responders who carried a previous diagnosis of CMV retinitis but had inactive disease at the onset of antiretroviral therapy. The median time from ART initiation to IRV was (43 weeks) [63] . Another large prospective surveillance study [64] identified 374 patients with a history of CMV retinitis involving 539 eyes. Thirtyone of 176 ART responders (17.6%) were diagnosed with IRU. Male gender, use of ART, higher CD4 cell counts, and involvement of the posterior retinal pole as factors associated with a reduced risk of developing IRU, whereas prior use of intravitreous injections of cidofovir, large retinal lesions, and adequate immune recovery on ART were associated with increased risk.\nThe diagnosis of ocular manifestations of IRIS requires a high level of suspicion. In addition to signs of retinitis, inflammatory symptoms include vitritis, papillitis, and macular edema, resulting in symptoms of loss of visual acuity and floaters in affected eyes. Treatment of IRIS associated CMV retinitis and IRV may involve anti-CMV therapy with gancyclovir or valgancyclovir [17, 65] . However, the occurrence of IRU in patients receiving anti-CMV therapy draws its use into question [64, 66, 67] . The use of systemic corticosteroids has been successful, and IRV may require periocular corticosteroid injections [61, [68] [69] [70] . Due to its significant morbidity and varying temporal presentations, clinicians should maintain a high level of vigilance for ocular manifestations of CMV-associated IRIS.\nWith the introduction of protease inhibitors, increasing rates of herpes zoster were noted in HIV-infected patients. Two studies comparing ART and non-ART patients reported increased incident cases of zoster and rates estimated at 6.2-9.0 cases per 100 person-years, three to five times higher than rates observed in the pre-HAART era [18, 71] . While another study [72] reported no difference in overall incidence between HAART eras (3.2 cases per 100 person-years), the use of HAART was associated with increased odds of developing an incident zoster outbreak (OR = 2.19, 95% confidence interval: 1.49 to 3.20). These studies suggest that ART may play a role in increasing the risk of zoster, which is reflected in large observational IRIS cohorts, where dermatomal varicella zoster comprises 9-40% of IRIS cases [4, 32, 33] . Mean onset of disease from ART initiation was 5 weeks (range 1-17 weeks) [71] , and no cases occurred before 4 weeks of therapy [18] . Both studies identified significant increases in CD8 T cells as a risk factor for developing dermatomal zoster.\nAlthough complications such as encephalitis, myelitis, cranial and peripheral nerve palsies, and acute retinal necrosis can occur in immunocompromised HIV patients, the vast majority of patients exhibit typical or atypical dermatomal involvement without dissemination or systemic symptoms [18, 71, 73] .\nA randomized, controlled trial demonstrated oral acyclovir to be effective for dermatomal zoster in HIV-infected patients, facilitating healing and shortening the time of zoster-associated pain [74] . Its use in cases of varicella zoster IRIS appears to be of clinical benefit [18] . The benefit of corticosteroids in combination with acyclovir in acute varicella zoster has been demonstrated in two large randomized, controlled trials. The combination of corticosteroids and acyclovir decreased healing times, improved acute pain, and quality of life, but did not affect the incidence or duration of postherpetic neuralgia [75, 76] . The incidence of postherpetic neuralgia in immunocompetent individuals does not differ significantly from HIV-infected patients, but increases with increasing patient age [77] . Successful symptomatic management involving opioids, tricyclic antidepressants, gabapentin, and topical lidocaine patches individually or in combination has been shown to be beneficial [78] [79] [80] [81] [82] and should be attempted in HIV patients with postherpetic neuralgia as a complication of herpes zoster IRIS.\nAccurate incidence of C. neoformans-associated IRIS is unknown. It is infrequently reported in overall IRIS cohorts, and many cases appear as single case reports. \nA recent study [90] evaluated antifungal combination therapies in the treatment of C. neoformans meningitis in HIV patients. Although significant log reductions in colony forming units were observed with all combinations, substantial numbers of patients remained culture positive 2 weeks after therapy. It may be important to delay ART until CSF sterility can be achieved with effective antifungal combinations such as amphotericin B and flucytosine. However, the exact timing of ART and whether attaining CSF culture sterility is important in avoiding IRIS is unknown. This is illustrated by cases of reactivation cryptococcal meningitis described in four patients who had received at least four weeks of antifungal therapy prior to ART [13, 22, 83] . It is reasonable to administer systemic corticosteroids to alleviate unresponsive inflammatory effects, as anecdotal benefits have been observed in these patients [21, 84] . Furthermore, serial lumbar punctures may be required to manage persistent CSF pressure elevations in these patients [85, 86] . Although continuation of ART has been performed safely [13, 84] , interruption of antiviral therapy may be necessary in severe or unresponsive cases.\nOther less common infectious etiologies, as well as noninfectious etiologies, are listed in Table 1 . Because these other infectious and non-infectious etiologies are rare, no recommendations exist for their management.\nWhile exact estimates of incidence are not yet available, IRIS in patients initiating ART has been firmly established as a significant problem in both high and low income countries. Because of wide variation in clinical presentation and the still increasing spectrum of symptoms and etiologies reported, diagnosis remains problematic. Fur-thermore, no test is currently available to establish an IRIS diagnosis. Standardized disease-specific clinical criteria for common infectious manifestations of the disease should be developed to: 1) identify risk factors for developing the syndrome and 2) optimize the prevention, management of opportunistic infections. Results of trials addressing the optimal timing and duration of treatment of opportunistic infections will assist in developing guidelines for the prevention and management of IRIS. Treatment of IRIS will remain a clinical challenge due to the variety of clinical presentations and the presence of multiple pathogens capable of causing the syndrome. Until a greater understanding of the syndrome is achieved in different regions of the world, clinicians need to remain vigilant when initiating ART and individualize therapy according to known treatment options for the specific infectious agent. " }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: Influenza viruses cause serious global economic and public health burdens. Annual influenza epidemics resulted in more than 30,000 deaths a year in the United States during 1990-1999 [1, 2] . Periodic pandemics result in significantly higher death tolls. Emergence of new influenza A virus strains can be caused by ''antigenic shift,'' resulting from reassortment of gene segments, including H and/or N types [3, 4] , ''antigenic drift'' resulting from the continuing accumulation of mutations in the H and N genes [5] , or a pathogenic virus jumping species and acquiring the ability to infect and be transmitted among humans, as in the 1918 pandemic [6] . The recent outbreak of highly pathogenic H5N1 avian influenza virus (HPAI), which originated in Southeast Asia and has since spread globally, has resulted in 166 deaths (272 confirmed human cases) as of February 6, 2007 (http://www.who. int/en/). The global emergence of this virus has brought renewed urgency to the effort to track the spread and the evolution of influenza viruses.\nCurrently, rapid methods for influenza virus diagnosis rely on antigen-specific antibody probes [7] , or real-time reverse transcription PCR (RT-PCR) analysis of the matrix (M) gene for identification of the viral species [8, 9] followed by H and N subtype specific RT-PCR assays for determination of the viral subtypes [10, 11] . Since there are many H and N subtypes with significant intra-and inter-subtype sequence variations, these methods do not identify all H and N subtypes, nor are they likely to identify reassortants or newly emerging genetic variants. Further, none of the current surveillance methods provide information relevant to tracking antigenically novel strains that emerge each year or distinguish amongst multiple lineages of influenza viruses that can co-circulate and persist in a popula-tion [12] . Secondary genome sequence comparisons and phylogenetic analyses are necessary to fully understand the multiple lineages of viruses, recognize newly emergent influenza variants, and monitor global spread of these viruses [12, 13] . For instance, analysis of human influenza virus H3N2 sequences from 1999-2004 revealed that at least three major clades of influenza viruses were in circulation after the 2002-2003 influenza season [12] . The differences were due to multiple reassortment events though all shared a common H-gene lineage. Several similar wholegenome studies with avian influenza viruses have revealed the presence of multiple, region-specific sub-lineages of the HPAI H5N1 virus in Southeast Asia that are spreading to Europe and Africa [14] [15] [16] [17] [18] .\nWe have developed a method based on broad-range RT-PCR followed by electrospray ionization mass spectrometry (RT-PCR/ ESI-MS) for rapid and accurate detection of influenza virus, subspecies characterization, and early identification of genetic changes in circulating viruses. This method has previously been applied to detection of other pathogens in human clinical samples [19, 20, 21, 22] , but it has unique capabilities and advantages for influenza surveillance. Here, we show how a highthroughput assay incorporating eight parallel RT-PCR reactions followed by ESI-MS analysis can be used to simultaneously survey for all species of influenza viruses, provide clade-level resolution, identify mixed viral populations in the same sample, detect reassortants, and facilitate monitoring of viral evolution, all integral components of broad influenza surveillance.\nTo measure the breadth of coverage and resolution offered by the panel of primers described in Methods (details in Table S1 ), we tested 92 well-characterized influenza virus isolates collected from human, avian, and animal species. Despite the extensive genetic diversity of this sample set, the broad-range primers generated amplicons from all isolates, and base composition signatures distinguished the isolates (Figure 1 ). Most isolates showed base compositions consistent with expected signatures for the corresponding H/N sub-types based on bioinformatic analysis of existing sequence data. Two of the isolates, however, showed previously unknown base compositions at several primer loci suggesting these might be novel influenza virus types; these are noted as ''Unknown'' in Figure 1 .\nBase composition signatures provide a multidimensional fingerprint of the genomes of the various viruses, which can be used to determine clusters of related species/sub-types. One such representation ( Figure 2 ) shows base composition data derived from the PA, PB1, and NP gene segments analyzed on individual axes. Importantly, only three of the six influenza A primer pairs from Figure 1 are visualized on this three-dimensional plot. There was strong agreement between the bioinformatic analysis of sequence data from GenBank and experimental measurements of base composition signatures from Figure 1 . Human H3N2 and H1N1 viruses clustered independently from each other and from the avian/human H5N1 and H1N1 viruses.\nTo assess the utility of the RT-PCR/ESI-MS assay for surveillance of influenza virus in human populations, we analyzed 656 blinded clinical samples collected over a seven-year period (1999) (2000) (2001) (2002) (2003) (2004) (2005) (2006) . The results were compared with conventional analysis of the same samples by virus culture/serology and real-time RT-PCR methods. Two hundred forty-three samples were influenza positive both by RT-PCR/ESI-MS and conventional assays. Ten samples were positive only by RT-PCR/ESI-MS while eight samples were positive only by culture/real-time RT-PCR, corresponding to approximately 97% sensitivity and 98% specificity. Of the influenza-positive samples, RT-PCR/ESI-MS analysis identified 186 as influenza A virus and 67 as influenza B virus, in complete agreement with conventional typing methods.\nBase composition analysis of multiple RNA segments enables further categorization of isolates into previously established clades determined by sequencing (details shown in Table S2 ). Of the 186 influenza A samples, we determined that 149 of were H3N2 subtype and 34 were H1N1. The subtype of three samples could not be distinguished between H3N2 and H1N2 because these viruses probably arose from a recent reassortment of the H gene from an H1N1 virus with gene segments from an H3N2 virus [12] . Importantly, our predictions agreed completely with serology and direct RT-PCR analysis of the H and N segments from these samples. Nonetheless, although base composition analysis of the PB1, NP, M1, PA, and NS gene segments can be highly predictive of the H and N types, direct analysis of the H and N segments is necessary for unambiguous subtyping due to the potential for viral reassortment.\nIn addition to identification and species typing, RT-PCR/ESI-MS provided a quantitative estimate of the number of viral genome copies in the original patient sample. This was achieved by including a fixed amount (300 copies/well) of an internal RNA calibration standard in each PCR reaction [19] [20] [21] [22] . The genome copy numbers in the influenza samples ranged from low (,100 genome copies per well) to intermediate (100-2,500 copies/well) to high (.3,000 copies per well). Based on the samples within the linear range of the calibration standard, we estimated an average genome load of 750 copies/PCR reaction, representing an average viral load of ,1.5610 4 genomes from material extracted from the original swab or 200 mL of a nasal aspirate. Six of the samples required serial dilutions to obtain viral concentrations in the quantifiable spectrum and ranged from 1.5610 6 to 8.0610 7 genomes/swab. Thus, for influenza-positive patients with respiratory symptoms, we observed a range of five orders of magnitude in viral RNA shedding.\nTo demonstrate the capabilities of RT-PCR/ESI-MS to track the evolution of circulating influenza viruses, we created a tree representation of the H3N2 influenza virus sequences from Genbank ( Figure 3 , black) as described in Methods. The 104 experimentally determined H3N2 base compositions were mapped onto this tree (Figure 3 , blue). Analysis revealed a distribution very similar to the sequence-derived clades of Holmes et al. [12] . The branch terminating in clade A represents the dominant branch between years 1999-2004, with a clade B branch that co-circulates in the same time period. Strikingly, however, clade-A isolates were not detected after this time and Table S3 ) and confirmed the findings described above. A visual display of the most likely relationships among the isolates is shown in Figure 4 . Collectively, these results demonstrate the richness of the genetic information provided by direct RT-PCR/ESI-MS analysis of human clinical materials. Figures 5A and B show the spectra from samples containing a mixture of the ''parent'' BCtype AADFAA and single-nucleotide variations AAHFAA and AADFBB, respectively, providing a snapshot of enduring ''fit'' quasispecies contributors and of potential viral evolution in action.\nThe dynamic range for mixed RT-PCR/ESI-MS detections has previously been determined to be approximately 100:1 [20] , which allows for detection of viral variants with as low as 1% abundance in a mixed population. Figures 5C and 5D show results obtained with a clinical sample containing a mixture of viruses at the limit of detection for mixed populations. To demonstrate that these peaks truly represented mixed viral populations, the PCR amplicons were cloned and 450 independent colonies were sequenced. Nine (2%) of these 450 clones had the predicted mutations, correlating well with the measured amplitude of the low-abundance peaks.\nA total of 293 non-overlapping nucleotides, excluding the primer regions, were analyzed using the genetic loci targeted by the influenza A primers. This corresponds to 2.15% of the influenza A virus genome. Out of 174 human samples analyzed from the 2005-2006 season, only two showed evidence for mixed viral populations at one of the six loci, corresponding to 1.1% of the samples. Thus, assuming the same mutation rate for the broader viral genome as for the region analyzed by PCR/ESI-MS, about 50% of the human H3N2 virus samples would have a mixed population of viruses.\nChoosing amongst the various molecular methods available for pandemic influenza surveillance requires consideration of both practical issues (e.g., broad availability, convenience, cost, and throughput) and scientific issues relevant to public health (e.g., sensitivity, breath of coverage, and the depth and value of the information provided). At one end of the spectrum, a conventional RT-PCR test with specific primers and probes provides a highlyspecific, sensitive, rapid, convenient, quantitative, relatively inexpensive, and high-throughput format that can provide valuable surveillance information. However, these tests are not optimal for surveillance when the exact nature of the pandemic virus is not known. Moreover, without supplemental nucleic acid sequencing, conventional RT-PCR-based tests are not capable of signaling the appearance of new genetic variants, except by potentially demonstrating a loss of sensitivity. Further, a single RT-PCR test can achieve only a single presence/absence analysis limited to the specific target for which it was designed. Discrimination of all known variants of influenza at the level of resolution described here would require hundreds of independent RT-PCR reactions. At the other end of the spectrum, virus isolation using culture methods followed by complete genome sequencing does not require prior knowledge of the virus' sequence and provides cladelevel resolution and highly detailed information regarding virus evolution. Unfortunately, this method is slow, labor intensive, expensive, and low throughput, rendering it ineffective in public health arenas requiring rapid response.\nIn this work we describe a novel method that employs some of the best properties of each of the discussed techniques, and also supplies additional valuable information not provided by those techniques. For example, our method may identify mixed Black font: types determined through sequence analysis; blue font: experimentally determined base composition types; red font: experimentally determined base composition types for season 2005-06. Ten rare sequence types (,1.5%) were not uniquely discernable by the base composition analysis of the eight amplicons used in this analysis, as more than one subtype produced the same BC-type. These BC-types are indicated by asterisks. doi:10.1371/journal.pone.0000489.g003 populations of viruses, either as viral quasispecies as previously illustrated (i.e., development of ''drift'' strains) or co-infections with circulating strains (i.e., potential for development of ''shift'' strains). Recent advances in ESI-MS using bench-top mass spectrometers have enabled analysis of PCR amplicons with sufficient mass accuracy that the nucleotide base composition (the A, G, C, and T count) of the PCR amplicon can be unambiguously determined [23] . The approach outlined here provides two important advantages for surveillance. First, broad-range primers targeted to highly-conserved sites within the influenza virus family that flank highly variable, information-rich regions can be used to amplify sequences from highly diverse viruses in the same assay. The measured base compositions allow identification of the viral species with a high degree of resolution. For this approach, we selected primer pairs targeting the core gene segments (PB1, PB2, PA, M, NS, and NP) that have conserved regions spanning all known influenza viruses and used the resultant base compositions to infer the influenza virus H/N types. In silico analysis of influenza virus sequences from the GenBank database showed that this approach would detect all known influenza viruses and distinguish .90% of all species and types. This overcomes the limitations of directly targeting the H and N segments, which requires specific primer pairs for each known H/N type. Further, since the H/N segments evolve rapidly, newly emerging strains might not be readily detected by the traditional approach.\nSecond, mixed viral populations in the same sample that differ only by a single mutation and is present in as low as 1-2% of the virus population can be identified, providing early insights into viral evolution as an integral component of surveillance. This information-rich result is provided with the same throughput and consumable costs as conventional, sequence-specific RT-PCR assays.\nThe results from 174 influenza A H3N2 samples from the 2005-2006 season in the northern hemisphere were particularly interesting because they revealed viral evolution during a single season. The viruses detected appeared to have been seeded from two of the more abundant BC types circulating during the previous season in the southern hemisphere. The majority (97) of the samples had identical BC types and probably arose from a single founder from the previous season in the southern hemisphere. Most of the remaining samples differed from this founder BC type by one or two additional point mutations within the target regions described here. Surprisingly, when mutations occurred, they became fixed rapidly in the viral population, since only two of the 174 samples from the 2005-2006 season showed evidence of mixed populations. Sequencing revealed that both mutations were silent transitions in third codon positions.\nIn summary, the RT-PCR/ESI-MS method has the capacity to provide rapid diagnosis of human influenza in individual patients with respiratory symptoms, as well as public health surveillance of emerging, potentially pandemic strains, including novel reassortants. The use of RT-PCR/ESI-MS for human as well as avian/ animal surveillance offers the potential for new insights into viral evolution on a scale and at a cost previously not possible. Benchtop mass spectrometers are capable of analyzing complex PCR products at a rate of approximately one reaction product/minute, making the RT-PCR/ESI-MS technology practical for large-scale analysis of clinical specimens or for animal surveillance. Further, as we have demonstrated with influenza viruses, this method provides sensitive detection directly from patient specimens with specificity approximating sequence-level resolution. The ability to quantitate virus shedding, detect low-abundance, mixed infections, and identify new genetic variants without prior knowledge of viral sequence also make this technology ideally suited for monitoring the emergence of drug-resistance mutations during therapy or for identifying newly emerging antigenic variants. Sample processing Viral stock samples consisting of cultured virus or stocks obtained from ATCC were prepared for analysis using the Qiagen QiaAmp Virus kit (Valencia, CA). Both manual (mini spin) kits and BioRobot kits were used. Robotic-based isolations were done on both the Qiagen MDx and Qiagen BioRobot 8000 platforms. Clinical swab samples were stored in Viral Transport Media (VTM). VTM solution (1 ml) was passed over a 0.2 micron filter, which was then subjected to bead beating in a small amount of lysis buffer. The resulting viral lysate was then used following the same protocol as above.\nBased upon analysis of multiple influenza sequence alignments, pan-influenza virus RT-PCR primer sets were developed that were capable of amplifying all three influenza virus species (A, B, and C) and subtypes (HxNy) from different animal hosts (e.g., human, avian, and swine) and distinguishing essential molecular features using base composition signatures. Additional primer pairs were designed that broadly amplified all known members of a particular species, but that did not cross-amplify members of different species (e.g., pan-influenza A and pan-influenza B primers). A surveillance panel of eight primer pairs (Table S1 ) was selected comprising one pan-influenza primer pair targeting the PB1 segment, five pan-influenza A primer pairs targeting NP, M1, PA, and the NS segments, and two pan-influenza B primer pairs targeting NP and PB2 segments. All primers used in this study had a thymine nucleotide at the 59 end to minimize addition of non-templated adenosines during amplification using Taq polymerase [24] . The sensitivity of each RT-PCR primer pair was determined using known quantities of a synthetic calibrant RNA template as described previously [20] . Each of the primer pairs was sensitive to as few as twenty copies of the calibrant RNA and several primers were sensitive to five copies (Table S1 ).\nOne The following RT-PCR cycling conditions were used: 60uC for 5 min, 4uC for 10 min, 55uC for 45 min, 95uC for 10 min, followed by 8 cycles of 95uC for 30 seconds, 48uC for 30 seconds, and 72uC for 30 seconds, with the 48uC annealing temperature increasing 0.9uC each cycle. The PCR was then continued for 37 additional cycles of 95uC for 15 seconds, 56uC for 20 seconds, and 72uC for 20 seconds. The RT-PCR cycle ended with a final extension of 2 min at 72uC followed by a 4uC hold.\nThe RT-PCR products were analyzed using the Ibis T5000 universal biosensor platform (Ibis Biosciences, Inc., Carlsbad, CA; http://www.ibisbiosciences.com), which performs automated post-PCR desalting, ESI-MS signal acquisition, spectral analysis, and data reporting as described previously [23] . Briefly, the steps were as follows: 15 mL aliquots of each PCR reaction were desalted and purified using a weak anion exchange protocol as described elsewhere [25] . Accurate mass (61 ppm), high-resolution (M/dM.100,000 FWHM) mass spectra were acquired for each sample using high-throughput ESI-MS protocols described previously [20] . For each sample, approximately 1.5 mL of analyte solution was consumed during the 74-second spectral acquisition. Raw mass spectra were post-calibrated with an internal mass standard and deconvolved to monoisotopic molecular masses. Unambiguous base compositions were derived from the exact mass measurements of the complementary single-stranded oligonucleotides [26] . Quantitative results are obtained by comparing the peak heights with an internal PCR calibration standard present in every PCR well at 100 molecules [20] .\nTo demonstrate the capabilities of RT-PCR/ESI-MS to track the evolution of circulating influenza viruses, we used a bioinformatic approach to develop a framework on which to display the RT-PCR/ ESI-MS results obtained with H3N2 viruses. Complete genome sequences of all H3N2 human influenza viruses available in GenBank were analyzed. A total of 731 genomes were included, from which we inferred the phylogeny of H3N2 influenza virus since 1996. Using the 565-nucleotide concatenated sequence of the six loci that we analyzed by RT-PCR/ESI-MS, we constructed a nonredundant alignment of 105 sequence types. Base compositions were determined for each genome segment (i.e., locus) analyzed and each unique base composition at each of these loci was assigned a letter according to decreasing number of occurrences (therefore, the letter A represents the most common allele identified at each locus). The concatenation of the six base-composition letters from the PB1, NP, M1, PA, NS1, and NS2 loci from the sequences of 731 H3N2 viruses (1996) (1997) (1998) (1999) (2000) (2001) (2002) (2003) (2004) (2005) available in GenBank yielded 95 six-letter codes referred to as base composition types (BC types). The predominant type is labeled AAAAAA. The topology of the tree was then deduced from the alignment of non-redundant sequences using the programs dnadist and neighbor from the Phylip package (http://evolution. genetics.washington.edu/phylip.html). Since the sequence types differ mostly by discrete single mutations, the original computergenerated tree was then extensively edited using graphics tools to place the labels of intermediate types within the tree itself in lieu of zero-length branches. Readability was further improved by sorting parallel branches chronologically. Table S1 Influenza virus primer pairs used in this study. Genbank reference sequence for each segment is indicated; however, the primer sequences are not identical to the reference sequence as described in the Methods. The limits of detection for each RT-PCR primer pair were determined using known quantities of a synthetic calibrant RNA template. Found at: doi:10.1371/journal.pone.0000489.s001 (0.06 MB PDF) Table S2 Distribution of BC-types observed in influenza A H3N2 positive human respiratory samples. Unique base compositions at each genome segment locus analyzed were assigned letter codes and concatenation of letter codes across the six loci analyzed yielded BC-types. H1N1 samples were not assigned a BC-type. Experimentally determined BC-types (marked RT-PCR/ESI-MS Analysis results) were compared to BC-type signature information of sequences currently available in GenBank and the closest matching strain is shown (right pane). Last column shows comparison to clade designation described in Holmes et al [12] . Found at: doi:10.1371/journal.pone.0000489.s002 (0.24 MB PDF) " }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: The 2001 FMD epidemic in the UK had a substantial cost in human, animal health and economic terms (Alexandersen et al. [1] , Kao [2] ). Understanding the risk factors underlying the transmission dynamics of that epidemic and evaluating the effectiveness of the control measures are essential to minimise the scale and cost of any future outbreak. Epidemic modelling [3, 4, [5] [6] [7] proved critical to decision making about control policies which were (in some cases controversially) adopted to control the 2001 epidemic [8] [9] [10] . Modelling now has a 'peace-time' contingency planning role.\nOne weakness of the modelling studies undertaken in 2001 was the relatively ad-hoc nature of the parameter estimation methods employed. In their first paper, Ferguson et al. [4] used maximum likelihood methods to fit to the observed incidence time series, but did not attempt to fit to the spatio-temporal pattern of spread. In their later work, the same authors developed a more robust method for estimating species-specific susceptibility and infectiousness parameters and spatial kernel parameters (see Supplementary Information to [3] ), but at the time the statistical basis for the methods developed was lacking. In retrospect, the methods developed turned out to be closely related to those developed during the SARS epidemic by Wallinga and Teunis [11] , although the earlier work incorporated population denominator data to allow for spatial-and species-based heterogeneity in disease transmission. Nevertheless, the methods employed had the limitation of not being fully parametric, meaning they could not be extended to fit arbitrary transmission models to the observed data. Keeling et al. [5] used maximum likelihood methods to estimate transmission parameters, but it was also supplemented by more ad hoc least-squares matching to regional incidence time series.\nTherefore there remains a need to develop rigorous modern statistical approaches for parameter estimation of non-linear models for the 2001 FMD outbreak. Bayesian Markov Chain Monte Carlo (MCMC) techniques are the best established such methods and have been successfully employed in the analysis of a range of spatiotemporal outbreak data in the past [12] [13] [14] , as well as to purely temporal incidence data [15, 16] . Here we develop MCMC-based inference models for the 2001 FMD epidemic in GB. The models examine: the extent to which transmission was spatially localised and the temporal variation in transmission, species-specific variation in susceptibility, infectious-ness and heterogeneity in contact rates between and within species.\nWe take the farm as the unit of our study and ignore the possible impact of within-farm epidemic dynamics. Thus we implicitly assume disease spread within a farm is so rapid as to be practically instantaneous, with all animals on a farm becoming infectious at the same time.\nOur data consists of information on all the farms in the UK listed in the 2000 agricultural census [see http://www.defra.gov. uk/footandmouth/cases/index.htm ]. There were a total of 134,986 farms listed in that dataset and uniquely identified by their County/Parish/Holding (CPH) number. Their spatial coordinates are provided together with the number of animals by species within each farm. A partition of all GB farms according to the animal types represented is shown in Figure 1a . Their geographical distribution is represented in Figure 1b as the number of farms per 565 km. Notice the high density areas in the North West (Cumbria), South West (Devon), Wales and Scotland where the main epidemic foci developed. There is also an area of high density in the Shetland Islands corresponding to very small crofter smallholdings. Figure 1c and d show the numbers of sheep and cattle kept per 565 km square.\nDuring the 2001 FMD outbreak, a total of 2026 infected premises (IPs) were recorded -farms where FMD was diagnosed, and which were subsequently culled. The IP dataset contains, for each farm, the estimated date of infection (determined by a clinical evaluation of the age of lesions on affected animals), and the dates of disease reporting, confirmation and culling.\nA total of 7457 other (non-IP) farms were also culled -mostly as contiguous premises (CPs, about 3103) or dangerous contacts (DCs, about 1287), but some under other local culling policies used in Cumbria and Scotland. For instance about 1846 (79%) out of a total of 2342 sheep farms in Cumbria had all sheep culled under the ''local 3 km radial sheep cull'' policy adopted there. Some of the farms (about 30) were recorded both as DCs and CPs. Multiple records per farm were often found in the disease control management system dataset, and it was often unclear whether this was due to data entry errors or as a result of sequential species-specific culls on the same farm. In our analysis we therefore considered the whole farm to be culled at the last recorded date of culling.\nThe most frequent species are cattle and sheep (see Figure 1a ). There are less than 3% farms with pigs only and only 10 farms with just pigs were diagnosed as IPs in 2001 (less than 1% of all the IPs). This indicates a-priori that pigs contributed far less to the 2001 outbreak than many other FMD outbreaks (despite their high levels of shedding [1, 17] ), and we therefore decided to discard pigs-only farms from the current study to simplify the analysis. The Sensitivity Analysis section shows that this simplification does not significantly affect estimates of other epidemiological parameters. We discarded another three IPs due to missing information or possible mistakes regarding their location or number of animals, leaving a total of 2013 IPs in our analysed dataset.\nWe model the epidemic as a space-time survival process [18] . The total observation time T is the 240 days between 7 th February and 5 th October 2001. Each farm i at the location (x i , y i ) is associated with an infection time t i (if infected), a removal time r i (if slaughtered) and two integers n c i and n s i representing, respectively, the number of cattle and sheep on the farm. S c and S s represent per-capita cattle and sheep susceptibility, respectively, while I c and I s represent per capita cattle and sheep infectivity. The susceptibility is a relative measure of animal sensitivity to the disease whereas infectivity represents the infectious risk posed by an animal to others. We use a continuous kernel to describe how the probability of contact between farms scaled with distance. Transmission is naturally assumed to decrease with the distance between farms according to the power law\nwhere d ij represents the Euclidian distance between the infected farm i and the susceptible farm j. The parameters a (kernel offset) and c (kernel power) are to be estimated. The kernel captures all forms of movement and contact between farms and as such, the use of a simple 2 parameter function is inevitably a highly simplified representation of the true complexity of inter-farm contacts. We examined other functional forms for the kernel (such as those used in some other analyses [19] ) but the resulting model fits were much poorer than found using the power-law kernel above.\nGiven the susceptibility and infectiousness parameters and the kernel, the infection hazard from an infected farm j to a susceptible farm i is then quantified by \nThis model is over-specified as stated, so we arbitrarily assume S s = 1 throughout, meaning S c represents the ratio of cattle-tosheep susceptibility. For a constant (distance-independent) kernel this is just a mass-action closed epidemic model with heterogeneous susceptibility and infectiousness. This model assumes susceptibility and infectiousness parameters scale linearly with the number of animals of different species on the farm, a relatively strong assumption imposed for model parsimony reasons. The mixing matrix embedded in (2) quantifies the 4 species-specific mixing rates between animals on different farms: cattle-to-cattle (S c I c ) sheep-to-cattle (S c I s ), cattle-to-sheep (S s I c ) and sheep-to-sheep (S s I s ). This model formulation is identical to that used by Keeling et al. [5] , except for the functional form of kernel used. The force of infection on a susceptible farm i at time t depends on the whole history of events and is just\nwhere\n, if the farm i is susceptible and the farm j is infectious at the time t 0, otherwise\nBy default, we assume a latent period of 1 day (latency is represented within the function L); i.e. farms are infectious the day after they are infected. However, we test the sensitivity of our estimates to the assumption by also examining latent periods of 2 and 3 days. The probability density function that farm i is infected at time t is then given by\nHence, the contribution that a farm i, observed to be infected at time t, makes to the log likelihood is just:\nA farm which is not infected contributes to the overall likelihood the probability that it escapes infection during the observation period, i.e. until the time it is culled (r i ) or for the duration of the epidemic T, whichever is shorter. Its contribution to the log likelihood is therefore\nThe total log likelihood of the model can be written as\nWe then extend the simple model above by introducing an additional parameter to understand to what extent the transmission within species is altered by between species transmission. The parameter r quantifies the degree to which mixing between species is assortative -with r,1 representing assortative mixing and r.1 disassortative mixing. The interaction model still assumes constant parameters with respect to time along the whole observation period T. The mixing matrix defined in equation (2) becomes S c I c rS c I s rS s I c S s I s ð8Þ\nwhere we again fix S s to be 1 to avoid model over specification. The force of infection (3) and model log likelihood equation (7) change accordingly. Assuming transmission parameters were constant in time throughout the epidemic is obviously a crude simplification. However, allowing infectivity to vary continuously in time results in an over-specified model and problems of parameter identification and confounding. We therefore examined two sets of models in which changes in transmission parameter were restricted to 2 significant points in time denoted by T cut , namely 23 rd February (when the national ban on animal movements was introduced) and 31 st March (when control measures were intensified and the so called 24/48 hour IP/CP culling policy was introduced). Models were respectively fitted to the individual case data from the start of the epidemic (conditioning on the first infection) or from after 23rd February (conditioning on the 54 farms that were already infected by that date). A detailed history of the epidemic is given by Kao [9] . We separately fitted model variants which assumed a discrete change in parameters on 23 rd February and on 31 st March. Confounding meant that only a very limited number of parameters could be varied in time, so we examined the effect of varying infectiousness and kernel parameters separately. We fitted four separate time-varying model variants: (i) varying the cattle infectivity by a factor and keeping sheep infectivity constant through time (Cattle Infectivity model); (ii) varying sheep infectivity by a factor but not cattle infectivity (Sheep Infectivity model); (iii) varying both cattle and sheep infectivity by the same ratio (Cattle & Sheep Infectivity model); (iv) varying the kernel parameters (Time Varying Kernel model). For the last model variant we also fitted a version which includes non-assortative mixing between species (see equation (8)). Hence the most general mathematical expression of the transmission model is:\nwhere\nThe scripts pre and post are self-explanatory for time varying parameters. When fitting models with time varying infectivity parameters we actually fit I post and the ratio m = I pre /I post we called infectivity factor. This is a within species ratio, a parameter directly fitted by the models, unlike the between species infectivity ratio additionally calculated as explained later in the text (see Parameter estimates section). Note that all models above treat the epidemic as fully observed, i.e. infection times are assumed to be known (when in fact only estimated infection times are known -see Sensitivity Analysis section), and only IPs are assumed to be infectious.\nWe adopt a Bayesian framework for statistical inference and use MCMC methods for fitting the model to individual case data. This is not strictly necessary, given our simplifying assumption that the epidemic was completely observed, but it provides a more consistent and robust framework within which to relax that assumption in future work.\nWe obtained parameter estimates and equal-tailed 95% credible intervals from the marginal posterior distributions of the fitted parameters. For the basic model for instance we estimated the relative cattle susceptibility, S c , two infectivity parameters (I c (t);I c and I s (t);I s for all t) and two kernel parameters (c(t);c post ;c pre ;c and a(t);a post ;a pre ;a for all t).\nWe used the posterior mean deviance as a Bayesian measure of fit or model adequacy as defined by Spiegelhalter et al. [20] . The posterior density deviance is defined as:\nwhere log{P(y|h)} is the log-likelihood function for the observed data vector y given the parameter vector h and C is a constant which does not need to be known for model-comparison purposes (being a function of the data alone). The smaller the mean posterior deviance, the better the corresponding model fits the data.\nIf the posterior deviance distributions for two different models overlap significantly, it is necessary to use additional criteria to compare model fit -namely a comparison of the relative complexity of the models. The Deviance Information Criterion (DIC) is perhaps the most general of such methods, being a generalisation of the Akaike information criterion for Bayesian hierarchical models [20] . We define the complexity of a model by its effective number of parameters, p D , defined as\nwhere E[ ] represents taking expectations (the posterior average).\nThe DIC is then defined as\nA lower value of DIC corresponds to a better model. This criterion offers flexibility for comparing non-nested models [20] and it is straightforwardly computed within an MCMC algorithm.\nWe applied the classic random walk Metropolis Hastings algorithm [21, 22] and a block-sampling of parameters due to the computationally expensive form of the likelihood [23, 24] . A log scale has been used for sampling as the parameters were all positive definite and were expected to potentially vary by orders of magnitude. However, linear scale sampling yielded similar results. The convergence of the chains was also very much improved (see Robert [25] for more on perfect sampling and reparameterization issues) compared with sampling on a linear scale. The model was coded in C and parallelized using OpenMP 2.0.\nThe MCMC sampler was allowed to equilibrate with convergence being evaluated visually from the likelihood and parameter traces. For the simpler models, 5,000 iterations were sufficient for equilibration, while this increased to 20,000 for the most complex models. Also, using log scale sampling, we verified that the chains were able to converge even if started with initial parameter values far from the final posterior mean values. Posterior distributions were estimated from 100,000 iterations. The rate of the acceptance varies from model to model. For the baseline model we achieved a 25% rate of acceptance and for the most complex model (8 parameters), a rate of approx 10%. These values compare well with the ''golden'' acceptance rate for Random Walk Metropolis Hastings of 23% (Roberts [26] ).\nWe did not encounter common problems in MCMC estimation like slow convergence and slow mixing (O'Neill [27] ). There were some correlations between parameters, mostly having biological explanations (cattle and sheep infectivity for instance), but a careful parameterization lowers them. We verified parameter estimates were not dependent on parameterization choices -e.g. no difference was seen whether we fitted species infectivity individually, or just fitted sheep infectivity and then the ratio of cattle-to-sheep infectivity. Table 1 lists the parameter estimates we obtained for a set of fitted models conditioned only on the first infection whereas Table 2 presents the estimates for models conditioned on infections occurring up to 23 rd February. The posterior deviances for each set of models are plotted in Figure 2a and Figure 2b , respectively. Figure 2a illustrates some clear conclusions. Of the two models without time variation in parameters, the interaction model fits significantly better than the baseline model without heterogeneous mixing between species. However, fitting the interaction model broadened the credible intervals of the infectivity parameter estimates (Table 1) , indicating (unsurprisingly) slight confounding between the 4 infectivity and susceptibility parameters.\nOf the models which allowed infectivity to vary on 23 rd February, allowing only cattle infectivity variation gave a slightly better fit than varying sheep infectivity or both. However, of the models with parameters which vary on 23 rd February, the model variants which allow the 2 kernel parameters to vary at that time point fit substantially better (by both deviance and DIC criteria, see Table 1 ) than those which just allow a species-specific variation in infectivity. This is encouraging for the inference procedure, as the main control measure initiated on that date was the banning of (Figure 3a and Figure 3b ). The parameter estimates are less precise before 23 rd February (Table 1) due to the relatively small number of IPs (about 57) before that date. Looking at the most complex model (namely the interaction model with time varying kernel), cattle were estimated to be 5.7fold (4.6, 6.8) more susceptible than sheep (see Figure 3c and Table 1 ). Rather than mentioning animals' specific infectivity (see Figure 3d and Table 1 ), it is more informative to comment on the cattle:sheep infectivity ratio parameter for the most complex fit (this ratio does dot appear in the tables as it is not a model parameter). We calculated it within the MCMC algorithm as the ratio of the two species infectiousness for each sampled parameter point. The most complex model suggests that cattle are 5.95-fold (4.54, 7.63) more infectious than sheep (Figure 3e) .\nThe parameter quantifying assortativity in mixing was estimated at r = 0.45 (0.31, 0.61) -well below 1, the level at which mixing between species is random (Figure 3f ). By comparison with the model with a time varying kernel but random mixing between species, the effect of heterogeneous mixing between species modified the between-species transmission as given by matrix (1.9) as indicated below. \nCattle-to-cattle and sheep-to-sheep transmission is higher (by 19% and 54% respectively) for the model with non-random mixing, whereas the sheep-to-cattle and cattle-to-sheep transmissions dropped by 41% and 37 % respectively. Conditioned on 23 rd February, 7 model variants have been considered (Table 2 and Figure 2b) . We examined the baseline and interaction models (no change in parameters over time), allowing cattle infectivity to vary on 31 st March and both cattle and sheep infectivity to vary by the same factor after 31 st March (with and without heterogeneity in mixing) and allowing both kernel parameters to vary on 31 st March.\nUnsurprisingly, the kernel parameters were not significantly different if allowed to be different before and after 31 st March, neither did this model prove to be the best fit. Overall, while the variations in mean deviance (Figure 2b ) seen between model variants were much smaller than for the models conditioned on the first infection (Figure 2a ), the interaction model allowing for time varying cattle infectivity gave the most adequate fit (measured by both mean deviance and DIC, see Table 2 ).\nWe cannot statistically compare the two sets of models in Table 1 and Table 2 , as the data used are different for the two cases. However, the parameter estimates from the best-fitting models of each table are largely consistent. Each post-23 rd February estimated value from the best-fit model in Table 1 is included in the corresponding pre-31 st March 95% credible interval of the best fit model in Table 2 (and vice-versa).\nThe most important message from the second set of models is that all models with cattle time varying infectivity (best fit) indicated higher values of infectivity after 31 st March than before (m = 0.73 (0.63, 0.83)) ( Table 2 ). This may seem paradoxical but reflects the fact that while culling (the effect of which is explicitly included in the input data) dramatically reduced case incidence in April, from May to September 2001, case incidence maintained itself at a low level -but almost entirely within cattle farms. This increase in cattle infectivity may therefore really reflect the impact of reduced biosecurity and/or increased non-compliance with movement controls.\nIt is informative to examine what our parameter estimates imply in terms of geographic variation in transmission potential. Given the parameter estimates for each model, we can define the relative risk of transmission an infectious farm j would pose to all susceptible farms in the country r j : r j~Ic n c j zI s n s j P i=j Sc Ss n c i zn s\nThis quantity multiplied by the average duration of infectiousness of a farm (time from end of latency to culling) gives the reproduction number R 0j of the farm j. We divided the UK into 5 km squares and then calculated the average transmission risk of all farms in each square (local R 0 ). Figure 4 shows how geographic risk changed before and after 23 rd February for our best fit model conditioned on the first infection. The kernel shape has a major influence on the average risk distribution throughout the country. Figure 5 shows the corresponding risk maps for the estimates inferred from our best fit model conditioned on 23 rd February. A slightly higher risk is predicted after 31 st March by the model conditioned on 23 rd February due to the increase in the cattle infectivity after this date. The risk estimates after 23 rd February from the first set of models appear consistent with those obtained from the models conditioned on 23 rd February, though a rigorous statistical comparison is not appropriate. \nWe have made the strong assumption for this study that the only infected farms during the 2001 epidemic were the reported IPs, and hence that any farms which were infected but culled before clinical diagnosis were not responsible for causing any infections. It is therefore interesting to calculate how many of the proactively culled farms our model predicts might have been infected (but, by definition, not diagnosed).\nTo calculate the probability p i that a particular proactively culled farm i was infected, we need to adjust the infection hazard by the probability that the farm would have not been reported as a clinical case before its culling date T i c . From the outbreak data, we calculate the probability density of the time from infection to report for reported IPs and hence the cumulative probability distribution of the time from infection to report, denoted by F. Then, with l i (t) being the force of infection on a proactively culled farm i at time t (from the best fit model conditioned on 23 rd February), the probability that that farm gets infected and escapes reporting between its potential infection time and culling time T i c is\nWe calculate the expected number of infections in different classes (e.g. DCs, CPs) of proactively culled farms culled within a particular time interval (T i c [ T 0 ,T 1 ½ ). For instance, the expected number of CPs culled at the time T i c [ T 0 ,T 1 ½ which are predicted to have been infected can be formally written as\nThis is a simplification, as in reality the delay from infection to report almost certainly depends on the size and species mix on a farm, but the result is nevertheless indicative of the expected level of infection in proactive culling. Also, at this stage, the calculations are made as if culling was a non-informative censoring process. This is a reasonable assumption for all proactively culled farms except for DCs (which by definition had been identified by veterinarian as having had a high risk of exposure) but our method may underestimate the infection rate. In calculating the infection to report delay distributions, we divided the epidemic after 23 rd February into 3 time periods: 23 rd February-31 st March, 31 st March-1 st May and 1 st May-5 th October. In these intervals a total of 1332, 4498 and 1627 farms were slaughtered, respectively. Our best fit model conditioned on 23 rd February predicts different infectivity regimes before and after 31 st March (see Parameter Estimates and Table 2 ) but we split further the second period of time due to different delays in reporting to culling. The infection to report delay is 8.6 and 8.8 days for the last two periods of time respectively but the infection to cull delay drops from 9.4 and 8.8 days respectively. Applying this approach to the interaction model with time varying cattle infectivity which conditioned on the 23 rd of February, we calculated the expected proportion of proactively culled farms which were infected. We estimate that approximately 1.3% (1%, 1.6%) of 7457 culled non-IP farms may have been infected -97 in total (Figure 6a ). Of the 1332 farms culled between 23 rd February and 31 st March, 1.7% (1%, 2.4%) may have been infected (23 farms). Of the 4498 farms culled between 31 st March and 1 st May, we estimate 0.7% (0.5%, 1%) were infected (34 farms). In the period 1 st May to 5 th October, we estimate that 1.6% (1%, 2.3%) of 1627 farms culled were infected (27 farms).\nThe proportion of CPs estimated to have been infected is 2% (1.5%, 2.5%), equating to 62 farms (Figure 6b ). Over the whole epidemic, we estimated 1.5% (0.8%, 2.1%) of farms designated as DCs were infected (19 farms). This estimate (Figure 6c ) does not allow for higher risk of infection implied by the veterinary judgement that led to those DCs being identified, which may mean that a higher proportion were in fact infected. If we assume that DCs were 3 times more likely to be infected due to their status than the model would predict, then the incidence of infection in DCs goes up accordingly, i.e. to 4.6% or 59 farms.\nFarms culled neither as DCs or CPs (typically those culled under the 3 km and local sheep cull policies in the Cumbria, Dumfries and Galloway areas) had the lowest estimated rate of infectiona mere 0.5 % (0.2%, 0.8%) or 16 out of 3067 farms.\nIn this section we examine the sensitivity of our results to a number of factors: leaving pigs out of the analysis, possible errors in the estimated IP infection dates, and the assumed latent period.\nTo justify the simplification of the analysis by discarding the number of pigs in a farm, we present some more detailed statistics regarding this variable. We also fit the simplest model conditioned the last two farms are exclusively pig farms. We denote by n p i ,S p ,I p the number of pigs in farm i, pigs susceptibility and pigs infectivity respectively. The simplest model similar to (1.2) conditioned on the first infection has been fitted, reducing the number of parameters in the same manner.\nIn addition we estimated pig:sheep susceptibility ratio and pig infectivity, assuming all parameters constant through time. We found that cattle:sheep susceptibility ratio is 6. Table 1 shows parameter estimates for cattle and sheep are largely unaffected by ignoring the pig population, with none of the estimates from the two analyses being significantly different. We conclude that including pigs would not change the conclusions presented in Table 1 regarding cattle and sheep (given the very small number of IPs which had pigs) but it would decrease the power of the analysis and increase model complexity.\nTo understand to what extent our estimates are affected by the assumption that the infection dates have been accurately observed, we randomized the estimated infection dates by adding a Gaussian noise with zero mean and a standard deviation of 2 days. This is motivated by the substantial proportion in the observed standard deviation (73.5% less or equal than 2 days) of the distribution time from the estimated infection date to the report date of IPs. We then fitted the simplest model (conditioned on both first infection and 23 rd February) to 10 such randomised datasets. The average estimates across them are given in Table 3 . They lie well within the confidence intervals we predicted in Table 1 . The average cattle:sheep infectivity ratio is also very close to the values estimated using the original data.\nThe average estimates across 10 randomized datasets using the most appropriate model conditioned on 23 rd February (i.e. cattle infectivity and interaction model) are also in Table 3 . The values are within the 95%CI presented in Table 2 . We assessed a sensitivity analysis for the estimated proportion of infections in proactively culled farms (see the previous section) with respect to infection times. Using the predicted parameters for each dataset, we calculated the average proportions across all of them, for each category of proactively culled farms. The average proportion of infections between DC farms is 1.37% (2%, 0.78% and 0.72% for each period of time, respectively). For CP farms, the same quantities evaluate to 1.9% with 1.8%, 1.3% and 1.98%, respectively. Overall proactively culled farms, we obtained an average percentage of 1.25% with 1.64%, 0.81% and 1.6% for each considered period of time. All the values are well within the 95%CIs predicted by the original data (see the previous section and Figure 6 ).\nAll the results presented above assume a fixed latent period of 1 day. We tested the sensitivity of parameter estimates to this assumption by examining latent periods of 2 and 3 days. Overall, we would expect infectiousness parameters to increase to compensate for the shorter infectious period, and thus slightly increased generation time (namely the mean time from infection of one case and the time of infection of the cases that case generates). Interestingly, however, it is the kernel parameter estimates which are altered as the latent period is varied with the kernel becoming slightly less local with increasing latent period. For two and three days latent period, pre 23 rd February, the values of c dropped from 1.69 (Table 1) \nThis paper has presented a statistical analysis of the spatiotemporal evolution of the 2001 foot and mouth outbreak in GB. Qualitatively, the results agree with those obtained by Keeling et al. [5] in identifying cattle as being the key species in the 2001 epidemic. Using the interaction model conditioned on 23 rd February with time varying cattle infectivity, we estimated that 88% of IPs between 23 rd Feb-31 st March were infected by cattle and only 12% by sheep. Sheep-to-sheep transmission only accounts for 3.1% of IPs in that period. After 31 st March (when we estimated that cattle infectivity increased slightly, see Table 2 ) Allowing for non-random mixing between species indicates contacts between farms are assortative on the basis of species composition of the farm; i.e. like species mix with like. This agrees with intuition about the nature of farming practices (e.g. sharing of personnel and equipment is likely to be more common if 2 farms have the same livestock species). The implications of the moderate degree of assortativity we found for control measures remains to be explored.\nWe did not use data collected during the epidemic on traced contacts between farms to fix the spatial kernel function in our analysis, since in the final version of the FMD epidemic data warehouse [http://www.defra.gov.uk/footandmouth/cases/index.htm] very few of the contacts apparently identified early in the epidemic remain confirmed. Also we shared the concern of earlier work that the distribution of contact distances in traced contacts may well be biased [3] . We therefore estimated the kernel function, using an offset power-law functional form. The higher value of the kernel power parameter we estimated after 23 rd February (2.67 vs. 1.70 before - Figure 3a) is consistent with the expected dramatic shortening in the typical contact distance following the national movement ban. This localized spread together with the higher estimated level of infectivity in cattle after 31 st March explains the long tail of the epidemic seen in 2001.\nIn estimating the transmission risk between farms, we assumed a dependence on the Euclidian distance between them. In reality, other metrics (e.g. the time required to travel between two farms) might be more reasonable, and should be examined in future work. We also did not include information on landscape (e.g. height above sea-level, location of rivers, trees etc).\nThe estimated risk maps (Figure 4 and Figure 5 ) match the areas of the country where highest case incidence rates were seen -with the notable exception of Wales. The discrepancy between the high predicted risk in Wales and the small number of cases observed may reflect inaccuracies in the input data set -Keeling et al. [5] reduced farm-level sheep population numbers by 30% in Wales and obtained a better geographic match to the data (Matt Keeling, personal communication). However, the discrepancy may also reflect model inadequacy. We have not here allowed for other farm-level risk factors, such as the farm fragmentation index considered by Ferguson et al. [3] . We have not explored more complex non-linear models of the dependence of susceptibility and infectiousness on the number of animals on a farm or relaxed our implicit assumption that contact rates between farms scale linearly with the local density of farms. All these assumptions are being relaxed in ongoing work.\nThe most important issue to be revised in future work is to allow for proactively culled farms which were not diagnosed as IPs to be potentially infected and infectious to other farms. This requires modification of the inference model used to allow for an arbitrary number of unobserved infections. The very low numbers of proactively culled farms we estimated as infected suggested that the effect of this model refinement may be limited. It should be noted though that these infection prevalence estimates are in part a result of the relatively non-local kernel estimated simultaneously. If kernel estimates change in a refined analysis -and if DCs were attributed a much higher risk of infection than estimated here due to their status -then it is possible that estimated infection rates in DCs and other proactively culled farms may increase somewhat.\nHowever, even if these factors increased our estimated infection prevalence among proactively culled farms 5 fold (which seems unlikely from ongoing work), it would still mean that only a small proportion (,10%) of DCs and CPs culled were infected. This does not imply that proactive culling had no effect on the epidemic -as the largest expected effect of such culling is via the targeted depletion of susceptible animals. In this regard, proactive culling has the same epidemiological impact as vaccination. Future work will revisit past estimates of exactly how important such culling was for the control of the 2001 FMD epidemic." }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: Pneumonia virus of mice (PVM) infection in mice was originally described by Horsfall and colleagues [1, 2] , but until relatively recently, the sole interest in this virus was as a pathogen of laboratory rodent colonies [3] [4] [5] . Over the past several years, we and others have built on Horsfall's early studies, and have developed and characterized an in vivo model of severe respiratory virus infection using PVM [reviewed in [6, 7] ]. Among our findings, we have shown that a minimal, physiologically relevant inoculum of PVM (typically <100 pfu) results in robust virus replication in lung tissue, accompanied by influx of granulocytes in response to local production of specific proinflammatory chemokines [8] . The pathophysiology of PVM bronchiolitis leading to pneumonia and acute respiratory distress syndrome (ARDS) is similar to that observed in response to severe respiratory syncytial virus (hRSV) infection in human infants [9] .\nWhile PVM clearly replicates efficiently in mouse lung tissue, the in vitro propagation of this pathogen is significantly less straightforward. The primate BS-C-1 epithelial cell line supports minimal rates of PVM replication in vitro [10] . The BS-C-1 cell line has been used for traditional plaque assays, but PVM-induced plaques develop slowly, have relatively indistinct borders, and are difficult to eval-uate quantitatively [see Figure 1A ]. Furthermore, from an evolutionary perspective, one would prefer to perform molecular studies of virus pathogenesis in cells from a relevant species, i.e...a rodent cell type or cell line. We have demonstrated that PVM replicates in the mouse LA4 respiratory epithelial cell line [11] , but virus growth is similarly slow, even at temperatures permissive for virus propagation in vitro.\nIn this work, we explore PVM replication in several independent cell lines and identify the mouse macrophage \nThe rodent L2, LA4, RAW 267.4, J774A.1, RLE and 3T3 and primate A549, BS-C-1, and HEp-2 cell lines obtained from American Type Culture Collection (Manassas, VA) were maintained in Iscove's Modified Dulbecco's medium with 10% heat-inactivated fetal calf serum, 2 mM glutamine and penicillin-streptomycin at 5% CO 2 and 32°C (permissive for virus growth in culture) unless otherwise indicated. Mouse-passaged PVM prepared as described was stored in liquid nitrogen at ~10 6 pfu/ml [12] . Virus replication in RAW 264.7 cells was determined by both Q-RT-PCR detection of the virus SH gene [see reference [13] ] for complete method] and by western blot [14] probed with a 1:200 dilution of polyclonal anti-PVM N peptide antibody prepared against sequence SQQLN-IVDDTPDDDI encoding amino acids 379 -393 of the PVM N protein. Proinflammatory cytokines in culture were evaluated by ELISA (R&D Systems, Minneapolis, MN). Q-RT-PCR detection of interferon-β was via standard methods using primer -probe set Mm00439546_s1 (ABI, Columbia, MD) normalized as described [13] on RNA prepared from infected and control uninfected cells in culture (RNazol B, Friendship, TX).\nThe cell lines evaluated for the ability to support virus replication included rat epithelial L2 and RLE, mouse epithelial LA4, mouse macrophage RAW 267.4 and J774A.1, and primate epithelial A549, BS-C-1, and HEp-2. All were inoculated with PVM on day 0 (MOI = 0.02, 10 4 pfu per 5 × 10 6 cells). On day 7, virus titer in the culture supernatants was determined by standard plaque assay [12] . Although pneumoviruses maintain strict host-pathogen specificity in vivo, we determined that PVM replicated to a limited extent in vitro (< 10 3 pfu/ml supernatant) in each of the aforementioned cell lines. The mouse monocyte/ macrophage RAW 264.7 cell line (established from a tumor induced by Abelson murine leukemia virus) generated the highest virus titers (10 4 pfu/ml) under culture conditions described. Cells of the RAW 264.7 line also support replication of other unrelated virus pathogens, including murine hepatitis virus and Japanese encephalitis virus [15] [16] [17] .\nTo evaluate the kinetics of virus replication and production of proinflammatory mediators in the RAW 264.7 cell line, cells at 50% confluence were inoculated with PVM (MOI 0.1) on day 0 and harvested on days 2 -5 thereafter. RAW 264.7 is a semi-adherent cell line, and is not well-suited for plaque assays. Here, virus replication was examined qualitatively on western blot of cellular homogenates probed with PVM-specific antisera [ Figure 1B ]. Virus was first detected in infected cultures on day 3 post-inoculation, and then in increasing amounts through day 5. No immunoreactive PVM N protein was detected in uninfected control cultures.\nVirus replication was also examined quantitatively by Q-RT-PCR using the virus SH gene as a target sequence [13] , [ Figure 1C ]. PVM replication was readily detected in inoculated RAW 264.7 cells, reaching ~4 × 10 5 copies per microgram total RNA on day 5 of infection. No copies of the virus SH gene were detected in uninfected cells.\nRAW 264.7 cells respond to infection with PVM by producing a variety of proinflammatory mediators. Transcription of interferon-β in response to virus infection was detected by Q-RT-PCR [ Figure 1D ]. Cytokines MIP-2, TNF-α, MIP-1α, and MIP-1β were detected in culture supernatants by ELISA [ Figure 1E ]. Interestingly, MIP-1α and MIP-2 are among the most prominent mediators detected in BAL fluid of infected mice; MIP-1α levels correlate directly with the severity of pneumovirus disease in both PVM and hRSV infection [18, 19] . In parallel to our findings, hRSV replicates in the human monocytic THP-1 cell line [20] , and several groups have provided evidence consistent with hRSV and bovine RSV (bRSV) replication in alveolar macrophages, although this point remains controversial [21] [22] [23] [24] [25] . Furthermore, hRSV infection of the human monocytic U937 cell line was associated with production of the proinflammatory mediator, platelet-activating factor (PAF) [26] .\nIn summary, PVM has recently emerged as a useful novel model for the study respiratory disease in mice [7, [27] [28] [29] [30] ; this has provided significant incentive toward identifying tissue culture systems for virus propagation. The mouse RAW 264.7 cell line supports efficient replication of PVM in vitro and responds to infection by augmenting production of cytokines implicated in the pathogenesis of respiratory disease. Use of this ex vivo model of PVM infection will permit further study of biological responses associated with virus infection and the cellular and molecular level. " }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: Since 2001, state and local health departments in the US have accelerated efforts to prepare for bioterrorism and other high-impact public health emergencies. These activities have been spurred by federal funding and guidance from the US Centers for Disease Control and Prevention (CDC) and the Health Resources and Services Administration (HRSA) [1] [2] [3] . Over time, the emphasis of this guidance has expanded from bioterrorism to include \"terrorism and non-terrorism events, including infectious disease, environmental and occupational related emergencies\" [4] as well as pandemic influenza [5] .\nFor any locality, the rarity of major public health emergencies necessitates the use of practice-based exercises to simulate real life experiences in order to develop and improve skills and to assess response capabilities over time. The US Federal Emergency Management Agency (FEMA) describes six levels of exercises, increasing in complexity from informational seminars that minimally exercise response capacities to simulations that mimic reality and exercise participants' capacity to implement emergency response functions [6] .\nIntermediate in this progression is the tabletop exercise, which FEMA describes as a \"facilitated group analysis of an emergency situation.\" As practiced in public health, there is considerable variability in how tabletop exercises are designed and conducted. Tabletop exercises may be structured discussions of evolving events or unstructured reactions to short scenarios; participants may be limited to public health staff or involve representatives from partner agencies or organizations; scenarios may range from simple to complex; and facilitation may range from being minimally directive, allowing participants to assume responsibility for managing the discussion through \"role play,\" to highly directive, enabling the facilitator to assure that specific questions are addressed.\nRecognizing the need to exercise public health emergency response, and enabled by funding and directives from CDC and HRSA, health departments throughout the US have implemented exercise programs. These exercise programs have had varying goals, including building relationships among stakeholders [7, 8] , training staff [9] [10] [11] , and evaluating preparedness levels [12, 13] , and they have been used for a variety of purposes, including to identify gaps in preparedness [14] , make recommendations for improving preparedness [15] , and identifying variations in preparedness across health departments [16] . These exercises have involved diverse groups of stakeholders involved in public health preparedness, such as representatives from public health [17] , health care [18] , agriculture [19] , and emergency medical services [20] . Despite the commonality of preparedness and response compo-nents across a variety of biological threats, most exercises have been designed for single use and focus on single disease, such as smallpox [21] , pandemic influenza [22] , or a novel virus [23] .\nThese exercises have focused attention on the interaction between preparedness goals and exercise strategies, and have illuminated strengths and vulnerabilities in public health emergency decision making and response capacities. The increased utilization of tabletop exercises by health departments has not been accompanied by a parallel increase in knowledge sharing about lessons learned from them, either with regard to identifying common challenges that confront health departments or strategies for effective exercise design and management. Further, the literature dealing with tabletop exercises to date consists almost entirely of case studies and descriptions of a single exercise or a single disease. This paper describes lessons learned by public health researchers at RAND, and their collaborators, about the process of developing and conducting tabletop exercises in collaboration with state and local health departments in the US and their implications for public health emergency preparedness.\nData for this paper come from four related projects conducted from 2003-2006. Taken together, these projects involved developing, conducting, and evaluating 31 tabletop exercises with state and local health departments of different sizes and structures in 13 different states across the northeast, south, mid-west, and west regions of the country (Table 1) . Participating health departments did not incur any expenses through their involvement in these exercises other than the staff time required to participate.\nTwo of these projects, one in California and the other in Georgia, involved the conduct of exercises in multiple jurisdictions in the same state. In California, the Little Hoover Commission, a bipartisan, independent state body, asked RAND to assess California's public health infrastructure. A key component of the project, described in greater detail elsewhere [16] , was the development of a tabletop exercise that simulated a smallpox outbreak. This exercise was conducted in seven local health departments across California. In Georgia, RAND collaborated with the Georgia Division of Public Health and the Rollins School of Public Health at Emory University to develop, conduct, and evaluate a series of tabletop exercises focusing on different biologic agents in seven local health departments across Georgia, as well as one exercise focused at the state level.\nThe two remaining projects were funded by US Department of Health and Human Services (HHS) and involved the participation of multiple local health departments.\nThe first project involved developing ten different tabletop exercise templates and formats focusing on the local public health response to bioterrorist agents. These were tested in 13 local health departments in 12 different states. The second project involved developing a tabletop exercise to examine the interface between local health departments and health care systems in a hypothetical influenza pandemic. This exercise was tested in three local health departments in different states. Greater detail on the structure for these tabletop exercises as well as the tabletop exercise templates themselves can be found elsewhere [7, 17] .\nAll exercises focused on at least one of three related objectives: training, relationship-building, and evaluation. The structure and design of the tabletop exercises varied from project to project because their objectives were somewhat different. The key domains covered are outlined in Table 2 . The level of facilitator involvement varied with the exercise objectives. At one extreme, the facilitator's role was limited to introducing the exercise scenario and periodically interjecting updates. During these exercises, the participants were encouraged to lead the discussion themselves, based on their respective roles in their agency or organization. At the other extreme, the facilitator took a very active role by leading the discussion and interjecting questions or prompts. In between were exercises in which the facilitator turned the discussion over to participants but occasionally joined the discussion to request clarifications from the participants or assure that issues critical to the exercise objectives were discussed.\nDespite these differences, all of the exercises shared common elements, including: evolving hypothetical scenarios, facilitated group discussions, and some level of collective decision making by participants emphasizing *< 100,000 = small, 100,000-1,000,000 = medium, > 1,000,000 = large; **Mild involvement-most of exercises was role played by participants, with very little intervention or direction from facilitators; Moderate involvement-most of exercises was role played or issue discussion, with the facilitator inserting additional probes and ensuring the discussion stayed on track; Active-most of the exercise was more discussion based, with facilitator asking questions or identifying issues that were subsequently discussed.\nthe role of local health departments in recognizing and initiating a response to an emergency. The scenarios typically began with a single case report or series of case reports that heralded a nascent disease outbreak and required a public health assessment. These situations exercised the internal communication and coordination across disciplines within health departments as well as the communication and coordination with partner agencies and organizations such as health care facilities and emergency medical service agencies. Several exercises extended beyond this initial response and included scenarios that progressed days or weeks into an outbreak, requiring greater interactions between local-and state-level authorities and attention to health care surge capacity.\nEvery exercise concluded with a \"hot wash\" in which participants discussed their collective performance, identified strengths and weaknesses, and when relevant, related their performance to experience with actual outbreaks or crises. In the latter exercises, participants were prompted to develop an initial 'action plan' that addressed key vulnerabilities identified in the exercise. The facilitators subsequently generated a written \"After Action Report\" (AAR) that summarized the exercise experience and highlighted the observed strengths and areas for improvement. In addition, participants completed exercise evaluation forms. These consisted of a series of structured and semistructured questions that asked participants to discuss what they learned during the exercise and to evaluate aspects of the exercise structure and conduct. For example, participants were asked to identify key gaps in preparedness that occurred during the exercise and to identify the most useful thing they learned during the tabletop exercise. The observations reported here are based on reviewing the after action reports, participant evaluations, as well as internal team discussions and consensus following the exercise debriefings.\nThe performance of health departments that participated in our tabletop exercises varied from agency to agency. However, there were consistent themes that emerged across the agencies, regardless of the structure or the biologic agent/disease discussed; nearly all agencies struggled with a common set of challenges. These challenges, summarized in Table 3 and described below, represent critical dimensions of an outbreak response.\nMany local health departments did not have a structured process for notifying or soliciting case reports from health care providers in the community other than those in hospitals, largely because they did not have reliable contact information for private providers or a sure means to reach them rapidly. In most instances local health departments had good relations with staff in local hospitals (e.g., emergency department staff and infection control practitioners) but did not appear to have similar working relationships with non-hospital based practitioners.\nLocal public health officials were sometimes unsure about their direct role in following up with suspected ill patients and collecting and shipping clinical samples for laboratory testing. For example, there was frequently confusion around whether it was the responsibility of the local health department, the state health department, or the medical personnel at the hospital to collect laboratory samples. Once the samples were collected there was often confusion around whose responsibility it was to transport the samples, and in a few sites, local law enforcement were surprised to find out that they were the responsible party. A related issue was the ability of health departments to realistically generate enough surge capacity in their public health workforce to investigate or respond to a large event, especially one that encompassed multiple jurisdictions in the same state, thereby limiting the state health department's ability to shift manpower and resources from one jurisdiction to the next.\nFew of the health departments in which we conducted exercises were proactive in their contacts with the media, and most waited until they were contacted by the media to begin communicating with the public. One consequence of this passive approach was that public health officials often responded defensively to early and sometimes unexpected media requests and in turn, had trouble quickly formulating an initial message to the public that was clear, informative, and alleviated anxiety.\nHealth departments consistently expressed uncertainty about how to effectively communicate with vulnerable or underrepresented population groups in their jurisdictions, and few had well established relationships with community leaders or organizations that could serve as messengers or communications channels to these groups.\nIn several sites, law enforcement and EMS personnel present in exercises had greater familiarity with these groups and could help identify trusted community messengers. Further, in some communities, health departments had limited language capacities or were not sufficiently familiar with community leaders to communicate effectively with these groups.\nCommunicating fully and effectively with response partners (e.g., law enforcement and EMS) about their occupational health risks and personal protection was also a challenge for local public health. In particular, while public health officials were usually quick to notify response partners soon after determining an event to be significant, response partners in many cases felt that public health officials were slow to provide them with critical information about the disease in question, what their risks might be, or what actions they should take to protect themselves. As a result, response partners frequently reported feeling either left out of the process or expressed concerns about continuing to work unless the risks to them were clarified and more was done to ensure their safety on the job.\nThe use of the National Incident Management System (NIMS) and its associated Incident Command Structure (ICS) structure is relatively new to public health. This was evident in the exercises, in that nearly all health departments had difficulties deciding if and when to implement the ICS process and in identifying the party who would serve as incident commander. Similar challenges were seen in the decision and processes related to opening an Emergency Operations Center (EOC). As a result, in many exercises, local public health officials delayed taking these steps and preferred maintaining a more informal management process. This approach was preferred even as the outbreak became progressively larger, thereby stressing these informal networks.\nAs outbreaks evolved, there was often a lack of clarity about whether and when local health departments should hand off control to the state health department, how responsibilities should be jointly shared between local and state authorities, and whether or when federal agencies, such as CDC, should become involved. In many of the exercises, state health departments were surprised by the level of assistance requested by their local health departments especially in the early stages of the outbreak; in other more rare examples, state health departments surprised local health departments by assuming roles and responsibilities local health departments regarded as their own. Regardless, the general consensus among local public health participants in most exercises was that CDC staff would be on the ground to help them fairly quickly, particularly in situations where bioterrorism was considered likely.\nMost local health departments articulated some type of plan for increasing medical surge capacity by developing alternative care sites. In most instances however, these plans were unable to hold up to even a modest amount of scrutiny during the exercise because they were superficial and lacked sufficient detail necessary for rapid implementation. Related to this issue, local health departments frequently reported that there were not enough local health care workers to manage these sites even if they could create them. For example one participant noted, \"We have pop-up tents and beds to increase capacity, we just don't have pop-up people to staff them.\"\nEven obtaining a census of available staff members turned out to be challenging as many health care participants noted that some staff would likely be double-counted, particularly nurses and security officers who might work in several institutions. Increasing staff capacity through the use of community volunteers, including retired medical personnel, while often recognized as one potential solution to staffing shortages, proved to be extremely difficult to actually implement. Public health participants universally recognized the importance of volunteers, but learned that their plans to recruit, train, and mobilize large numbers of volunteers were too vague and lacked concrete actionable steps for realistic application during a real emergency.\nIn nearly all exercises, we also identified a number of strengths within participating health departments. However, there was far less commonality in these strengths than we observed with the areas for improvement. Universally, we observed public health leaders and staff who were committed and struggling to 'do the right thing.' The most commonly observed strengths were strong relationships between epidemiologists and hospital infection control practitioners and between public health workers and other emergency coordinators. In some instances, prior experience with emergency planning or response, such as involvement of health departments and emergency service agencies in coastal areas in preparing for or responding to hurricanes, was associated with stronger and more facile interactions between health department officials and partner agencies.\nOur exercises were conducted over a period of several years. While we did not conduct exercises with any health department more than once and did not employ an experimental design to assess changes over time, we were struck by how the performance of health departments overall improved over time. First, compared to earlier exercises, local health departments appeared far more sophisticated about their early internal processes related to notification, enhanced surveillance and large outbreak investigations. In addition, by the end of the exercise period, health departments had considered plans for surge capacity, and participating hospitals had explicit plans for cancelling emergency surgery and discharging less severely ill patients. They also appeared more acutely aware of the challenges in assuring adequate numbers of staff to provide care.\nThe large number of tabletop exercises we conducted allowed us to test and compare different strategies for designing and conducting tabletop exercises. These comparisons enabled us to modify our exercises over time to build upon lessons learned from previous exercises. Below we briefly highlight five lessons we learned from this experience.\nExercises should be designed to achieve a specific objective When first developing the tabletop exercises, our assumption was that a single exercise could achieve multiple objectives, such as training, relationship building, and evaluation. While these objectives are interrelated and opportunities often exist to achieve them concomitantly in the same exercise, it is critical to define the priority objective for the exercise because different objectives have different implications for exercise design. For example, if exercise participants outlined a response that was flawed or problematic, in an exercise primarily focused around the objective of training it would be appropriate for the facilitator to pause and help the participants re-think their approach. On the other hand, if the objective of the exercise is evaluation, this type of facilitator involvement can lead the participants to choose a different course of action and therefore bias the overall outcome being evaluated. Taken further, in an exercise designed to build relationships and links across disciplines or agencies, a facilitator intervention implying that a participant had made a mistake could be embarrassing or diminish that person's credibility, depending on the level of trust among participants.\nExercises should be as realistic as possible while remaining logistically feasible Taken together, the optimal mix of design elements represents a balance between exercise objectives and logistic feasibility. The ideal balance is one that assures sufficient realism to provide a meaningful experience while minimizing distractions associated with the necessary artifice of exercise scenarios. Some departures from reality may be inadvertent if scenarios are developed with insufficient attention to local routines, forcing participants to sidestep usual procedures. Even seemingly minor design errors, such as using an outdated name for a hospital, or a time course for a disease that is inconsistent with its known epidemiology, can undermine the credibility of the exercise and can distract participants enough to take them out of their roles, thus disrupting the flow of the exercise.\nThe desire for a realistic exercise scenario can lead to the development of tabletop exercises around scenarios rather than issue areas based on local preparedness needs and priorities. This does not ensure that the participants will address the important issue areas. A broad mix of challenges related to a given scenario must be addressed, often simultaneously, ranging from conducting epidemiologic or environmental investigations, implementing and modifying interventions as information becomes available, communicating within and across agencies, and communicating with political leaders and the public. Introducing this full set of tasks into an exercise scenario in a way that meaningfully exercises relevant capacities is unlikely to align with the exercise's objective. Moreover, different stakeholders may want to address different issue areas and may become frustrated if their expectations are not met. It is therefore important for stakeholders to agree on a limited number of priority issue areas for the exercise and then to focus the design of the scenario around these areas.\nFor example, one set of exercises we designed focused on pandemic influenza preparedness in local health departments. Because it was infeasible to exercise the entire pandemic plan around a single scenario and in a single exercise, we developed the scenario and then the exercise by first meeting with local stakeholders to decide on the issue areas that would be covered in the exercise. These issue areas included disease surveillance, medical surge capacity, non-pharmacological disease control, and the use of antiviral medications. The scenario was then customized to unfold to deal with each of these issue areas.\nKey decisions for each discussion point were then developed, as well as facilitator probes and instructions based on the specific objective of the exercise.\nIf not designed or facilitated properly, an exercise can lack focus and resolution, leaving participants to wonder what exactly was accomplished during the exercise. Therefore, it is paramount that exercises are designed to focus on issue areas that require concrete decisions over a limited period of time. For example, an exercise dealing with a simulated smallpox outbreak that unfolds over time might involve a discussion period dealing with movement restrictions in which participants are asked one or more questions such as, \"Should schools be closed at this point?\" Participants should then be given a limited amount of time to discuss this issue and make a decision. It is the facilitator's job to keep the discussion focused on the issue area and the specific question(s) at hand and to ensure that at the end of discussion, participants have collectively made the decision(s) they were tasked to make. Exercises can be designed to have multiple such issue area discussions as the scenario unfolds.\nDepending on the goals and objectives of an exercise, an exercise can involve a narrow or wide range of potential participants. While broader inclusion would likely be more realistic, such inclusiveness needs to be weighed against the logistics of effectively managing a larger number of participants and the potential adverse effects of inclusion. For example, participants may be less comfortable discussing ideas, taking risks, or making mistakes, depending on who is in the room. Such constraints may impede the exercise process and undermine achievement of exercise objectives.\nOne solution to this problem would be to sequentially stage the involvement of different participants or to physically separate different groups in a way that more closely mimics actual situations. For example, some conversations that involve airing uncertainties or weighing difficult alternatives may normally involve a limited group of people, and members of that group may be more comfortable exercising such a conversation apart from colleagues from other agencies or organizations.\nThe disadvantage of this approach is that it is substantially more difficult logistically and it diminishes the opportunity for people from different groups to gain an understanding of one another's role and approach to problems. In those exercises where certain participants, notably law enforcement, joined the scenarios at different stages, the feedback was generally critical, and participants felt that staging participation diminished learning and teambuilding opportunities. Another solution is to split exercise participants into two or more groups that allow everyone to participate, often placing people at similar levels of responsibility in the same group, and to conclude the exercise with a session that brings everyone together to share what they learned.\nThe exercises described in this report represented collaborations between people familiar with local circumstances and people from outside the participating jurisdictions who had expertise in exercise design and facilitation. Because we did not test an alternative approach that exclusively involved local personnel, it is difficult to generalize from this experience about the value of engaging people from outside the participating agencies. Nonetheless, it was our impression that at certain points in the development, facilitation, and feedback steps, there was value in involving people who were not personally invested in local relationships or situations and who could offer seemingly independent advice or perspectives.\nTabletop exercises can provide useful insights into both strengths and vulnerabilities in public health preparedness. It is important to recognize, however, that exercise outcomes are influenced by the way they are designed and conducted. The exercises described in this report emphasized varying dimensions of public health preparedness, reflecting differences in state or local priorities for prioritizing exercise objectives. For example, some emphasized the early response to initial reports of suspect illness while others emphasized management of surges in demand for health care services that are likely to occur later emergency scenarios. Given the intellectual and emotional demands of participation in an exercise, participants (and facilitators) may be less energetic during later rather than earlier stages of an exercise scenario, affecting perceived capacity to execute different elements of a response. Potential gaps between observed and actual preparedness should be considered in interpreting after-action reports and evaluating exercises themselves.\nThe utility of tabletop exercises as tools to identify areas for improvement and make improvements on these problems is still evolving. Our ability to evaluate exercise performance is hampered by the lack of an evidence base about what constitutes optimal performance and by the lack of standards for assessing public health preparedness. There is a need to move beyond qualitative performance measures to ones that are quantifiable and can be measured over time. These quantifiable measures can range from simple checklists to Likert rating scales to scorecards. For example, in a series of our exercises we used checklists to assess the performance of health departments related to surveillance, risk communication and other functions.\nOne fairly consistent observation was that health departments identified gaps that had been identified in prior exercises or actual experience, but had not yet been addressed. Reasons for this included lack of time, and lack of knowledge about how to make change. We now conclude exercises by having health departments prioritize the challenges observed during the exercise and then have them develop initial action plans related to up to three priority items.\nThere are important limitations to our work and its interpretation that must be recognized. First, the nature of our exercises changed over time on a number of important dimensions, including the scenario, priority objectives, facilitation, exercise designers and facilitators, and attention to beginning an action plan after the hot wash. As a result of this variation, we are unable to provide a numerical tabulation of the numbers of health departments that struggled with each gap or displayed given strengths. Second, because we did not employ a methodology that could conclusively assess change over time, we cannot be certain that the improvements we identified were truly reflective of improvement, and not due to the inclusion of more sophisticated health departments in the latter part of our exercise period. We doubt this is the case, however, given the national emphasis on preparedness and planning and the ways in which health departments participating in later years qualitatively described their improvement. Furthermore, similar observations regarding improvements in public health preparedness during the same time period have recently been reported by others [24, 25] . We also cannot asses the potential influences that external events (e.g., hurricanes, outbreaks) may have had on health departments during the time period of our work, but it is noteworthy that all exercises were concluded before Hurricane Katrina struck. In addition, our exercises were not conducted in a random sample of health departments, and the findings may not be generalizable to all health departments. Finally, as discussed above, the evidence base for determining best practices in the design and conduct of exercises is extremely thin. We share our experience in the hope that it will help others, but do not propose that our recommendations constitute best, proven practices.\nDeveloping, conducting, and evaluating tabletop exercises requires considerable planning and the perspectives of a variety of stakeholders. While these tabletop exercises identified both strengths and vulnerabilities in emergency preparedness, additional work is needed to develop reliable metrics to gauge exercise performance, inform followup action steps, and to develop re-evaluation exercise designs that assess the impact of post-exercise interventions." }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]

[ { "from": "human", "value": "Full Text: Microarray technology was invented to rapidly profile the quantities of mRNA transcripts in a particular cellular context [1] [2] [3] . Its application has become universal in biomedical researches. Although it is mRNA that is actually detected by microarray experiments, and it is mRNA that has the direct relationship with protein, the methodology and algorithms for data analysis are commonly gene based. As evident in the probe annotation file provided by Affymetrix, gene-level annotation is widely accepted even though it fails to discriminate multiple mRNAs transcribed from the same gene. As a result, the analysis results are usually summarized at the gene level, such as differentially expressed genes [4] [5] [6] or gene-sets [7, 8] . Even in the recent works that integrate protein-protein interaction data and microarray data [9] [10] [11] , the analysis unit is reduced to the gene instead of mRNA. This practice could be attributed to the fact that most of the functional knowledge is at gene level instead of transcript level, and the functional differences between mRNA variants transcribed from the same gene are seldom clear. Although the gene-level analysis ignores the difference among mRNAs variants, this strategy is still biologically meaningful considering that the diversity of genes is much higher than that of transcripts encoded by the same gene.\nIt has been well established that alternative splicing increases mRNA diversity, and over 60% of human genes are involved in this mechanism [12] . In addition, other RNA processing events, such as RNA editing, also account for the increased diversity at the mRNA level [13] . Since these events enable one gene to encode multiple proteins which might be functionally heterogeneous, we feel it necessary to separate transcript-level synonymous probesets from gene-level synonymous ones. The probesets that hybridize to more than one transcript variant of the same gene are referred to as gene-level synonymous probesets; while the ones that hybridize to a single variant are named as transcript-level synonymous probesets. It has been noticed that transcript-level synonymous probesets tend to have similar expression profiles, while gene-level synonymous probesets may have distinct expression profiles [14, 15] . In fact, individual reports demonstrated that the expression of transcript variants could be totally different [16, 17] . These phenomena indicate that the gene-level strategy of microarray data analysis is imprecise enough that one may overlook the expressional inconsistencies among gene-level synonymous probesets.\nAs a matter of fact, Affymetrix suffixes their probeset ID according to the probeset's specificity. For example, probesets that recognize unique transcript variants are suffixed with _at, and probesets that recognize multiple alternative transcripts from a single gene are suffixed with _a_at or _s_at, and so on. This suffix system gives a hint on the varied specificities of the probesets, and could be considered as an endeavor trying to do away with customers' worry about the gene-level data analysis strategy. However, the correctness of the suffix system has been in doubt [14, 18] . Therefore it is not reliable to perform transcriptlevel analysis on the basis of this imperfect suffix system. As the standard annotation files and most of the analysis algorithms are gene-oriented, analysts often average out the expression heterogeneity of the same gene when dealing with probeset level data [19] .\nIn this paper, we linked the probesets of two widely used Affymetrix arrays with the International Protein Indexes (IPIs) [20, 21] through proper association and rigorous alignment procedures, and demonstrated the statistically significant advantage of interpreting microarray data at the transcript-level or protein-level. Our results can be viewed as a more precise annotation of Affymetrix array's probesets, with which one may achieve a more reliable interpretation of their experimental data. Moreover, the application of this new annotation substantially increased the expression correlation between interacting proteins.\nTwo Affymetrix arrays, MOE430A_2 and HG-U133A, were chosen for this study, in which 21,097 and 16,213 putative protein-coding probesets for the two arrays were subject to the alignment investigation (Table 1) . Candidate probeset-mRNA relationships were compiled based on the probeset-gene mapping information specified in the standard Affymetrix annotation files and gene-mRNA mapping relationships provided by separate mRNA transcript sources. Rigorous blast procedure similar to that described in [22] were performed to filter these candidate probeset-mRNA relationships. Finally, the mRNA targets passing the filtering criteria were linked to protein IDs in the IPI database.\nThrough the rigorous association and alignment, we obtained precise annotations for 18,894 and 15,288 probesets in Affymetrix arrays MOE430A_2 and HG-U133A respectively (see Additional file 1). These annotations discriminate alternative mRNA variants transcribed from a same gene, thus are at transcript level as opposed to the standard gene-level annotation files provided by Affymetrix. It is worth noting that since the transcript data we used were quite redundant, a conceptual transcript variant may be represented by multiple redundant transcript accessions in the transcript database. In our transcriptlevel annotation file, each conceptual transcript is identified with one IPI ID, as we only investigated the probesets associated with protein-coding transcripts.\nStatistics on the non-control, investigated and annotated probesets, together with the number of involved genes and proteins, are shown in Table 1 . It is evident that the proportion of genes covered by our annotated probesets to those covered by all non-control probesets ('gene retaining percentage' in Table 1 ), 95.2% for MOE430A_2 and 85.4% for HG-U133A, are higher than the corresponding probeset retaining percentages, 83.5% and 68.8%, indicating that the gene coverage has only been slightly reduced by our filtering procedures. This observation is in support of our primary goal, that is to refine gene-level probeset annotations to transcript-level, but not to simply remove the poor-quality gene-level annotations.\nFurthermore, we classified the probesets based on the way they linked to proteins. By checking the alignment results, the probesets in our annotation tables were divided into two groups, namely one-to-one and one-to-many. In our annotation table, a one-to-one probeset was linked to only a single protein, while a one-to-many probeset was linked to multiple proteins due to alternative splicing of one gene. The rest of the investigated probesets that were not linked to any protein were categorized into the third group of \"one-to-null\". The statistics of these three groups are shown in Table 2 . As we know, Affymetrix suffixes their probeset ID according to the probeset's specificity. The over ten types of probeset suffixes can be categorized into three groups: transcript-specific, with '_at', gene-specific, with '_a_at' or '_s_at', and other suboptimal probesets that may cross-hybridize with unrelated sequences. The first two groups are comparable to our one-to-one and one-to-many probesets respectively. However, we found that, out of a total of 21,097 and 16,213 investigated probesets in MOE430A_2 and HG-U133A respectively (Table 1) , our one-to-one type accounts for only 53.7% and 43.3%, which were significantly lower than that of _at probesets, 74.0% and 72.6%, in the respective arrays. This suggests that some of _at probesets are not really specific for a transcript.\nTo further clarify the issue of probeset specificity, we grouped the investigated probesets with the varied Affymetrix suffixes as well as our own categorizing system (Table 3 ). It is evident that overall one-to-one and one-tomany take up the majority of the _at group and the _a_at/ _s_at group respectively, but there are some disagreements between the two classifications. For example, the Affymetrix _at group has 30.1% one-to-many probesets and 11.7% one-to-null probesets, suggesting that these so-called 'transcript-specific' probesets do not have the expected high specificity, and that they should be treated with caution in data analysis. On the other hand, the Affymetrix _a_at/_s_at group contains 42.0% one-to-one probesets. We attributed this mainly to the trimming of Table 2 : One-to-one and one-to-many probesets in our annotation tables.\nAnnotated probesets One-to-one probesets 11327 (53.7% of investigated) 7014 (43.3% of investigated) One-to-many probesets 7567 8274\nSum 18894 15288\nOne-to-null probesets 2203 925\nTotal (Investigated probesets) 21097 16213 the originally false transcripts during the update of sequence information, as they may lead to the absence of the transcript targets of some probesets. Similarly, there are a large number of one-to-one probesets in the socalled Affy others group, 1,206 out of 2,619 for MOE430A_2 and 476 out of 1,555 for HG-U133A. These probesets, however, might not be really one-to-one mapping to the identified transcript targets, as our strategy was based on the premise that most probesets were specific for certain genes, and our blast was limited to the candidate transcripts associated with the probesets, but not the entire transcript pool.\nThese subgroups of the investigated probesets with different Affymetrix suffixes indicate the imperfection of the Affymetrix suffix system, thus affirming the necessity of our transcript-level or protein-level probeset annotations. In fact, several other research groups have addressed the misleading nature of the Affymetrix suffix system and the imperfection of its standard annotation file, including some re-annotation works for array HG-U133 [14, 18, 19, 23] . We will discuss these related works in detail in next section.\nSince the probesets were linked to transcripts and proteins through rigorous association and alignment procedures, the expression profiles of transcript-level synonymous probesets were supposed to be more consistent than those of gene-level synonymous probesets [14, 15] . This was taken as the basis for the evaluation of our annotations.\nWe downloaded 30 expression datasets assayed with MOE430A_2 from the Gene Expression Omnibus database (GEO) [24, 25] , and calculated the Pearson correlation coefficients (PCCs) of expression profiles of synonymous probesets at gene level and transcript level. Since transcript-level synonymous probesets in our work recognized a single protein-coding transcript variant identified with a unique IPI ID, transcript-level synonymous probesets were also named as protein-level synonymous ones. Practically, we grouped gene-level synonymous probesets according to gene ID, and protein-level ones according to IPI ID. The probeset-protein mapping tables provided at NetAffx, the official protein-level annotation of Affymetrix probesets [26] , was used as comparison. Probesets corresponding to a single protein according to NetAffx were designated as 'Affy-protein' level synonymous probesets, which was a third level of synonymy.\nWithin each synonymous group, all pair-wise PCCs were calculated and then summarized to one value indicating the expression correlation of this group. The expression correlations of all synonymous groups at one level were then averaged into an overall value for a dataset (see Method section for details). The correlations of synonymous groups for 30 datasets at three different levels were depicted in parallel in Figure 1A . It is noticeable that the synonymous probesets at Affy-protein level show higher correlations than those at gene level, but the synonymous ones based on our protein level annotation show even higher correlations consistently over 30 datasets (p < 0.05 for 30 datasets under student's t-test, see Additional file 2 for details). The comparison proves the rationality and necessity of our protein-level probeset annotation.\nThese results support the argument that it is more reliable to interpret microarray data at transcript level than at gene level. The inferiority of the Affy-protein level annotation to our annotation could be attributed to the technical details in their alignment and association procedures [26] . First, they used the representative mRNA sequence ('consensus' or 'exemplar' sequence of each probeset), instead of the probe sequences themselves, as the query sequence in the alignment. Second, they aligned against the GenBank non-redundant protein database, rather than a comprehensive pool of mRNA sequences. In microarray experiment hybridization takes place between the probe sequences immobilized on the array and the cDNA sequences from the sample, so one can deduce that the alignment between the representative mRNA sequences and the protein sequences cannot precisely simulate the hybridization between probes and mRNAs. Finally, NetAffx filtered the blast results according to a cut- off of E-value, which indicates the likelihood of the observed alignment by chance [27] . Although frequently adopted for sequence homology analysis in closely related species, E-value is not sensitive enough to grade the many well aligned targets from the same species. In our practice, we took the probe sequences as the query and the mRNA sequences as the alignment targets, and adopted the matching nucleotide proportion as the filtering criterion (see Methods).\nA similar comparison was conducted for HG-U133A array, involving another transcript-level annotation by Harbig et al. [14] . Across the whole 28 datasets, the annotation by Harbig et al. showed advantage over Affy-protein-level annotation, while our transcript-level annotation performed best (p < 0.05 for 18 datasets under Student's t-test, see Figure 1B and Additional file 2). As our work was done two years later than Harbig et al.'s, the updated mRNA sequences in the probe-mRNA alignment is one of factors contributing to the increased performance. The other contributing factor is different approach we used to identify the mapping of probes to mRNA targets. Harbig et al. performed a two-phase blast: first, blast probeset target sequences against mRNA sequence pool, and then blast probe sequences against the retrieved mRNA sequences. Efficient as it was, this two-phase-blast strategy reduced the alignment precision as compared to our direct probe-against-mRNA blast strategy. Moreover, Harbig et al. accepted the mRNA with the highest average probe matches as the target of a probeset, even if the highest value could be suboptimal.\nAccording to our data, the puzzling fact that probesets for one gene may show variable expression profiles can be clarified when viewing the data at the protein level. Such genes are likely to involve alternative transcript variants which are constitutively different in expression levels. We looked at two genes as examples. Shown in Figure 2A , three probesets in GDS1277 dataset, 1448556_at, 1421382_at and 1451844_at, were mapped to the mouse Prlr gene (GeneID: 19116), with the first probeset correlating to protein IPI00321091, or PRL-R3, and the other two correlating to protein IPI00408593, or PRL-R2. The Prlr gene was reported to encode at least seven isoforms of prolactin receptor precursor in mouse [16] . The two probesets corresponding to the same protein IPI00408593, showed similar expression profiles as expected, with a PCC value of 0.6738 (P = 4.57e-6); while the probeset corresponding to another isoform, IPI00321091, showed a significantly different expression profile, with PCCs of 0.2530 and -0.0331 separately for 1421382_at and 1451844_at ( Figure 2A ). These data were consistent with the previous report that these two isoforms were predominantly expressed in liver and kidney, where PRL-R3 was highly expressed and PRL-R2 was weakly expressed [16] . We noticed that the shape of the expression curve of PRL-R3 seemed to be somewhat comparable to those of PRL-R2, although PRL-R3 probeset presented an overall higher expression level. These phenomena may suggest that PRL-R3 and PRL-R2 are partially co-regulated so that they show a comparable expression pattern, but they are expressed at different levels probably due to diverse roles of the regulatory elements.\nShown in Figure 2B , three probesets in GDS1076 dataset, 1419114_at, 1419115_at and 1419116_at, were mapped to the mouse gene Alg14 (GeneID: 66789), with the former two corresponding to IPI00132168 and the latter one corresponding to IPI00405947. Both proteins were indicated in IPI as homologs of yeast asparagine-linked glycosylation 14 without any further information. We notice two interesting phenomena in this case. First, the two probesets correlating to a same protein do not show similar expression profiles. Instead, they behave like the probesets correlating to different variants characterized by a PCC value of 0.4080 (P = 0.0415). This issue might be due to some factors causing microarray hybridization efficiency shift. Secondly, according to our calculation, the expressions of these two variants are negatively correlated with PCC values of -0.7740 (P = 5.04e-5) for 1419116_at and 1419115_at, and -0.4402 (P = 0.0296) for 1419116_at and 1419114_at. So far there are no reports on expression regulation of these two transcript variants of gene Alg14, and no function reports of the corresponding protein isoforms. This negative correlation suggested that these two proteins might perform different roles thus should be distinctly annotated.\nIn recent years, considerable efforts have been devoted to identifying and characterizing protein-protein interaction (PPI). Besides investigations on the molecular events involved in PPI, functional annotation of an unclassified protein according to its interacting partners is also an important topic [28] . Since it is too bold to infer protein functions according to the \"majority rule\" that utilizes only the PPI network structure [29, 30] , many studies integrate other data sources into the functional characterization of PPI, among which the gene expression data is the favorite [9, 31, 32] . All these works assumed that interacting protein pairs were characterized with higher expression correlation than random ones. However, previous investigations indicated that the relationship between expression correlation and PPI was weak on a genomic scale [33] [34] [35] although a recent work strengthened the association by integrating cross-species conservation information [10] .\nWe noticed that in these genome-scale studies PPI information was always first converted to gene pairs, after which the Pearson correlations of the probeset pairs corresponding to the gene pairs were evaluated. That is, the analysis targets were expanded from real interacting protein pairs to all possible cross-gene protein pairs for which interaction may not always exist. As illustrated in Figure 3 , suppose we have gene a (abbreviated to Ga) and gene b (Gb), with Ga encoding protein a1 (Pa1) and protein a2 (Pa2), Gb encoding protein b1 (Pb1) and protein b2 (Pb2). Among these protein variants, only proteins Pa2 and Pb1 interact with each other, while the other three possible cross-gene interactions, including Pa1-Pb1, Pa1-Pb2 and Pa2-Pb2, do not really happen. The four probesets, Pst_a1, Pst_a2, Pst_b1, and Pst_b2, recognize transcript variants Ta1, Ta2, Tb1 and Tb2 respectively, producing proteins Pa1, Pa2, Pb1 and Pb2. In the conventional genome-scale studies mentioned above, besides the probeset pair (Pst_a2, Pst_b1) corresponding to the real interacting protein pair, the other three cross-gene pairs, (Pst_a1, Pst_b1), (Pst_a1, Pst_b2) and (Pst_a2, Pst_b2), were also included, which would blunt the expression cor-Expression profiles of synonymous probesets Figure 2 Expression profiles of synonymous probesets. A) Three probesets on the array MOE430A_2 are associated with the mouse gene Prlr. Probeset 1448556_at correlates to protein IPI00321091, or PRL-R3; probesets 1421382_at and 1451844_at both point to protein IPI00408593, or PRL-R2. GEO dataset GDS1277 was analyzed. B) Three probesets on the array MOE430A_2 are associated with the mouse gene Alg14. Probesets 149114_at and 149115_at both point to protein IPI00132168; probeset 1419116_at correlates to protein IPI00405947. GEO dataset GDS1076 was analyzed.\nrelation between the real interacting entities according to our preceding observations. We propose that this might partly explain the weak coherency between PPI and expression correlation.\nIn order to validate our proposition, we investigated the relationship between protein interactions and expression correlation at both gene-level and protein-level perspectives, using 1,037 interacting protein pairs from the HPRD [36] and 28 microarray datasets assayed with HG-U133A from the GEO. As depicted in Figure 3 , the probeset pairs corresponding to all possible cross-gene protein pairs are termed as GGI pairs, out of which only the probeset pair corresponding to the real PPI, such as Pst_a2-Pst_b1, is a PPI pair. We calculated PCCs of both PPI pairs and GGI pairs for all available 1,037 interactions, evaluated the statistical significances of these PCC values under one-tailed t-test, and estimated the corresponding false discovery rates (FDR) using the SPLOSH FDR estimation method [37] .\nFor all datasets, we observed strengthened expression correlations between interacting proteins under the PPI schema relative to the GGI schema. Taking the GDS987 dataset including 41 arrays as an example ( Figure 4A 153, respectively, and the former was significantly smaller than the latter (Kolmogorov-Smirnov test p value is 9.9E-12). Summarizing the comparisons at the positive side and the negative side, we conclude that the expression correlation between interacting proteins is strengthened when the non-interacting protein pairs are excluded from the interacting gene pairs, that is, with the PPI PCC calculation in place of the conventional GGI PCC calculation.\nIn Figure 4A , we notice that the negative correlation is also strengthened by the PPI PCC calculation as well as the positive correlation. That is to say, PPI pairs seem to be either positively correlated or negatively correlated, but not exclusively 'co-expressed' as previous publications reported [10] . This phenomenon is more evident when we examine the PCC values for each coupled PPI pair and GGI pair. Figure 4B shows a scatter plot of the PPI PCC values versus the corresponding GGI PCC values. It is evident that most points fall into the 1st and the 3rd quadrants, indicating that each pair of PPI PCC value and GGI PCC value tends to have the same signs. The scatter plot suggests a linear relationship between the PPI PCCs and GGI PCCs, and indeed we get a linear regression formula, y = 0.5612x + 0.0046, at high confidence (p < 2e-16).\nSince the estimated coefficient, 0.5612, is far less than 1, we may conclude that the absolute PCC values of PPI pairs are often larger than those of the corresponding GGI pairs. So the PPI PCC calculation preserves the original positive or negative correlation tendency revealed by the conventional GGI PCC calculation, and strengthens it with larger absolute correlation values. Such correlation tendencies between interacting proteins, especially those negative ones, would very likely be submerged under the Figure 3 Illustration of Entities and terminologies involved in protein-protein interaction. A) Relationship among genes, transcripts, proteins and probesets. B) Two types of pairs mentioned in the text. PPI pair indicates the probeset pair correlating to a real interacting protein pair; while GGI pairs are those probeset pairs correlating to all possible cross-gene pairs. PPI may not exist in all cross-gene pairs.\nbackground correlations of random pairs if the non-interacting protein pairs are included in the analysis.\nSimilar observations were made over all datasets (see Additional file 4 and Additional file 5). Given the results from all 28 datasets, we were also able to compare the PPI PCC calculation and GGI PCC calculation at a higher level. Under a FDR threshold of 0.1, the list of significant PPI pairs or GGI pairs from each dataset was determined. Based on these lists, we counted the significantly correlated PPIs or GGIs (termed 'significant PPIs' or 'significant GGIs') over each dataset, and the datasets on which a PPI or GGI demonstrates significant correlation (termed 'PPIsignificant datasets' or 'GGI-significant datasets'). The former is a 'twenty-eight by two' table, shown in Table 4 ; the latter is a '1,037 by two' table, shown fully in Additional file 4 and partly in Table 5 . In both summary tables, we find that the statistics of PPI PCC calculation are mostly larger than the counterparts of GGI PCC calculation, indicating that PPI PCC calculation can detect more correlated PPI pairs in a certain experiment setting ( Table 4 ), and that it can detect the correlation tendency across more experiment settings ( Table 5 ).\nThe same experiments were also implemented with 274 PPI pairs extracted from the IntAct database [38] , and sim- Protein pairs with the false discovery rate of PCC value less than 0.1 were deemed as significantly correlated pairs. 'Significant PPIs' are pairs detected by the PPI calculation; 'Significant GGIs' are those detected by the GGI calculation. ilar conclusions were obtained. More details can be found in Additional file 6 and Additional file 7.\nIn this work, we re-annotated the probesets of two widely used Affymetrix arrays, MOE430A_2 and HG-U133A, via proper association and rigorous alignment procedures in a transcript perspective, and demonstrated the necessity and advantage of exploring microarray data at the transcript or protein level, instead of the conventional gene level.\nAlthough Affymetrix utilized the most complete information available at the time of array design, tremendous progress in genome sequencing and annotation in recent years renders existing probeset designs and target identifications suboptimal. In recent years, there have been continuous reports on systematic false expression signals of Affymetrix probesets [39] , spurious expression correlation caused by cross hybridization [18] , and expressional inconsistency among different microarray platforms or even different generations of one platform [40] [41] [42] [43] . A few research groups performed probe-against-mRNA blast similar to ours [22, 42, 44] , but mostly they centered around UniGene [45] and therefore improved the accuracy of annotation only at gene level. A major trend among these efforts was to redefine probesets so that probes matching the same molecular target were placed into custom probesets, as proposed by [19, 23, 39, 42] , but as the authors of [19] pointed out, 'these transcript-targeted probesets are not transcript-specific, as probesets targeting transcripts from the same gene may share many or even all probes'. Thus the probe re-organization strategy may be used to make distinction at the level of genes, but not at the level of transcripts or splice variants [18] . Besides, this strategy takes the probe-level intensity file (the CEL file) as a prerequisite, however only around half of the expression datasets deposited in public databases like GEO were found with CEL files.\nIn order to make distinction precisely at the transcript level, we preserved the classical Affymetrix probesets, but distinguished them among their alternatively spliced transcript targets according to the consistent alignments of probes against up-to-date mRNA sequences. Our annotation table clearly divides the Affymetrix probesets into three groups with increased transcript-level specificity (reliability): one-to-null probesets that do not recognize any transcript, one-to-many probesets that hybridize to multiple alternative transcript variants of the intended gene, and one-to-one probesets that hybridize to unique alternative transcript variants of the intended gene. We IPI1 IPI2 Gene1 Gene2 PPI-significant datasets GGI-significant datasets IPI00003326 IPI00015161 ARL2 PDE6D 15 7 IPI00003894 IPI00019930 RNF11 UBE2D1 17 1 IPI00007411 IPI00021831 AKAP11 PRKAR1A 15 2 IPI00008529 IPI00008527 RPLP2 RPLP1 25 5 IPI00011118 IPI00026689 RRM2 CDC2 17 16 IPI00013871 IPI00011118 RRM1 RRM2 14 10 IPI00015952 IPI00029012 EIF4G2 EIF3S10 14 3 IPI00016910 IPI00290460 EIF3S8 EIF3S4 16 10 IPI00018350 IPI00184330 MCM5 MCM2 16 15 IPI00021700 IPI00026689 PCNA CDC2 14 13 IPI00022865 IPI00026689 CCNA2 CDC2 16 15 IPI00026689 IPI00015105 CDC2 CKS2 15 13 IPI00027462 IPI00007047 S100A9 S100A8 23 17 IPI00028266 IPI00026689 CCNB2 CDC2 17 15 IPI00165506 IPI00025616 POLDIP2 POLD2 14 2 IPI00246058 IPI00025277 PDCD6IP PDCD6 16 1 IPI00290461 IPI00029012 EIF3S1 EIF3S10 15 4 IPI00291006 IPI00018206 MDH2 GOT2 16 11 IPI00294696 IPI00026689 CCNB1 CDC2 17 14 IPI00306708 IPI00026689 PBK CDC2 16 14 IPI00328118 IPI00026689 SPAG5 CDC2 15 10 IPI00604664 IPI00291006 NDUFS1 MDH2 18 10 IPI00647217 IPI00552920 SKIV2L2 EXOSC8 18 2 A PPI-significant dataset is a dataset where the false discovery rate of the PCC value for the PPI pair is less than 0.1, while a GGI-significant dataset is a dataset where the false discovery rate of the PCC value for the GGI pair is less than 0.1. Only PPI pairs with more than 14 'PPI-significant datasets' datasets are shown. The full table is provided in Additional file 4.\ndiscriminate the intended alternative transcript variants of Affymetrix probesets based on the NetAffx's gene-level annotation for the first time. Given the fact that existing solutions are accompanied with imperfections and no consensus has been reached on an overwhelming strategy, our alternative solution to the problematic standard annotation points out a new way to improve the interpretation and exploitation of Affymetrix microarray data.\nAlthough the transcript collections were not identical and the reannotation strategies differed more or less, we made out some similar discoveries to previous reports. For example, Harbig et al. found that a number of probesets did not detect any transcript and attributed this phenomenon to the elimination of the target sequence in the process of sequence update [14] . In our study, altogether 16.5% and 31.2% of non-control probesets in the MOE430A_2 and HG-U133A arrays were not found with any transcript targets in the pool of GenBank, RefSeq and Ensembl. Using newer and larger collection of transcript sequences, we even obtained a quite similar statistics of the percentage of 'multiple-targeting' probesets to that estimated in a foregoing study [18] , specifically 54.6% for MOE430A_2 and 54.1% HG-U133A (see Table 2 ). The significant mutual agreement among the related researches justifies the necessity to set up an improved annotation mechanism of the Affymetrix probes in the face of the continual growth of genomic and transcriptomic knowledge, ideally at transcript or protein level.\nOver the past few years, the analysis of alternative splicing has emerged as an important new field in bioinformatics, and several recent large-scale studies have shown that alternative splicing can be analyzed in a high-throughput manner using DNA-microarray methods [46, 47] . Most of these studies used arrays particularly manufactured for analyzing alternative splicing, such as genomic tiling array and exon-exon junction array. Constructed without any priori knowledge of the possible exon content of a genomic sequence, the genomic tiling array [48, 49] is in principle capable of detecting novel alternative splicing events of diverse types, but it is in doubt whether their data will be readily interpretable as successful experiences remain insufficient [46] . On the other hand, although designed particularly to address the alternative splicing issue, exon and exon-exon junction arrays [49] were reported to be plagued by problematic probe specificity and unsatisfying hybridization efficiency because of the necessity of probe coverage across the full length of the gene (including 5' end) [5] . Many questions about the reproducibility of the amplification protocol, the quantitative accuracy, and the data analysis need to be addressed as a prerequisite to reliable quantitative analysis using these splicing arrays [50] . Given the current imperfection of splicing array techniques and inconvenience in deci-phering their generated data, it is an economic way to do large-scale investigations of alternative transcribing events with standard gene expression arrays, provided that the recognizing targets of the probes can be rigorously defined at the transcript level. Hu et al. proposed a primitive analysis method to explore alternative splicing with Affymetrix 3' gene expression arrays, though they regretted that only alternative splicing biased toward the 3'end of the gene can be detected in their way [51] . In the present paper, we conducted a large-scale alignment of the probe sequences in traditional gene expression arrays against the currently most comprehensive collection of transcript sequences, highlighting the probesets mapping to unique alternative transcripts unambiguously. For each of the two Affymetrix expression arrays tested in this study, we found over 40% of all probesets could be mapped to transcripts in a oneto-one manner, so our work strongly validate that it is feasible to analyze alternative splicing using traditional gene expression arrays. While the foregoing work contributed by Hu et al. remains as a qualitative analysis method aiming at detecting novel alternative splicing events, our work gives explicitly the relationship between the probesets and the currently known alternative transcript variants, which can be immediately exploited to facilitate quantitative analysis of alternative variants. As our mapping relationships are defined for the standard probesets of the traditional gene expression arrays, they can be conveniently exploited as the standard NetAffx annotation information, without any ad hoc influence on the widely applied experiment protocols or the routine data processing algorithms. In the demonstrative implementations of the novel annotation tables, we actually observed several examples of negatively correlated alternative variants (see Figure 2B for one of them), which will shed light on further studies of expression regulation of alternative transcript variants.\nTo sum up, we re-annotated two popular Affymetrix gene expression arrays, MOE430_2 and HG-U133A, in a transcript-level perspective, aiming at identifying probesets' detecting targets precisely at the transcript level. Although previous works addressed similar issues [14, 15, 18, 19, 22, 23] , we are the first to rigorously link existing Affymetrix probesets to their specific transcript targets and their corresponding proteins. Armed with this new annotation, we re-examined a number of previous studies, 30 datasets for MOE430_2 and 28 datasets for HG-U133A from GEO, and revealed increased expression consistency among synonymous probesets and closer expression correlation among interacting proteins. This transcript-level annotation of Affymetrix probesets allows for a more reliable gene expression data analysis and a more accessible protein-level correlation study.\nThe Affymetrix 3' eukaryotic gene expression analysis arrays MOE430A_2 and HG-U133A were selected for this study. Probe sequence files and corresponding annotation files, 'Mouse430A_2_annot_csv.zip' (annotated on 2005-12-19) and 'HG-U133A_annot_csv.zip' (annotated on 2006-04-11), were downloaded from Affymetrix website [52] . Also downloaded there were the NetAffx probesetprotein mapping files for MOE430A_2 (file 'Mouse430A_2_blast_csv', updated on 2005-12-18) and HG-U133A ('HG-U133A.na21.blast.csv.zip', updated 2006-04-11), which were the blast results of the representative mRNA sequence of probes against protein sequence databases [26] . Ensembl transcripts: 37,854 mouse transcript sequences were obtained from the Ensembl database (release 38) [56] . Mapping tables between Ensembl Gene ID, Ensembl Transcript ID, and Ensembl Peptide ID were obtained from Ensembl martview [57] .\nIPI entries and their mappings to external protein accession numbers were acquired from the International Protein Index (IPI) database [21] (release 3.17). Also obtained there were the mapping relations between IPI numbers and transcript IDs (GenBank, RefSeq, and Ensembl). The counterpart file for human was downloaded there too (release 3.16).\nMicroarray datasets were downloaded from the Gene Expression Ominibus on April 15, 2006 . Array MOE430A_2, indexed as GPL339, was associated with 2,276 samples in GEO, ranking the second among all registered Affymetrix mouse arrays. All 31 GDS datasets profiled with MOE430A_2 were used in the analyses except for GDS1057, which contains only two samples. Array HG-U133A, indexed as GPL96, was associated with 8,698 samples in GEO, ranking the first among all registered Affymetrix human arrays. For our analysis, we downloaded 31 GDS datasets with largest sample sizes, and used 28 of them in our analyses, excluding GDS534, GDS1329, and GDS1324 as they are in a data format inconsistent with the others. Details about the used datasets can be found in our Additional file 8. \nOut of the total 22,690 and 22,283 probesets in arrays MOE430A_2 and HG-U133A, respectively, 64 and 68 control probesets were firstly removed. The remaining probesets were associated with genes according to the probeset-gene mapping information provided in Affymetrix's standard annotation file. The probeset-transcript mapping relationships were obtained based on the gene-mRNA mapping tables from GeneBank, RefSeq and Ensembl. In the process, we only included probesets that were identified with one Entrez Gene ID or one Ensembl gene ID. We ignored the probesets that were associated with multiple entities or no entity in the two gene-centric databases, since their gene-level specificity appears doubtful in the standard annotation file. This filtered out 3.2% and 5.2% of non-control probesets in MOE430A_2 and HG-U133A, respectively. For the rest of the probesets, we linked the candidate transcript targets to their corresponding protein entries in IPI database. IPI is currently the least redundant yet most complete protein database for featured species, with one protein sequence matching each transcript variant. Those probesets of which transcript tar-gets do not have any protein counterparts were also excluded from the following blast validation in order to focus our attention to the transcripts with well-characterized functions at protein level. The remaining probesets, 21,097 for MOE430A_2 and 16,213 for HG-U133A, were selected for the BLAST procedure.\nWe then filtered the candidate probeset-mRNA mapping relationships by aligning probe sequences in these probesets against their corresponding transcripts. Probes were blasted against their candidate mRNA targets using the bl2seq program [61] , and the probe to transcript matches were accepted if no more than one mismatch was found. At the level of probesets, the probeset to transcript matches were accepted only if more than 90% of all probes within a probeset (over 10 probes for the typical 11-probeset) were mapped to the transcript in the same orientation.\nThe probeset-transcript-protein links related to the above probesets passing BLAST filter were retrieved. After reducing the redundancy information of multiple transcripts corresponding to the same IPI, we finally obtained rigorous probeset annotation files for Affymetrix arrays MOE430A_2 and HG-U133A. There are two types of probesets in the new annotation file: one-to-one probesets, where one probeset maps to only one IPI ID; and one-to-many probesets, where one probeset maps to two or more IPI IDs. Only the one-to-one probesets were used in the subsequent analyses.\nWe grouped gene-level synonymous probesets according to gene ID (gene-level), and protein-level ones according to IPI ID (protein-level). Additionally, probesets corresponding to a single protein according to NetAffx probeset-protein mapping tables (see Materials) are grouped as 'Affy-protein' level synonymous probesets. In the case of the HG-U133A array, we included a fourth level, the 'Harbig-protein level', for comparison. The Harbig-protein level reflects the probeset-protein association transformed from a recent alternative annotation of the Affymetrix U133 plus 2.0 array [14] , also proposed in a transcript-level perspective. The downloaded annotation file mapped 33,579 probesets to 287,791 GenBank mRNA accessions, among which 21,669 were found on HG-U133A array, mapped to 186,085 GenBank mRNA accessions. The probeset-mRNA associations related to HG-U133A array were further linked to IPI IDs, finally giving rise to 26,960 probeset-IPI mapping relationships among which 12,146 were one-to-one. The following treatments were the same as those implemented to the standard gene-level annotation, the NetAffx Protein annotations, and our novel annotations. 30 and 28 expression datasets were selected from GEO respectively for MOE430A_2 and HG-U133A, and the original intensity data within each GEO dataset (GDS) were transformed to log 2 base and normalized to a constant median across all samples. For a synonymous group, if the expression values of all probesets in all samples were no larger than the constant median value, the probesets in this group were regarded not moderately expressed, and their expression profiles not informative enough. Therefore, we only kept the synonymous groups with at least one expression value above the constant median value, similar to the filtering procedure used by Tian et al. [62] . For the remaining synonymous groups, Pearson correlation coefficients (PCCs) were calculated for the expression profiles of each probeset pair. The minimum value of these PCCs was taken as a measurement of the expression coherence of this group. We used the minimum aggregation because the gene level synonymous probesets gave rise to within-protein PCCs (which are theoretically higher) and across-protein PCCs (which are theoretically lower), and the former was identical to the result of the protein-level synonymous group. In such a setting, the maximum did not result in any difference, and the average aggregation was not as sensitive as the minimum in terms of differentiating the two groups, so we adopted the minimum aggregation.\nFinally, the mean of the expression coherences of all synonymous groups over a dataset was calculated. In this way we obtained an evaluation of expression consistencies within synonymous probesets for a microarray dataset, and may compare the expression consistencies at the three levels over different microarray datasets.\nGiven protein-protein interaction data from HPRD or IntAct, we first transformed the binary relations of protein accessions to IPI-IPI pairs, and also got the corresponding Gene-Gene pairs. For each PPI, we assembled the PPI probeset pairs and the GGI probeset pairs as illustrated in Figure 3 , where PPI pairs are those associated with the interacting IPI IDs while GGI pairs are those associated with the corresponding Gene IDs. For all probeset pairs associated with the IPI-IPI pair (PPI pairs) and those associated with the corresponding gene-gene pair (GGI pairs), the PCCs were calculated and averaged into a PPI PCC and GGI PCC, respectively. These PCCs of interacting pairs were further calculated to obtain the accompanying false discovery rates using the SPLOSH FDR estimation method.\nThe distributions of the PPI PCCs and the GGI PCCs were plotted in a same figure to show the contrast ( Figure 4A ). In addition, a background distribution of the PCCs of ran-dom probeset pairs was overlaid on the same figure. We let the number of random pairs equal to the number of PPI or GGI pairs, but repeated the process of calculating random PCC distribution 20 times and averaged over the 20 separate random distributions in order to cut down on random fluctuation. Within each run of calculating random PCC distribution, we randomly compiled 2 × n (n = 1037 or 274, for HPRD or IntAct, respectively) pairs of probesets, where each two probeset pairs formed a group. The two PCCs of each group were firstly averaged into a group-level PCC, and the distribution was calculated over the n group-level PCCs. The group-level averaging was devised to mimic the counterpart operation in PPI PCC or GGI PCC calculation." }, { "from": "gpt", "value": "Summary: [Summary placeholder]" } ]