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800 | Interferon-Induced Ifit2/ISG54 Protects Mice from Lethal VSV Neuropathogenesis | Interferon protects mice from vesicular stomatitis virus (VSV) infection and pathogenesis; however, it is not known which of the numerous interferon-stimulated genes (ISG) mediate the antiviral effect. A prominent family of ISGs is the interferon-induced with tetratricopeptide repeats (Ifit) genes comprising three members in mice, Ifit1/ISG56, Ifit2/ISG54 and Ifit3/ISG49. Intranasal infection with a low dose of VSV is not lethal to wild-type mice and all three Ifit genes are induced in the central nervous system of the infected mice. We tested their potential contributions to the observed protection of wild-type mice from VSV pathogenesis, by taking advantage of the newly generated knockout mice lacking either Ifit2 or Ifit1. We observed that in Ifit2 knockout (Ifit2 (−/−)) mice, intranasal VSV infection was uniformly lethal and death was preceded by neurological signs, such as ataxia and hind limb paralysis. In contrast, wild-type and Ifit1 (−/−) mice were highly protected and survived without developing such disease. However, when VSV was injected intracranially, virus replication and survival were not significantly different between wild-type and Ifit2(−/−) mice. When administered intranasally, VSV entered the central nervous system through the olfactory bulbs, where it replicated equivalently in wild-type and Ifit2 (−/−) mice and induced interferon-β. However, as the infection spread to other regions of the brain, VSV titers rose several hundred folds higher in Ifit2 (−/−) mice as compared to wild-type mice. This was not caused by a broadened cell tropism in the brains of Ifit2 (−/−) mice, where VSV still replicated selectively in neurons. Surprisingly, this advantage for VSV replication in the brains of Ifit2(−/−) mice was not observed in other organs, such as lung and liver. Pathogenesis by another neurotropic RNA virus, encephalomyocarditis virus, was not enhanced in the brains of Ifit2 (−/−) mice. Our study provides a clear demonstration of tissue-, virus- and ISG-specific antiviral action of interferon. |
801 | Immunogenetic Mechanisms Driving Norovirus GII.4 Antigenic Variation | Noroviruses are the principal cause of epidemic gastroenteritis worldwide with GII.4 strains accounting for 80% of infections. The major capsid protein of GII.4 strains is evolving rapidly, resulting in new epidemic strains with altered antigenic potentials. To test if antigenic drift may contribute to GII.4 persistence, human memory B cells were immortalized and the resulting human monoclonal antibodies (mAbs) characterized for reactivity to a panel of time-ordered GII.4 virus-like particles (VLPs). Reflecting the complex exposure history of the volunteer, human anti-GII.4 mAbs grouped into three VLP reactivity patterns; ancestral (1987–1997), contemporary (2004–2009), and broad (1987–2009). NVB 114 reacted exclusively to the earliest GII.4 VLPs by EIA and blockade. NVB 97 specifically bound and blocked only contemporary GII.4 VLPs, while NBV 111 and 43.9 exclusively reacted with and blocked variants of the GII.4.2006 Minerva strain. Three mAbs had broad GII.4 reactivity. Two, NVB 37.10 and 61.3, also detected other genogroup II VLPs by EIA but did not block any VLP interactions with carbohydrate ligands. NVB 71.4 cross-neutralized the panel of time-ordered GII.4 VLPs, as measured by VLP-carbohydrate blockade assays. Using mutant VLPs designed to alter predicted antigenic epitopes, two evolving, GII.4-specific, blockade epitopes were mapped. Amino acids 294–298 and 368–372 were required for binding NVB 114, 111 and 43.9 mAbs. Amino acids 393–395 were essential for binding NVB 97, supporting earlier correlations between antibody blockade escape and carbohydrate binding variation. These data inform VLP vaccine design, provide a strategy for expanding the cross-blockade potential of chimeric VLP vaccines, and identify an antibody with broadly neutralizing therapeutic potential for the treatment of human disease. Moreover, these data support the hypothesis that GII.4 norovirus evolution is heavily influenced by antigenic variation of neutralizing epitopes and consequently, antibody-driven receptor switching; thus, protective herd immunity is a driving force in norovirus molecular evolution. |
802 | Induction of GADD34 Is Necessary for dsRNA-Dependent Interferon-β Production and Participates in the Control of Chikungunya Virus Infection | Nucleic acid sensing by cells is a key feature of antiviral responses, which generally result in type-I Interferon production and tissue protection. However, detection of double-stranded RNAs in virus-infected cells promotes two concomitant and apparently conflicting events. The dsRNA-dependent protein kinase (PKR) phosphorylates translation initiation factor 2-alpha (eIF2α) and inhibits protein synthesis, whereas cytosolic DExD/H box RNA helicases induce expression of type I-IFN and other cytokines. We demonstrate that the phosphatase-1 cofactor, growth arrest and DNA damage-inducible protein 34 (GADD34/Ppp1r15a), an important component of the unfolded protein response (UPR), is absolutely required for type I-IFN and IL-6 production by mouse embryonic fibroblasts (MEFs) in response to dsRNA. GADD34 expression in MEFs is dependent on PKR activation, linking cytosolic microbial sensing with the ATF4 branch of the UPR. The importance of this link for anti-viral immunity is underlined by the extreme susceptibility of GADD34-deficient fibroblasts and neonate mice to Chikungunya virus infection. |
803 | Targets and Intracellular Signaling Mechanisms for Deoxynivalenol-Induced Ribosomal RNA Cleavage | The trichothecene mycotoxin deoxynivalenol (DON), a known translational inhibitor, induces ribosomal RNA (rRNA) cleavage. Here, we characterized this process relative to (1) specific 18S and 28S ribosomal RNA cleavage sites and (2) identity of specific upstream signaling elements in this pathway. Capillary electrophoresis indicated that DON at concentrations as low as 200 ng/ml evoked selective rRNA cleavage after 6 h and that 1000 ng/ml caused cleavage within 2 h. Northern blot analysis revealed that DON exposure induced six rRNA cleavage fragments from 28S rRNA and five fragments from 18S rRNA. When selective kinase inhibitors were used to identify potential upstream signals, RNA-activated protein kinase (PKR), hematopoietic cell kinase (Hck), and p38 were found to be required for rRNA cleavage, whereas c-Jun N-terminal kinase and extracellular signal-regulated kinase were not. Furthermore, rRNA fragmentation was suppressed by the p53 inhibitors pifithrin-α and pifithrin-μ as well as the pan caspase inhibitor Z-VAD-FMK. Concurrent apoptosis was confirmed by acridine orange/ethidium bromide staining and flow cytometry. DON activated caspases 3, 8, and 9, thus suggesting the possible coinvolvement of both extrinsic and intrinsic apoptotic pathways in rRNA cleavage. Satratoxin G (SG), anisomycin, and ricin also induced specific rRNA cleavage profiles identical to those of DON, suggesting that ribotoxins might share a conserved rRNA cleavage mechanism. Taken together, DON-induced rRNA cleavage is likely to be closely linked to apoptosis activation and appears to involve the sequential activation of PKR/Hck →p38→p53→caspase 8/9→caspase 3. |
804 | Pilot Evaluation of RT-PCR/Electrospray Ionization Mass Spectrometry (PLEX-ID/Flu assay) on Influenza-Positive Specimens | The PLEX-ID/Flu assay has been recently developed to enable the detection and typing of influenza viruses based on the RT-PCR/electrospray ionization mass spectrometry technology. This novel assay was evaluated for typing performance on 201 positive influenza A or B nasopharyngeal swab specimens (NPS) detected by real-time RT-PCR during the 2010-2011 season. The PLEX-ID/Flu assay detected and characterized 91.3% and 95.3% of all influenza A and B samples, respectively. All non-typeable influenza A and B specimens by the assay showed low viral loads with threshold cycle values ≥ 33. Taken together, and although our results need to be confirmed by further prospective studies, the PLEX-ID/Flu assay detected positively and gave a typing result for 93% of all NPS detected positively by real-time RT-PCR, thus suggesting a potential role for influenza virus surveillance among other techniques. |
805 | Evaluation of atherosclerotic lesions using dextran- and mannan–dextran-coated USPIO: MRI analysis and pathological findings | Magnetic resonance imaging (MRI) can detect atherosclerotic lesions containing accumulations of ultrasmall superparamagnetic iron oxides (USPIO). Positing that improved USPIO with a higher affinity for atherosclerotic plaques would yield better plaque images, we performed MRI and histologic studies to compare the uptake of dextran- and mannan–dextran-coated USPIO (D-USPIO and DM-USPIO, respectively) by the atherosclerotic walls of rabbits. We intravenously injected atherosclerotic rabbits with DM-USPIO (n = 5) or D-USPIO (n = 5). Two rabbits were the controls. The doses delivered were 0.08 (dose 1) (n = 1), 0.4 (dose 2) (n = 1), or 0.8 (dose 3) (n = 3) mmol iron/Kg. The dose 3 rabbits underwent in vivo contrast-enhanced magnetic resonance angiography (MRA) before and 5 days after USPIO administration. Afterwards, all animals were euthanized, the aortae were removed and subjected to in vitro MRI study. The signal-to-noise ratio (SNR) of the aortic wall in the same region of interest (ROI) was calculated in both in vivo and in vitro studies. Histological assessment through measurement of iron-positive regions in Prussian blue-stained specimens showed that iron-positive regions were significantly larger in rabbits injected with DM- rather than D-USPIO (P < 0.05) for all doses. In vivo MRA showed that the SNR-reducing effect of DM- was greater than that of D-USPIO (P < 0.05). With in vitro MRI scans, SNR was significantly lower in rabbits treated with dose 2 of DM-USPIO compared with D-USPIO treatment (P < 0.05), and it tended to be lower at dose 3 (P < 0.1). In conclusion, we suggest that DM-USPIO is superior to D-USPIO for the study of atherosclerotic lesions in rabbits. |
806 | IL-15 Participates in the Respiratory Innate Immune Response to Influenza Virus Infection | Following influenza infection, natural killer (NK) cells function as interim effectors by suppressing viral replication until CD8 T cells are activated, proliferate, and are mobilized within the respiratory tract. Thus, NK cells are an important first line of defense against influenza virus. Here, in a murine model of influenza, we show that virally-induced IL-15 facilitates the trafficking of NK cells into the lung airways. Blocking IL-15 delays NK cell entry to the site of infection and results in a disregulated control of early viral replication. By the same principle, viral control by NK cells can be therapeutically enhanced via intranasal administration of exogenous IL-15 in the early days post influenza infection. In addition to controlling early viral replication, this IL-15-induced mobilization of NK cells to the lung airways has important downstream consequences on adaptive responses. Primarily, depletion of responding NK1.1+ NK cells is associated with reduced immigration of influenza-specific CD8 T cells to the site of infection. Together this work suggests that local deposits of IL-15 in the lung airways regulate the coordinated innate and adaptive immune responses to influenza infection and may represent an important point of immune intervention. |
807 | Screening for Influenza A(H1N1)pdm09, Auckland International Airport, New Zealand | Entry screening for influenza A(H1N1)pdm09 at Auckland International Airport, New Zealand, detected 4 cases, which were later confirmed, among 456,518 passengers arriving April 27–June 22, 2009. On the basis of national influenza surveillance data, which suggest that ≈69 infected travelers passed through the airport, sensitivity for screening was only 5.8%. |
808 | Immunologic Changes during Pandemic (H1N1) 2009, China | We analyzed changes in immunologic values over time for 28 hospitalized patients with pandemic (H1N1) 2009. Levels of interleukin-6, interferon-γ, and interleukin-10 increased 1 day after illness onset and then decreased to baseline levels. Levels of virus-specific antibody were undetectable 1 day after illness onset and peaked 36 days later. |
809 | Pandemic (H1N1) 2009 Risk for Frontline Health Care Workers | To determine whether frontline health care workers (HCWs) are at greater risk for contracting pandemic (H1N1) 2009 than nonclinical staff, we conducted a study of 231 HCWs and 215 controls. Overall, 79 (17.7%) of 446 had a positive antibody titer by hemagglutination inhibition, with 46 (19.9%) of 231 HCWs and 33 (15.3%) of 215 controls positive (OR 1.37, 95% confidence interval 0.84–2.22). Of 87 participants who provided a second serum sample, 1 showed a 4-fold rise in antibody titer; of 45 patients who had a nose swab sample taken during a respiratory illness, 7 had positive results. Higher numbers of children in a participant’s family and working in an intensive care unit were risk factors for infection; increasing age, working at hospital 2, and wearing gloves were protective factors. This highly exposed group of frontline HCWs was no more likely to contract pandemic (H1N1) 2009 influenza infection than nonclinical staff, which suggests that personal protective measures were adequate in preventing transmission. |
810 | Increased Urinary Angiotensin-Converting Enzyme 2 in Renal Transplant Patients with Diabetes | Angiotensin-converting enzyme 2 (ACE2) is expressed in the kidney and may be a renoprotective enzyme, since it converts angiotensin (Ang) II to Ang-(1-7). ACE2 has been detected in urine from patients with chronic kidney disease. We measured urinary ACE2 activity and protein levels in renal transplant patients (age 54 yrs, 65% male, 38% diabetes, n = 100) and healthy controls (age 45 yrs, 26% male, n = 50), and determined factors associated with elevated urinary ACE2 in the patients. Urine from transplant subjects was also assayed for ACE mRNA and protein. No subjects were taking inhibitors of the renin-angiotensin system. Urinary ACE2 levels were significantly higher in transplant patients compared to controls (p = 0.003 for ACE2 activity, and p≤0.001 for ACE2 protein by ELISA or western analysis). Transplant patients with diabetes mellitus had significantly increased urinary ACE2 activity and protein levels compared to non-diabetics (p<0.001), while ACE2 mRNA levels did not differ. Urinary ACE activity and protein were significantly increased in diabetic transplant subjects, while ACE mRNA levels did not differ from non-diabetic subjects. After adjusting for confounding variables, diabetes was significantly associated with urinary ACE2 activity (p = 0.003) and protein levels (p<0.001), while female gender was associated with urinary mRNA levels for both ACE2 and ACE. These data indicate that urinary ACE2 is increased in renal transplant recipients with diabetes, possibly due to increased shedding from tubular cells. Urinary ACE2 could be a marker of renal renin-angiotensin system activation in these patients. |
811 | PepMapper: A Collaborative Web Tool for Mapping Epitopes from Affinity-Selected Peptides | Epitope mapping from affinity-selected peptides has become popular in epitope prediction, and correspondingly many Web-based tools have been developed in recent years. However, the performance of these tools varies in different circumstances. To address this problem, we employed an ensemble approach to incorporate two popular Web tools, MimoPro and Pep-3D-Search, together for taking advantages offered by both methods so as to give users more options for their specific purposes of epitope-peptide mapping. The combined operation of Union finds as many associated peptides as possible from both methods, which increases sensitivity in finding potential epitopic regions on a given antigen surface. The combined operation of Intersection achieves to some extent the mutual verification by the two methods and hence increases the likelihood of locating the genuine epitopic region on a given antigen in relation to the interacting peptides. The Consistency between Intersection and Union is an indirect sufficient condition to assess the likelihood of successful peptide-epitope mapping. On average from 27 tests, the combined operations of PepMapper outperformed either MimoPro or Pep-3D-Search alone. Therefore, PepMapper is another multipurpose mapping tool for epitope prediction from affinity-selected peptides. The Web server can be freely accessed at: http://informatics.nenu.edu.cn/PepMapper/ |
812 | Autophagy: More Than a Nonselective Pathway | Autophagy is a catabolic pathway conserved among eukaryotes that allows cells to rapidly eliminate large unwanted structures such as aberrant protein aggregates, superfluous or damaged organelles, and invading pathogens. The hallmark of this transport pathway is the sequestration of the cargoes that have to be degraded in the lysosomes by double-membrane vesicles called autophagosomes. The key actors mediating the biogenesis of these carriers are the autophagy-related genes (ATGs). For a long time, it was assumed that autophagy is a bulk process. Recent studies, however, have highlighted the capacity of this pathway to exclusively eliminate specific structures and thus better fulfil the catabolic necessities of the cell. We are just starting to unveil the regulation and mechanism of these selective types of autophagy, but what it is already clearly emerging is that structures targeted to destruction are accurately enwrapped by autophagosomes through the action of specific receptors and adaptors. In this paper, we will briefly discuss the impact that the selective types of autophagy have had on our understanding of autophagy. |
813 | The Sigma Class Glutathione Transferase from the Liver Fluke Fasciola hepatica | BACKGROUND: Liver fluke infection of livestock causes economic losses of over US$ 3 billion worldwide per annum. The disease is increasing in livestock worldwide and is a re-emerging human disease. There are currently no commercial vaccines, and only one drug with significant efficacy against adult worms and juveniles. A liver fluke vaccine is deemed essential as short-lived chemotherapy, which is prone to resistance, is an unsustainable option in both developed and developing countries. Protein superfamilies have provided a number of leading liver fluke vaccine candidates. A new form of glutathione transferase (GST) family, Sigma class GST, closely related to a leading Schistosome vaccine candidate (Sm28), has previously been revealed by proteomics in the liver fluke but not functionally characterised. METHODOLOGY/PRINCIPAL FINDINGS: In this manuscript we show that a purified recombinant form of the F. hepatica Sigma class GST possesses prostaglandin synthase activity and influences activity of host immune cells. Immunocytochemistry and western blotting have shown the protein is present near the surface of the fluke and expressed in eggs and newly excysted juveniles, and present in the excretory/secretory fraction of adults. We have assessed the potential to use F. hepatica Sigma class GST as a vaccine in a goat-based vaccine trial. No significant reduction of worm burden was found but we show significant reduction in the pathology normally associated with liver fluke infection. CONCLUSIONS/SIGNIFICANCE: We have shown that F. hepatica Sigma class GST has likely multi-functional roles in the host-parasite interaction from general detoxification and bile acid sequestration to PGD synthase activity. |
814 | Discriminating Active from Latent Tuberculosis in Patients Presenting to Community Clinics | BACKGROUND: Because of the high global prevalence of latent TB infection (LTBI), a key challenge in endemic settings is distinguishing patients with active TB from patients with overlapping clinical symptoms without active TB but with co-existing LTBI. Current methods are insufficiently accurate. Plasma proteomic fingerprinting can resolve this difficulty by providing a molecular snapshot defining disease state that can be used to develop point-of-care diagnostics. METHODS: Plasma and clinical data were obtained prospectively from patients attending community TB clinics in Peru and from household contacts. Plasma was subjected to high-throughput proteomic profiling by mass spectrometry. Statistical pattern recognition methods were used to define mass spectral patterns that distinguished patients with active TB from symptomatic controls with or without LTBI. RESULTS: 156 patients with active TB and 110 symptomatic controls (patients with respiratory symptoms without active TB) were investigated. Active TB patients were distinguishable from undifferentiated symptomatic controls with accuracy of 87% (sensitivity 84%, specificity 90%), from symptomatic controls with LTBI (accuracy of 87%, sensitivity 89%, specificity 82%) and from symptomatic controls without LTBI (accuracy 90%, sensitivity 90%, specificity 92%). CONCLUSIONS: We show that active TB can be distinguished accurately from LTBI in symptomatic clinic attenders using a plasma proteomic fingerprint. Translation of biomarkers derived from this study into a robust and affordable point-of-care format will have significant implications for recognition and control of active TB in high prevalence settings. |
815 | Novel Evidence of HBV Recombination in Family Cluster Infections in Western China | Two hepatitis B virus (HBV) C/D recombinants were isolated from western China. No direct evidence indicates that these new viruses arose as a result of recombination between genotype C and D or a result of convergence. In this study, we search for evidence of intra-individual recombination in the family cluster cases with co-circulation of genotype C, D and C/D recombinants. We studied 68 individuals from 15 families with HBV infections in 2006, identified individuals with mixed HBV genotype co-infections by restriction fragment length polymorphism and proceeded with cloning and DNA sequencing. Recombination signals were detected by RDP3 software and confirmed by split phylogenetic trees. Families with mixed HBV genotype co-infections were resampled in 2007. Three of 15 families had individuals with different HBV genotype co-infections in 2006. One individual (Y2) had a triple infection of HBV genotype C, D and C/D recombinant in 2006, but only genotype D in 2007. Further clonal analysis of this patient indicated that the C/D recombinant was not identical to previously isolated CD1 or CD2, but many novel recombinants with C2, D1 and CD1 were simultaneously found. All parental strains could recombine with each other to form new recombinant in this patient. This indicates that the detectable mixed infection and recombination have a limited time window. Also, as the recombinant nature of HBV precludes the possibility of a simple phylogenetic taxonomy, a new standard may be required for classifying HBV sequences. |
816 | Identifying Live Bird Markets with the Potential to Act as Reservoirs of Avian Influenza A (H5N1) Virus: A Survey in Northern Viet Nam and Cambodia | Wet markets are common in many parts of the world and may promote the emergence, spread and maintenance of livestock pathogens, including zoonoses. A survey was conducted in order to assess the potential of Vietnamese and Cambodian live bird markets (LBMs) to sustain circulation of highly pathogenic avian influenza virus subtype H5N1 (HPAIV H5N1). Thirty Vietnamese and 8 Cambodian LBMs were visited, and structured interviews were conducted with the market managers and 561 Vietnamese and 84 Cambodian traders. Multivariate and cluster analysis were used to construct a typology of traders based on their poultry management practices. As a result of those practices and large poultry surplus (unsold poultry reoffered for sale the following day), some poultry traders were shown to promote conditions favorable for perpetuating HPAIV H5N1 in LBMs. More than 80% of these traders operated in LBMs located in the most densely populated areas, Ha Noi and Phnom Penh. The profiles of sellers operating at a given LBM could be reliably predicted using basic information about the location and type of market. Consequently, LBMs with the largest combination of risk factors for becoming virus reservoirs could be easily identified, potentially allowing control strategies to be appropriately targeted. These findings are of particular relevance to resource-scarce settings with extensively developed LBM systems, commonly found in South-East Asia. |
817 | Porcine Noroviruses Related to Human Noroviruses | Detection of genogroup II (GII) norovirus (NoV) RNA from adult pigs in Japan and Europe and GII NoV antibodies in US swine raises public health concerns about zoonotic transmission of porcine NoVs to humans, although no NoVs have been detected in US swine. To detect porcine NoVs and to investigate their genetic diversity and relatedness to human NoVs, 275 fecal samples from normal US adult swine were screened by reverse transcription–polymerase chain reaction with calicivirus universal primers. Six samples were positive for NoV. Based on sequence analysis of 3 kb on the 3´ end of 5 porcine NoVs, 3 genotypes in GII and a potential recombinant were identified. One genotype of porcine NoVs was genetically and antigenically related to human NoVs and replicated in gnotobiotic pigs. These results raise concerns of whether subclinically infected adult swine may be reservoirs of new human NoVs or if porcine/human GII recombinants could emerge. |
818 | Clinical Relevance and Discriminatory Value of Elevated Liver Aminotransferase Levels for Dengue Severity | BACKGROUND: Elevation of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) is prominent in acute dengue illness. The World Health Organization (WHO) 2009 dengue guidelines defined AST or ALT≥1000 units/liter (U/L) as a criterion for severe dengue. We aimed to assess the clinical relevance and discriminatory value of AST or ALT for dengue hemorrhagic fever (DHF) and severe dengue. METHODOLOGY/PRINCIPAL FINDINGS: We retrospectively studied and classified polymerase chain reaction positive dengue patients from 2006 to 2008 treated at Tan Tock Seng Hospital, Singapore according to WHO 1997 and 2009 criteria for dengue severity. Of 690 dengue patients, 31% had DHF and 24% severe dengue. Elevated AST and ALT occurred in 86% and 46%, respectively. Seven had AST or ALT≥1000 U/L. None had acute liver failure but one patient died. Median AST and ALT values were significantly higher with increasing dengue severity by both WHO 1997 and 2009 criteria. However, they were poorly discriminatory between non-severe and severe dengue (e.g., AST area under the receiver operating characteristic [ROC] curve = 0.62; 95% confidence interval [CI]: 0.57–0.67) and between dengue fever (DF) and DHF (AST area under the ROC curve = 0.56; 95% CI: 0.52–0.61). There was significant overlap in AST and ALT values among patients with dengue with or without warning signs and severe dengue, and between those with DF and DHF. CONCLUSIONS: Although aminotransferase levels increased in conjunction with dengue severity, AST or ALT values did not discriminate between DF and DHF or non-severe and severe dengue. |
819 | Directed Fusion of Mesenchymal Stem Cells with Cardiomyocytes via VSV-G Facilitates Stem Cell Programming | Mesenchymal stem cells (MSCs) spontaneously fuse with somatic cells in vivo, albeit rarely, and the fusion products are capable of tissue-specific function (mature trait) or proliferation (immature trait), depending on the microenvironment. That stem cells can be programmed, or somatic cells reprogrammed, in this fashion suggests that stem cell fusion holds promise as a therapeutic approach for the repair of damaged tissues, especially tissues not readily capable of functional regeneration, such as the myocardium. In an attempt to increase the frequency of stem cell fusion and, in so doing, increase the potential for cardiac tissue repair, we expressed the fusogen of the vesicular stomatitis virus (VSV-G) in human MSCs. We found VSV-G expressing MSCs (vMSCs) fused with cardiomyocytes (CMs) and these fusion products adopted a CM-like phenotype and morphology in vitro. In vivo, vMSCs delivered to damaged mouse myocardium via a collagen patch were able to home to the myocardium and fuse to cells within the infarct and peri-infarct region of the myocardium. This study provides a basis for the investigation of the biological impact of fusion of stem cells with CMs in vivo and illustrates how viral fusion proteins might better enable such studies. |
820 | Design Novel Dual Agonists for Treating Type-2 Diabetes by Targeting Peroxisome Proliferator-Activated Receptors with Core Hopping Approach | Owing to their unique functions in regulating glucose, lipid and cholesterol metabolism, PPARs (peroxisome proliferator-activated receptors) have drawn special attention for developing drugs to treat type-2 diabetes. By combining the lipid benefit of PPAR-alpha agonists (such as fibrates) with the glycemic advantages of the PPAR-gamma agonists (such as thiazolidinediones), the dual PPAR agonists approach can both improve the metabolic effects and minimize the side effects caused by either agent alone, and hence has become a promising strategy for designing effective drugs against type-2 diabetes. In this study, by means of the powerful “core hopping” and “glide docking” techniques, a novel class of PPAR dual agonists was discovered based on the compound GW409544, a well-known dual agonist for both PPAR-alpha and PPAR-gamma modified from the farglitazar structure. It was observed by molecular dynamics simulations that these novel agonists not only possessed the same function as GW409544 did in activating PPAR-alpha and PPAR-gamma, but also had more favorable conformation for binding to the two receptors. It was further validated by the outcomes of their ADME (absorption, distribution, metabolism, and excretion) predictions that the new agonists hold high potential to become drug candidates. Or at the very least, the findings reported here may stimulate new strategy or provide useful insights for discovering more effective dual agonists for treating type-2 diabetes. Since the “core hopping” technique allows for rapidly screening novel cores to help overcome unwanted properties by generating new lead compounds with improved core properties, it has not escaped our notice that the current strategy along with the corresponding computational procedures can also be utilized to find novel and more effective drugs for treating other illnesses. |
821 | Prognosis of hospitalized patients with 2009 H1N1 influenza in Spain: influence of neuraminidase inhibitors | BACKGROUND: The H1N1 influenza pandemic strain has been associated with a poor prognosis in hospitalized patients. The present report evaluates the factors influencing prognosis. METHODS: A total of 813 patients hospitalized with H1N1 influenza in 36 hospitals (nationwide) in Spain were analysed. Detailed histories of variables preceding hospital admission were obtained by interview, validating data on medications and vaccine with their attending physicians. Data on treatment and complications during hospital stay were recorded. As definition of poor outcome, the endpoints of death and admission to intensive care were combined; and as a further outcome, length of stay was used. RESULTS: The mean age was 38.5 years (SD 22.8 years). There were 10 deaths and 79 admissions to intensive care (combined, 88). The use of neuraminidase inhibitors was reported by 495 patients (60.9%). The variables significantly associated with a poor outcome were diabetes (OR = 2.21, 95% CI = 1.21–4.02), corticosteroid therapy (OR = 3.37, 95% CI = 1.39–8.20) and use of histamine-2 receptor antagonists (OR = 2.68, 95% CI = 1.14–6.36), while the use of neuraminidase inhibitors (OR = 0.57, 95% CI = 0.34–0.94) was protective. Neuraminidase inhibitors within the first 2 days after the influenza onset reduced hospital stay by a mean of 1.9 days (95% CI = 4.7–6.6). CONCLUSIONS: The use of neuraminidase inhibitors decreases the length of hospital stay and admission to intensive care and/or death. |
822 | Veillonella montpellierensis Endocarditis | Veillonella spp. rarely cause infections in humans. We report a case of Veillonella endocarditis documented by isolating a slow-growing, gram-negative microbe in blood cultures. This microbe was identified as the newly recognized species Veillonella montpellierensis (100% homology) by 16S RNA gene sequence analysis. |
823 | Primate-to-Human Retroviral Transmission in Asia | We describe the first reported transmission to a human of simian foamy virus (SFV) from a free-ranging population of nonhuman primates in Asia. The transmission of an exogenous retrovirus, SFV, from macaques (Macaca fascicularis) to a human at a monkey temple in Bali, Indonesia, was investigated with molecular and serologic techniques. Antibodies to SFV were detected by Western blotting of serum from 1 of 82 humans tested. SFV DNA was detected by nested polymerase chain reaction (PCR) from the blood of the same person. Cloning and sequencing of PCR products confirmed the virus's close phylogenetic relationship to SFV isolated from macaques at the same temple. This study raises concerns that persons who work at or live around monkey temples are at risk for infection with SFV. |
824 | Application of Consensus Scoring and Principal Component Analysis for Virtual Screening against β-Secretase (BACE-1) | BACKGROUND: In order to identify novel chemical classes of β-secretase (BACE-1) inhibitors, an alternative scoring protocol, Principal Component Analysis (PCA), was proposed to summarize most of the information from the original scoring functions and re-rank the results from the virtual screening against BACE-1. METHOD: Given a training set (50 BACE-1 inhibitors and 9950 inactive diverse compounds), three rank-based virtual screening methods, individual scoring, conventional consensus scoring and PCA, were judged by the hit number in the top 1% of the ranked list. The docking poses were generated by Surflex, five scoring functions (Surflex_Score, D_Score, G_Score, ChemScore, and PMF_Score) were used for pose extraction. For each pose group, twelve scoring functions (Surflex_Score, D_Score, G_Score, ChemScore, PMF_Score, LigScore1, LigScore2, PLP1, PLP2, jain, Ludi_1, and Ludi_2) were used for the pose rank. For a test set, 113,228 chemical compounds (Sigma-Aldrich® corporate chemical directory) were docked by Surflex, then ranked by the same three ranking methods motioned above to select the potential active compounds for experimental test. RESULTS: For the training set, the PCA approach yielded consistently superior rankings compared to conventional consensus scoring and single scoring. For the test set, the top 20 compounds according to conventional consensus scoring were experimentally tested, no inhibitor was found. Then, we relied on PCA scoring protocol to test another different top 20 compounds and two low micromolar inhibitors (S450588 and 276065) were emerged through the BACE-1 fluorescence resonance energy transfer (FRET) assay. CONCLUSION: The PCA method extends the conventional consensus scoring in a quantitative statistical manner and would appear to have considerable potential for chemical screening applications. |
825 | 2,500-year Evolution of the Term Epidemic | The term epidemic (from the Greek epi [on] plus demos [people]), first used by Homer, took its medical meaning when Hippocrates used it as the title of one of his famous treatises. At that time, epidemic was the name given to a collection of clinical syndromes, such as coughs or diarrheas, occurring and propagating in a given period at a given location. Over centuries, the form and meaning of the term have changed. Successive epidemics of plague in the Middle Ages contributed to the definition of an epidemic as the propagation of a single, well-defined disease. The meaning of the term continued to evolve in the 19th-century era of microbiology. Its most recent semantic evolution dates from the last quarter of the 20th century, and this evolution is likely to continue in the future. |
826 | The Acute Environment, Rather than T Cell Subset Pre-Commitment, Regulates Expression of the Human T Cell Cytokine Amphiregulin | Cytokine expression patterns of T cells can be regulated by pre-commitment to stable effector phenotypes, further modification of moderately stable phenotypes, and quantitative changes in cytokine production in response to acute signals. We showed previously that the epidermal growth factor family member Amphiregulin is expressed by T cell receptor-activated mouse CD4 T cells, particularly Th2 cells, and helps eliminate helminth infection. Here we report a detailed analysis of the regulation of Amphiregulin expression by human T cell subsets. Signaling through the T cell receptor induced Amphiregulin expression by most or all T cell subsets in human peripheral blood, including naive and memory CD4 and CD8 T cells, Th1 and Th2 in vitro T cell lines, and subsets of memory CD4 T cells expressing several different chemokine receptors and cytokines. In these different T cell types, Amphiregulin synthesis was inhibited by an antagonist of protein kinase A, a downstream component of the cAMP signaling pathway, and enhanced by ligands that increased cAMP or directly activated protein kinase A. Prostaglandin E2 and adenosine, natural ligands that stimulate adenylyl cyclase activity, also enhanced Amphiregulin synthesis while reducing synthesis of most other cytokines. Thus, in contrast to mouse T cells, Amphiregulin synthesis by human T cells is regulated more by acute signals than pre-commitment of T cells to a particular cytokine pattern. This may be appropriate for a cytokine more involved in repair than attack functions during most inflammatory responses. |
827 | Allo-SCT for multiple myeloma: a review of outcomes at a single transplant center | Allogeneic stem cell transplant for multiple myeloma (MM) is one treatment associated with long-term disease-free survival. The high incidence of treatment-related mortality and relapses, however, are important reasons for controversy about the role of allografting in the management of MM. We reviewed our results of allografting for MM spanning a period of 34 years in order to better define long-term outcomes and identify areas of progress as well as areas requiring improvement. A total of 278 patients received allogeneic marrow or PBSCs after high-dose myeloablative (N=144) or reduced intensity, non-myeloablative (N=134) regimens. In multivariable analysis, adjusting for differences in patient groups, reduced intensity/non-myeloablative transplants were associated with significantly less acute GVHD, lower transplant mortality, better PFS and overall survival. There were no significant differences in relapse, progression or chronic GVHD, when adjusted. In multivariable analysis of patients receiving only non-myeloablative transplants, decreased overall survival and PFS were associated with relapse after a prior autograft and a β2 microglobulin >4.0. Transplant mortality was reduced and only influenced by a prior tandem autograft. |
828 | UNC93B1 Mediates Innate Inflammation and Antiviral Defense in the Liver during Acute Murine Cytomegalovirus Infection | Antiviral defense in the liver during acute infection with the hepatotropic virus murine cytomegalovirus (MCMV) involves complex cytokine and cellular interactions. However, the mechanism of viral sensing in the liver that promotes these cytokine and cellular responses has remained unclear. Studies here were undertaken to investigate the role of nucleic acid-sensing Toll-like receptors (TLRs) in initiating antiviral immunity in the liver during infection with MCMV. We examined the host response of UNC93B1 mutant mice, which do not signal properly through TLR3, TLR7 and TLR9, to acute MCMV infection to determine whether liver antiviral defense depends on signaling through these molecules. Infection of UNC93B1 mutant mice revealed reduced production of systemic and liver proinflammatory cytokines including IFN-α, IFN-γ, IL-12 and TNF-α when compared to wild-type. UNC93B1 deficiency also contributed to a transient hepatitis later in acute infection, evidenced by augmented liver pathology and elevated systemic alanine aminotransferase levels. Moreover, viral clearance was impaired in UNC93B1 mutant mice, despite intact virus-specific CD8+ T cell responses in the liver. Altogether, these results suggest a combined role for nucleic acid-sensing TLRs in promoting early liver antiviral defense during MCMV infection. |
829 | Bioinformatics analysis of rabbit haemorrhagic disease virus genome | BACKGROUND: Rabbit haemorrhagic disease virus (RHDV), as the pathogeny of Rabbit haemorrhagic disease, can cause a highly infectious and often fatal disease only affecting wild and domestic rabbits. Recent researches revealed that it, as one number of the Caliciviridae, has some specialties in its genome, its reproduction and so on. RESULTS: In this report, we firstly analyzed its genome and two open reading frameworks (ORFs) from this aspect of codon usage bias. Our researches indicated that mutation pressure rather than natural is the most important determinant in RHDV with high codon bias, and the codon usage bias is nearly contrary between ORF1 and ORF2, which is maybe one of factors regulating the expression of VP60 (encoding by ORF1) and VP10 (encoding by ORF2). Furthermore, negative selective constraints on the RHDV whole genome implied that VP10 played an important role in RHDV lifecycle. CONCLUSIONS: We conjectured that VP10 might be beneficial for the replication, release or both of virus by inducing infected cell apoptosis initiate by RHDV. According to the results of the principal component analysis for ORF2 of RSCU, we firstly separated 30 RHDV into two genotypes, and the ENC values indicated ORF1 and ORF2 were independent among the evolution of RHDV. |
830 | Oligonucleotide Based Magnetic Bead Capture of Onchocerca volvulus DNA for PCR Pool Screening of Vector Black Flies | BACKGROUND: Entomological surveys of Simulium vectors are an important component in the criteria used to determine if Onchocerca volvulus transmission has been interrupted and if focal elimination of the parasite has been achieved. However, because infection in the vector population is quite rare in areas where control has succeeded, large numbers of flies need to be examined to certify transmission interruption. Currently, this is accomplished through PCR pool screening of large numbers of flies. The efficiency of this process is limited by the size of the pools that may be screened, which is in turn determined by the constraints imposed by the biochemistry of the assay. The current method of DNA purification from pools of vector black flies relies upon silica adsorption. This method can be applied to screen pools containing a maximum of 50 individuals (from the Latin American vectors) or 100 individuals (from the African vectors). METHODOLOGY/PRINCIPAL FINDINGS: We have evaluated an alternative method of DNA purification for pool screening of black flies which relies upon oligonucleotide capture of Onchocerca volvulus genomic DNA from homogenates prepared from pools of Latin American and African vectors. The oligonucleotide capture assay was shown to reliably detect one O. volvulus infective larva in pools containing 200 African or Latin American flies, representing a two-four fold improvement over the conventional assay. The capture assay requires an equivalent amount of technical time to conduct as the conventional assay, resulting in a two-four fold reduction in labor costs per insect assayed and reduces reagent costs to $3.81 per pool of 200 flies, or less than $0.02 per insect assayed. CONCLUSIONS/SIGNIFICANCE: The oligonucleotide capture assay represents a substantial improvement in the procedure used to detect parasite prevalence in the vector population, a major metric employed in the process of certifying the elimination of onchocerciasis. |
831 | Prediction and Identification of T Cell Epitopes in the H5N1 Influenza Virus Nucleoprotein in Chicken | T cell epitopes can be used for the accurate monitoring of avian influenza virus (AIV) immune responses and the rational design of vaccines. No T cell epitopes have been previously identified in the H5N1 AIV virus nucleoprotein (NP) in chickens. For the first time, this study used homology modelling techniques to construct three-dimensional structures of the peptide-binding domains of chicken MHC class Ι molecules for four commonly encountered unique haplotypes, i.e., B4, B12, B15, and B19. H5N1 AIV NP was computationally parsed into octapeptides or nonapeptides according to the peptide-binding motifs of MHC class I molecules of the B4, B12, B15 and B19 haplotypes. Seventy-five peptide sequences were modelled and their MHC class I molecule-binding abilities were analysed by molecular docking. Twenty-five peptides (Ten for B4, six for B12, two for B15, and seven for B19) were predicted to be potential T cell epitopes in chicken. Nine of these peptides and one unrelated peptide were manually synthesized and their T cell responses were tested in vitro. Spleen lymphocytes were collected from SPF chickens that had been immunised with a NP-expression plasmid, pCAGGS-NP, and they were stimulated using the synthesized peptides. The secretion of chicken IFN-γ and the proliferation of CD8(+) T cells were tested using an ELISA kit and flow cytometry, respectively. The significant secretion of chicken IFN-γ and proliferation of CD8(+) T lymphocytes increased by 13.7% and 11.9% were monitored in cells stimulated with peptides NP(89–97) and NP(198–206), respectively. The results indicate that peptides NP(89–97) (PKKTGGPIY) and NP(198–206) (KRGINDRNF) are NP T cell epitopes in chicken of certain haplotypes. The method used in this investigation is applicable to predicting T cell epitopes for other antigens in chicken, while this study also extends our understanding of the mechanisms of the immune response to AIV in chicken. |
832 | Novel Paramyxoviruses in Free-Ranging European Bats | The zoonotic potential of paramyxoviruses is particularly demonstrated by their broad host range like the highly pathogenic Hendra and Nipah viruses originating from bats. But while so far all bat-borne paramyxoviruses have been identified in fruit bats across Africa, Australia, South America, and Asia, we describe the detection and characterization of the first paramyxoviruses in free-ranging European bats. Moreover, we examined the possible impact of paramyxovirus infection on individual animals by comparing histo-pathological findings and virological results. Organs from deceased insectivorous bats of various species were sampled in Germany and tested for paramyxovirus RNA in parallel to a histo-pathological examination. Nucleic acids of three novel paramyxoviruses were detected, two viruses in phylogenetic relationship to the recently proposed genus Jeilongvirus and one closely related to the genus Rubulavirus. Two infected animals revealed subclinical pathological changes within their kidneys, suggestive of a similar pathogenesis as the one described in fruit bats experimentally infected with Hendra virus. Our findings indicate the presence of bat-born paramyxoviruses in geographic areas free of fruit bat species and therefore emphasize a possible virus–host co-evolution in European bats. Since these novel viruses are related to the very distinct genera Rubulavirus and Jeilongvirus, a similarly broad genetic diversity among paramyxoviruses in other Microchiroptera compared to Megachiroptera can be assumed. Given that the infected bats were either found in close proximity to heavily populated human habitation or areas of intensive agricultural use, a potential risk of the emergence of zoonotic paramyxoviruses in Europe needs to be considered. |
833 | Lipid Rafts and Alzheimer’s Disease: Protein-Lipid Interactions and Perturbation of Signaling | Lipid rafts are membrane domains, more ordered than the bulk membrane and enriched in cholesterol and sphingolipids. They represent a platform for protein-lipid and protein–protein interactions and for cellular signaling events. In addition to their normal functions, including membrane trafficking, ligand binding (including viruses), axonal development and maintenance of synaptic integrity, rafts have also been implicated in the pathogenesis of several neurodegenerative diseases including Alzheimer’s disease (AD). Lipid rafts promote interaction of the amyloid precursor protein (APP) with the secretase (BACE-1) responsible for generation of the amyloid β peptide, Aβ. Rafts also regulate cholinergic signaling as well as acetylcholinesterase and Aβ interaction. In addition, such major lipid raft components as cholesterol and GM1 ganglioside have been directly implicated in pathogenesis of the disease. Perturbation of lipid raft integrity can also affect various signaling pathways leading to cellular death and AD. In this review, we discuss modulation of APP cleavage by lipid rafts and their components, while also looking at more recent findings on the role of lipid rafts in signaling events. |
834 | West Nile Virus Infection in Killer Whale, Texas, USA, 2007 | In 2007, nonsuppurative encephalitis was identified in a killer whale at a Texas, USA, marine park. Panviral DNA microarray of brain tissue suggested West Nile virus (WNV); WNV was confirmed by reverse transcription PCR and sequencing. Immunohistochemistry demonstrated WNV antigen within neurons. WNV should be considered in cases of encephalitis in cetaceans. |
835 | Differential Seroprevalence of Human Bocavirus Species 1-4 in Beijing, China | BACKGROUND: Four species of human bocaviruses (HBoV1-4) have been identified based on phylogenetic analysis since its first report in 2005. HBoV1 has been associated with respiratory disease, whereas HBoV2-4 are mainly detected in enteric infections. Although the prevalence of HBoVs in humans has been studied in some regions, it has not been well addressed globally. METHODOLOGY/PRINCIPAL FINDINGS: Cross-reactivity of anti-VP2 antibodies was detected between HBoV1, 2, 3, and 4 in mouse and human serum. The prevalence of specific anti-VP2 IgG antibodies against HBoV1-4 was determined in different age groups of healthy individuals aged 0-70 years old in Beijing, China, using a competition ELISA assay based on virus-like particles of HBoV1-4. The seroprevalence of HBoV1-4 was 50%, 36.9%, 28.7%, and 0.8%, respectively, in children aged 0-14 years (n = 244); whereas the seroprevalence of HBoV1-4 was 66.9%, 49.3%, 38.7% and 1.4%, respectively, in healthy adults (≥15 years old; n = 142). The seropositive rate of HBoV1 was higher than that of HBoV2, HBoV3, and HBoV4 in individuals older than 0.5 years. Furthermore, IgG seroconversion of HBoV1 (10/31, 32.3%), HBoV2 (8/31, 25.8%), and HBoV3 (2/31, 6.5%) was found in paired sera collected from children with respiratory tract infections who were positive for HBoV1 according to PCR analysis. CONCLUSIONS/SIGNIFICANCE: Our data indicate that HBoV1 is more prevalent than HBoV2, HBoV3, and HBoV4 in the population we sampled in Beijing, China, suggesting that HBoV species may play differential roles in disease. |
836 | Clarithromycin Suppresses Human Respiratory Syncytial Virus Infection-Induced Streptococcus pneumoniae Adhesion and Cytokine Production in a Pulmonary Epithelial Cell Line | Human respiratory syncytial virus (RSV) sometimes causes acute and severe lower respiratory tract illness in infants and young children. RSV strongly upregulates proinflammatory cytokines and the platelet-activating factor (PAF) receptor, which is a receptor for Streptococcus pneumoniae, in the pulmonary epithelial cell line A549. Clarithromycin (CAM), which is an antimicrobial agent and is also known as an immunomodulator, significantly suppressed RSV-induced production of interleukin-6, interleukin-8, and regulated on activation, normal T-cell expressed and secreted (RANTES). CAM also suppressed RSV-induced PAF receptor expression and adhesion of fluorescein-labeled S. pneumoniae cells to A549 cells. The RSV-induced S. pneumoniae adhesion was thought to be mediated by the host cell's PAF receptor. CAM, which exhibits antimicrobial and immunomodulatory activities, was found in this study to suppress the RSV-induced adhesion of respiratory disease-causing bacteria, S. pneumoniae, to host cells. Thus, CAM might suppress immunological disorders and prevent secondary bacterial infections during RSV infection. |
837 | Post-Transcriptional Control of Type I Interferon Induction by Porcine Reproductive and Respiratory Syndrome Virus in Its Natural Host Cells | Porcine reproductive and respiratory syndrome virus (PRRSV) is not only a poor inducer of type I interferon but also inhibits the efficient induction of type I interferon by porcine transmissible gastroenteritis virus (TGEV) and synthetic dsRNA molecules, Poly I:C. However, the mechanistic basis by which PRRSV interferes with the induction of type I interferon in its natural host cells remains less well defined. The purposes of this review are to summarize the key findings in supporting the post-transcriptional control of type I interferon in its natural host cells and to propose the possible role of translational control in the regulation of type I interferon induction by PRRSV. |
838 | Clinical review: Special populations - critical illness and pregnancy | Critical illness is an uncommon but potentially devastating complication of pregnancy. The majority of pregnancy-related critical care admissions occur postpartum. Antenatally, the pregnant patient is more likely to be admitted with diseases non-specific to pregnancy, such as pneumonia. Pregnancy-specific diseases resulting in ICU admission include obstetric hemorrhage, pre-eclampsia/eclampsia, HELLP (hemolysis, elevated liver enzymes, and low platelet count) syndrome, amniotic fluid embolus syndrome, acute fatty liver of pregnancy, and peripartum cardiomyopathy. Alternatively, critical illness may result from pregnancy-induced worsening of pre-existing diseases (for example, valvular heart disease, myasthenia gravis, and kidney disease). Pregnancy can also predispose women to diseases seen in the non-pregnant population, such as acute respiratory distress syndrome (for example, pneumonia and aspiration), sepsis (for example, chorioamnionitis and pyelonephritis) or pulmonary embolism. The pregnant patient may also develop conditions co-incidental to pregnancy such as trauma or appendicitis. Hemorrhage, particularly postpartum, and hypertensive disorders of pregnancy remain the most frequent indications for ICU admission. This review focuses on pregnancy-specific causes of critical illness. Management of the critically ill mother poses special challenges. The physiologic changes in pregnancy and the presence of a second, dependent, patient may necessitate adjustments to therapeutic and supportive strategies. The fetus is generally robust despite maternal illness, and therapeutically what is good for the mother is generally good for the fetus. For pregnancy-induced critical illnesses, delivery of the fetus helps resolve the disease process. Prognosis following pregnancy-related critical illness is generally better than for age-matched non-pregnant critically ill patients. |
839 | PaCO(2 )and alveolar dead space are more relevant than PaO(2)/FiO(2 )ratio in monitoring the respiratory response to prone position in ARDS patients: a physiological study | INTRODUCTION: Our aims in this study were to report changes in the ratio of alveolar dead space to tidal volume (VD(alv)/V(T)) in the prone position (PP) and to test whether changes in partial pressure of arterial CO(2 )(PaCO(2)) may be more relevant than changes in the ratio of partial pressure of arterial O(2 )to fraction of inspired O(2 )(PaO(2)/FiO(2)) in defining the respiratory response to PP. We also aimed to validate a recently proposed method of estimation of the physiological dead space (VD(physiol)/V(T)) without measurement of expired CO(2). METHODS: Thirteen patients with a PaO(2)/FiO(2 )ratio < 100 mmHg were included in the study. Plateau pressure (Pplat), positive end-expiratory pressure (PEEP), blood gas analysis and expiratory CO(2 )were recorded with patients in the supine position and after 3, 6, 9, 12 and 15 hours in the PP. Responders to PP were defined after 15 hours of PP either by an increase in PaO(2)/FiO(2 )ratio > 20 mmHg or by a decrease in PaCO(2 )> 2 mmHg. Estimated and measured VD(physiol)/V(T )ratios were compared. RESULTS: PP induced a decrease in Pplat, PaCO(2 )and VD(alv)/V(T )ratio and increases in PaO(2)/FiO(2 )ratios and compliance of the respiratory system (Crs). Maximal changes were observed after six to nine hours. Changes in VD(alv)/V(T )were correlated with changes in Crs, but not with changes in PaO(2)/FiO(2 )ratios. When the response was defined by PaO(2)/FiO(2 )ratio, no significant differences in Pplat, PaCO(2 )or VD(alv)/V(T )alterations between responders (n = 7) and nonresponders (n = 6) were observed. When the response was defined by PaCO(2), four patients were differently classified, and responders (n = 7) had a greater decrease in VD(alv)/V(T )ratio and in Pplat and a greater increase in PaO(2)/FiO(2 )ratio and in Crs than nonresponders (n = 6). Estimated VD(physiol)/V(T )ratios significantly underestimated measured VD(physiol)/V(T )ratios (concordance correlation coefficient 0.19 (interquartile ranges 0.091 to 0.28)), whereas changes during PP were more reliable (concordance correlation coefficient 0.51 (0.32 to 0.66)). CONCLUSIONS: PP induced a decrease in VD(alv)/V(T )ratio and an improvement in respiratory mechanics. The respiratory response to PP appeared more relevant when PaCO(2 )rather than the PaO(2)/FiO(2 )ratio was used. Estimated VD(physiol)/V(T )ratios systematically underestimated measured VD(physiol)/V(T )ratios. |
840 | Lung Function and Organ Dysfunctions in 178 Patients Requiring Mechanical Ventilation During The 2009 Influenza A (H1N1) Pandemic | INTRODUCTION: Most cases of the 2009 influenza A (H1N1) infection are self-limited, but occasionally the disease evolves to a severe condition needing hospitalization. Here we describe the evolution of the respiratory compromise, ventilatory management and laboratory variables of patients with diffuse viral pneumonitis caused by pandemic 2009 influenza A (H1N1) admitted to the ICU. METHOD: This was a multicenter, prospective inception cohort study including adult patients with acute respiratory failure requiring mechanical ventilation (MV) admitted to 20 ICUs in Argentina between June and September of 2009 during the influenza A (H1N1) pandemic. In a standard case-report form, we collected epidemiological characteristics, results of real-time reverse-transcriptase--polymerase-chain-reaction viral diagnostic tests, oxygenation variables, acid-base status, respiratory mechanics, ventilation management and laboratory tests. Variables were recorded on ICU admission and at days 3, 7 and 10. RESULTS: During the study period 178 patients with diffuse viral pneumonitis requiring MV were admitted. They were 44 ± 15 years of age, with Acute Physiology And Chronic Health Evaluation II (APACHE II) scores of 18 ± 7, and most frequent comorbidities were obesity (26%), previous respiratory disease (24%) and immunosuppression (16%). Non-invasive ventilation (NIV) was applied in 49 (28%) patients on admission, but 94% were later intubated. Acute respiratory distress syndrome (ARDS) was present throughout the entire ICU stay in the whole group (mean PaO(2)/FIO(2 )170 ± 25). Tidal-volumes used were 7.8 to 8.1 ml/kg (ideal body weight), plateau pressures always remained < 30 cmH(2)O, without differences between survivors and non-survivors; and mean positive end-expiratory pressure (PEEP) levels used were between 8 to 12 cm H(2)O. Rescue therapies, like recruitment maneuvers (8 to 35%), prone positioning (12 to 24%) and tracheal gas insufflation (3%) were frequently applied. At all time points, pH, platelet count, lactate dehydrogenase assay (LDH) and Sequential Organ Failure Assessment (SOFA) differed significantly between survivors and non-survivors. Lack of recovery of platelet count and persistence of leukocytosis were characteristic of non-survivors. Mortality was high (46%); and length of MV was 10 (6 to 17) days. CONCLUSIONS: These patients had severe, hypoxemic respiratory failure compatible with ARDS that persisted over time, frequently requiring rescue therapies to support oxygenation. NIV use is not warranted, given its high failure rate. Death and evolution to prolonged mechanical ventilation were common outcomes. Persistence of thrombocytopenia, acidosis and leukocytosis, and high LDH levels found in non-survivors during the course of the disease might be novel prognostic findings. |
841 | Comparative analysis of mycobacterium and related actinomycetes yields insight into the evolution of mycobacterium tuberculosis pathogenesis | BACKGROUND: The sequence of the pathogen Mycobacterium tuberculosis (Mtb) strain H37Rv has been available for over a decade, but the biology of the pathogen remains poorly understood. Genome sequences from other Mtb strains and closely related bacteria present an opportunity to apply the power of comparative genomics to understand the evolution of Mtb pathogenesis. We conducted a comparative analysis using 31 genomes from the Tuberculosis Database (TBDB.org), including 8 strains of Mtb and M. bovis, 11 additional Mycobacteria, 4 Corynebacteria, 2 Streptomyces, Rhodococcus jostii RHA1, Nocardia farcinia, Acidothermus cellulolyticus, Rhodobacter sphaeroides, Propionibacterium acnes, and Bifidobacterium longum. RESULTS: Our results highlight the functional importance of lipid metabolism and its regulation, and reveal variation between the evolutionary profiles of genes implicated in saturated and unsaturated fatty acid metabolism. It also suggests that DNA repair and molybdopterin cofactors are important in pathogenic Mycobacteria. By analyzing sequence conservation and gene expression data, we identify nearly 400 conserved noncoding regions. These include 37 predicted promoter regulatory motifs, of which 14 correspond to previously validated motifs, as well as 50 potential noncoding RNAs, of which we experimentally confirm the expression of four. CONCLUSIONS: Our analysis of protein evolution highlights gene families that are associated with the adaptation of environmental Mycobacteria to obligate pathogenesis. These families include fatty acid metabolism, DNA repair, and molybdopterin biosynthesis. Our analysis reinforces recent findings suggesting that small noncoding RNAs are more common in Mycobacteria than previously expected. Our data provide a foundation for understanding the genome and biology of Mtb in a comparative context, and are available online and through TBDB.org. |
842 | Proteasome-Dependent Disruption of the E3 Ubiquitin Ligase Anaphase-Promoting Complex by HCMV Protein pUL21a | The anaphase-promoting complex (APC) is an E3 ubiquitin ligase which controls ubiquitination and degradation of multiple cell cycle regulatory proteins. During infection, human cytomegalovirus (HCMV), a widespread pathogen, not only phosphorylates the APC coactivator Cdh1 via the multifunctional viral kinase pUL97, it also promotes degradation of APC subunits via an unknown mechanism. Using a proteomics approach, we found that a recently identified HCMV protein, pUL21a, interacted with the APC. Importantly, we determined that expression of pUL21a was necessary and sufficient for proteasome-dependent degradation of APC subunits APC4 and APC5. This resulted in APC disruption and required pUL21a binding to the APC. We have identified the proline-arginine amino acid pair at residues 109–110 in pUL21a to be critical for its ability to bind and regulate the APC. A point mutant virus in which proline-arginine were mutated to alanines (PR-AA) grew at wild-type levels. However, a double mutant virus in which the viral ability to regulate the APC was abrogated by both PR-AA point mutation and UL97 deletion was markedly more attenuated compared to the UL97 deletion virus alone. This suggests that these mutations are synthetically lethal, and that HCMV exploits two viral factors to ensure successful disruption of the APC to overcome its restriction on virus infection. This study reveals the HCMV protein pUL21a as a novel APC regulator and uncovers a unique viral mechanism to subvert APC activity. |
843 | Deciphering Novel Host–Herpesvirus Interactions by Virion Proteomics | Over the years, a vast array of information concerning the interactions of viruses with their hosts has been collected. However, recent advances in proteomics and other system biology techniques suggest these interactions are far more complex than anticipated. One particularly interesting and novel aspect is the analysis of cellular proteins incorporated into mature virions. Though sometimes considered purification contaminants in the past, their repeated detection by different laboratories suggests that a number of these proteins are bona fide viral components, some of which likely contribute to the viral life cycles. The present mini review focuses on cellular proteins detected in herpesviruses. It highlights the common cellular functions of these proteins, their potential implications for host–pathogen interactions, discusses technical limitations, the need for complementing methods and probes potential future research avenues. |
844 | Naturally-Occurring Genetic Variants in Human DC-SIGN Increase HIV-1 Capture, Cell-Transfer and Risk of Mother-To-Child Transmission | BACKGROUND: Mother-to-child transmission (MTCT) is the main cause of HIV-1 infection in children worldwide. Dendritic cell–specific ICAM-3 grabbing-nonintegrin (DC-SIGN, also known as CD209) is an HIV-1 receptor that enhances its transmission to T cells and is expressed on placental macrophages. METHODS AND FINDINGS: We have investigated the association between DC-SIGN genetic variants and risk of MTCT of HIV-1 among Zimbabwean infants and characterized the impact of the associated mutations on DC-SIGN expression and interaction with HIV-1. DC-SIGN promoter (p-336C and p-201A) and exon 4 (198Q and 242V) variants were all significantly associated with increased risk of intrauterine (IU) HIV-1 infection. Promoter variants decreased DC-SIGN expression both in vitro and in placental CD163(+) macrophages (Hofbauer cells) of HIV-1 unexposed infants but not of HIV-1 exposed infants. The exon 4 protein-modifying mutations increased HIV-1 capture and transmission to T cells in vitro. CONCLUSION: This study provides compelling evidence to support an important role of DC-SIGN in IU HIV-1 infection. |
845 | A New Model for Hendra Virus Encephalitis in the Mouse | Hendra virus (HeV) infection in humans is characterized by an influenza like illness, which may progress to pneumonia or encephalitis and lead to death. The pathogenesis of HeV infection is poorly understood, and the lack of a mouse model has limited the opportunities for pathogenetic research. In this project we reassessed the role of mice as an animal model for HeV infection and found that mice are susceptible to HeV infection after intranasal exposure, with aged mice reliably developing encephalitic disease. We propose an anterograde route of neuroinvasion to the brain, possibly along olfactory nerves. This is supported by evidence for the development of encephalitis in the absence of viremia and the sequential distribution of viral antigen along pathways of olfaction in the brain of intranasally challenged animals. In our studies mice developed transient lower respiratory tract infection without progressing to viremia and systemic vasculitis that is common to other animal models. These studies report a new animal model of HeV encephalitis that will allow more detailed studies of the neuropathogenesis of HeV infection, particularly the mode of viral spread and possible sequestration within the central nervous system; investigation of mechanisms that moderate the development of viremia and systemic disease; and inform the development of improved treatment options for human patients. |
846 | Ipomoelin, a Jacalin-Related Lectin with a Compact Tetrameric Association and Versatile Carbohydrate Binding Properties Regulated by Its N Terminus | Many proteins are induced in the plant defense response to biotic stress or mechanical wounding. One group is lectins. Ipomoelin (IPO) is one of the wound-inducible proteins of sweet potato (Ipomoea batatas cv. Tainung 57) and is a Jacalin-related lectin (JRL). In this study, we resolved the crystal structures of IPO in its apo form and in complex with carbohydrates such as methyl α-D-mannopyranoside (Me-Man), methyl α-D-glucopyranoside (Me-Glc), and methyl α-D-galactopyranoside (Me-Gal) in different space groups. The packing diagrams indicated that IPO might represent a compact tetrameric association in the JRL family. The protomer of IPO showed a canonical β-prism fold with 12 strands of β-sheets but with 2 additional short β-strands at the N terminus. A truncated IPO (ΔN10IPO) by removing the 2 short β-strands of the N terminus was used to reveal its role in a tetrameric association. Gel filtration chromatography confirmed IPO as a tetrameric form in solution. Isothermal titration calorimetry determined the binding constants (K(A)) of IPO and ΔN10IPO against various carbohydrates. IPO could bind to Me-Man, Me-Glc, and Me-Gal with similar binding constants. In contrast, ΔN10IPO showed high binding ability to Me-Man and Me-Glc but could not bind to Me-Gal. Our structural and functional analysis of IPO revealed that its compact tetrameric association and carbohydrate binding polyspecificity could be regulated by the 2 additional N-terminal β-strands. The versatile carbohydrate binding properties of IPO might play a role in plant defense. |
847 | Retroviral Env Glycoprotein Trafficking and Incorporation into Virions | Together with the Gag protein, the Env glycoprotein is a major retroviral structural protein and is essential for forming infectious virus particles. Env is synthesized, processed, and transported to certain microdomains at the plasma membrane and takes advantage of the same host machinery for its trafficking as that used by cellular glycoproteins. Incorporation of Env into progeny virions is probably mediated by the interaction between Env and Gag, in some cases with the additional involvement of certain host factors. Although several general models have been proposed to explain the incorporation of retroviral Env glycoproteins into virions, the actual mechanism for this process is still unclear, partly because structural data on the Env protein cytoplasmic tail is lacking. This paper presents the current understanding of the synthesis, trafficking, and virion incorporation of retroviral Env proteins. |
848 | Production, Characterization and Applications for Toxoplasma gondii-Specific Polyclonal Chicken Egg Yolk Immunoglobulins | BACKGROUND: Toxoplasma gondii may cause abortions, ocular and neurological disorders in warm-blood hosts. Immunized mammals are a wide source of hyperimmune sera used in different approaches, including diagnosis and the study of host-parasite interactions. Unfortunately, mammalian antibodies present limitations for its production, such as the necessity for animal bleeding, low yield, interference with rheumatoid factor, complement activation and affinity to Fc mammalian receptors. IgY antibodies avoid those limitations; therefore they could be an alternative to be applied in T. gondii model. METHODOLOGY/PRINCIPAL FINDINGS: In this study we immunized hens with soluble tachyzoite antigens of T. gondii (STAg) and purified egg yolk antibodies (IgY) by an inexpensive and simple method, with high yield and purity degree. IgY anti-STAg antibodies presented high avidity and were able to recognize a broad range of parasite antigens, although some marked differences were observed in reactivity profile between antibodies produced in immunized hens and mice. Interestingly, IgY antibodies against Neospora caninum and Eimeria spp. did not react to STAg. We also show that IgY antibodies were suitable to detect T. gondii forms in paraffin-embedded sections and culture cell monolayers. CONCLUSIONS/SIGNIFICANCE: Due to its cost-effectiveness, high production yield and varied range of possible applications, polyclonal IgY antibodies are useful tools for studies involving T. gondii. |
849 | Preparation of an antitumor and antivirus agent: chemical modification of α-MMC and MAP30 from Momordica Charantia L. with covalent conjugation of polyethyelene glycol | BACKGROUND: Alpha-momorcharin (α-MMC) and momordica anti-HIV protein (MAP30) derived from Momordica charantia L. have been confirmed to possess antitumor and antivirus activities due to their RNA-N-glycosidase activity. However, strong immunogenicity and short plasma half-life limit their clinical application. To solve this problem, the two proteins were modified with (mPEG)(2)-Lys-NHS (20 kDa). METHODOLOGY/PRINCIPAL FINDINGS: In this article, a novel purification strategy for the two main type I ribosome-inactivating proteins (RIPs), α-MMC and MAP30, was successfully developed for laboratory-scale preparation. Using this dramatic method, 200 mg of α-MMC and about 120 mg of MAP30 was obtained in only one purification process from 200 g of Momordica charantia seeds. The homogeneity and some other properties of the two proteins were assessed by gradient SDS-PAGE, electrospray ionization quadruple mass spectrometry, and N-terminal sequence analysis as well as Western blot. Two polyethylene glycol (PEG)ylated proteins were synthesized and purified. Homogeneous mono-, di-, or tri-PEGylated proteins were characterized by matrix-assisted laser desorption ionization-time of flight mass spectrometry. The analysis of antitumor and antivirus activities indicated that the serial PEGylated RIPs preserved moderate activities on JAR choriocarcinoma cells and herpes simplex virus-1. Furthermore, both PEGylated proteins showed about 60%–70% antitumor and antivirus activities, and at the same time decreased 50%–70% immunogenicity when compared with their unmodified counterparts. CONCLUSION/SIGNIFICANCE: α-MMC and MAP30 obtained from this novel purification strategy can meet the requirement of a large amount of samples for research. Their chemical modification can solve the problem of strong immunogenicity and meanwhile preserve moderate activities. All these findings suggest the potential application of PEGylated α-MMC and PEGylated MAP30 as antitumor and antivirus agents. According to these results, PEGylated RIPs can be constructed with nanomaterials to be a targeting drug that can further decrease immunogenicity and side effects. Through nanotechnology we can make them low-release drugs, which can further prolong their half-life period in the human body. |
850 | Evaluation of Four Different Systems for Extraction of RNA from Stool Suspensions Using MS-2 Coliphage as an Exogenous Control for RT-PCR Inhibition | Knowing when, and to what extent co-extracted inhibitors interfere with molecular RNA diagnostic assays is of utmost importance. The QIAamp Viral RNA Mini Kit (A); MagNA Pure LC2.0 Automatic extractor (B); KingFisher (C); and NucliSENS EasyMag (D) RNA extraction systems were evaluated for extraction efficiency and co-purification of inhibitors from stool suspensions. Real-Time Reverse Transcriptase Polymerase Chain Reaction (rRT-PCR) of MS-2 coliphage spiked into each system’s lysis buffer served as an external control for both. Cycle thresholds (Cts) of the MS2 were determined for RNA extracted from stool suspensions containing unknown (n = 93) or varying amounts of inhibitors (n = 92). Stool suspensions from the latter group were also used to determine whether MS-2 and enterovirus rRT-PCR inhibitions were correlated. Specifically 23 RNA extracts from stool suspensions were spiked with enterovirus RNA after extraction and 13 of these stool suspension were spiked with intact enterovirus before extraction. MS2 rRT-PCR inhibition varied for RNAs extracted by the different systems. Inhibition was noted in 12 (13.0%), 26 (28.3%), 7 (7.6%), and 7 (7.6%) of the first 93 RNA extracts, and 58 (63.0%), 55 (59.8%), 37 (40.2%) and 30 (32.6%) of the second 92 extracts for A, B, C, and D, respectively. Furthermore, enterovirus rRT-PCR inhibition correlated with MS2 rRT-PCR inhibition for added enterovirus RNA or virus particles. In conclusion, rRT-PCR for MS-2 RNA is a good predictor of inhibition of enterovirus RNA extracted from stool suspensions. EasyMag performed the best, however all four extraction methods were suitable provided that external controls identified problematic samples. |
851 | Analysis on the Pathogenesis of Symptomatic Pulmonary Embolism with Human Genomics | BACKGROUND: In the present study, the whole human genome oligo microarray was employed to investigate the gene expression profile in symptomatic pulmonary embolism (PE). METHODS: Twenty patients with PE and 20 age and gender matched patients without PE as controls were enrolled into the present study in the same period. The diagnosis of PE was based on the clinical manifestations and findings on imaging examinations. Acute arterial and/or venous thrombosis was excluded in controls. The whole human genome oligo microarray was employed for detection. Statistical analysis was performed with t test following analysis of very small samples of repeated measurements and Gene Ontology (GO) analysis. RESULTS: Genomic data showed no damage to vascular endothelial cells in PE patients. Genomic data only found increased mRNA expression of a small amount of coagulation factors in PE patients. In the PE group, anticoagulant proteins, Fibrinolytic system and proteins related to platelet functions only played partial roles in the pathogenesis of PE. In addition, the mRNA expressions of a fraction of adhesion molecules were markedly up-regulated. Gene Ontology analysis showed the genes with down-regulated expressions mainly explain the compromised T cell immunity. Symptomatic VTE patients have compromised T cell immunity. CONCLUSION: The damage to vascular endothelial cells is not necessary in the pathogenesis of VTE, and only a fraction of factors involved in the shared coagulation cascade are activated. Genomic results may provide a new clue for clinical diagnosis, treatment and prevention of VTE. |
852 | Diversity and roles of (t)RNA ligases | The discovery of discontiguous tRNA genes triggered studies dissecting the process of tRNA splicing. As a result, we have gained detailed mechanistic knowledge on enzymatic removal of tRNA introns catalyzed by endonuclease and ligase proteins. In addition to the elucidation of tRNA processing, these studies facilitated the discovery of additional functions of RNA ligases such as RNA repair and non-conventional mRNA splicing events. Recently, the identification of a new type of RNA ligases in bacteria, archaea, and humans closed a long-standing gap in the field of tRNA processing. This review summarizes past and recent findings in the field of tRNA splicing with a focus on RNA ligation as it preferentially occurs in archaea and humans. In addition to providing an integrated view of the types and phyletic distribution of RNA ligase proteins known to date, this survey also aims at highlighting known and potential accessory biological functions of RNA ligases. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00018-012-0944-2) contains supplementary material, which is available to authorized users. |
853 | Molecular Imaging Reveals a Progressive Pulmonary Inflammation in Lower Airways in Ferrets Infected with 2009 H1N1 Pandemic Influenza Virus | Molecular imaging has gained attention as a possible approach for the study of the progression of inflammation and disease dynamics. Herein we used [(18)F]-2-deoxy-2-fluoro-D-glucose ([(18)F]-FDG) as a radiotracer for PET imaging coupled with CT (FDG-PET/CT) to gain insight into the spatiotemporal progression of the inflammatory response of ferrets infected with a clinical isolate of a pandemic influenza virus, H1N1 (H1N1pdm). The thoracic regions of mock- and H1N1pdm-infected ferrets were imaged prior to infection and at 1, 2, 3 and 6 days post-infection (DPI). On 1 DPI, FDG-PET/CT imaging revealed areas of consolidation in the right caudal lobe which corresponded with elevated [(18)F]-FDG uptake (maximum standardized uptake values (SUVMax), 4.7–7.0). By days 2 and 3, consolidation (CT) and inflammation ([(18)F]-FDG) appeared in the left caudal lobe. By 6 DPI, CT images showed extensive areas of patchy ground-glass opacities (GGO) and consolidations with the largest lesions having high SUVMax (6.0–7.6). Viral shedding and replication were detected in most nasal, throat and rectal swabs and nasal turbinates and lungs on 1, 2 and 3 DPI, but not on day 7, respectively. In conclusion, molecular imaging of infected ferrets revealed a progressive consolidation on CT with corresponding [(18)F]-FDG uptake. Strong positive correlations were measured between SUVMax and bronchiolitis-related pathologic scoring (Spearman’s ρ = 0.75). Importantly, the extensive areas of patchy GGO and consolidation seen on CT in the ferret model at 6 DPI are similar to that reported for human H1N1pdm infections. In summary, these first molecular imaging studies of lower respiratory infection with H1N1pdm show that FDG-PET can give insight into the spatiotemporal progression of the inflammation in real-time. |
854 | The Dynamics, Causes and Possible Prevention of Hepatitis E Outbreaks | Rapidly spreading infectious diseases are a serious risk to public health. The dynamics and the factors causing outbreaks of these diseases can be better understood using mathematical models, which are fit to data. Here we investigate the dynamics of a Hepatitis E outbreak in the Kitgum region of northern Uganda during 2007 to 2009. First, we use the data to determine that [Image: see text] is approximately 2.25 for the outbreak. Secondly, we use a model to estimate that the critical level of latrine and bore hole coverages needed to eradicate the epidemic is at least [Image: see text] and [Image: see text] respectively. Lastly, we further investigate the relationship between the co-infection factor for malaria and Hepatitis E on the value of [Image: see text] for Hepatitis E. Taken together, these results provide us with a better understanding of the dynamics and possible causes of Hepatitis E outbreaks. |
855 | Human Bocaviruses Are Not Significantly Associated with Gastroenteritis: Results of Retesting Archive DNA from a Case Control Study in the UK | Gastroenteritis is a common illness causing considerable morbidity and mortality worldwide. Despite improvements in detection methods, a significant diagnostic gap still remains. Human bocavirus (HBoV)s, which are associated with respiratory infections, have also frequently been detected in stool samples in cases of gastroenteritis, and a tentative association between HBoVs, and in particular type-2 HBoVs, and gastroenteritis has previously been made. The aim of this study was to determine the role of HBoVs in gastroenteritis, using archived DNA samples from the case-control Infectious Intestinal Disease Study (IID). DNA extracted from stool samples from 2,256 cases and 2,124 controls were tested for the presence of HBoV DNA. All samples were screened in a real time PCR pan-HBoV assay, and positive samples were then tested in genotype 1 to 3-specific assays. HBoV was detected in 7.4% but no significantly different prevalence was observed between cases and controls. In the genotype-specific assays 106 of the 324 HBoV-positive samples were genotyped, with HBoV-1 predominantly found in controls whilst HBoV-2 was more frequently associated with cases of gastroenteritis (p<0.01). A significant proportion of HBoV positives could not be typed using the type specific assays, 67% of the total positives, and this was most likely due to low viral loads being present in the samples. However, the distribution of the untyped HBoV strains was no different between cases and controls. In conclusion, HBoVs, including HBoV-2 do not appear to be a significant cause of gastroenteritis in the UK population. |
856 | Structural Origins for the Loss of Catalytic Activities of Bifunctional Human LTA4H Revealed through Molecular Dynamics Simulations | Human leukotriene A4 hydrolase (hLTA4H), which is the final and rate-limiting enzyme of arachidonic acid pathway, converts the unstable epoxide LTA4 to a proinflammatory lipid mediator LTB4 through its hydrolase function. The LTA4H is a bi-functional enzyme that also exhibits aminopeptidase activity with a preference over arginyl tripeptides. Various mutations including E271Q, R563A, and K565A have completely or partially abolished both the functions of this enzyme. The crystal structures with these mutations have not shown any structural changes to address the loss of functions. Molecular dynamics simulations of LTA4 and tripeptide complex structures with functional mutations were performed to investigate the structural and conformation changes that scripts the observed differences in catalytic functions. The observed protein-ligand hydrogen bonds and distances between the important catalytic components have correlated well with the experimental results. This study also confirms based on the structural observation that E271 is very important for both the functions as it holds the catalytic metal ion at its location for the catalysis and it also acts as N-terminal recognition residue during peptide binding. The comparison of binding modes of substrates revealed the structural changes explaining the importance of R563 and K565 residues and the required alignment of substrate at the active site. The results of this study provide valuable information to be utilized in designing potent hLTA4H inhibitors as anti-inflammatory agents. |
857 | Molecular and Microscopic Analysis of Bacteria and Viruses in Exhaled Breath Collected Using a Simple Impaction and Condensing Method | Exhaled breath condensate (EBC) is increasingly being used as a non-invasive method for disease diagnosis and environmental exposure assessment. By using hydrophobic surface, ice, and droplet scavenging, a simple impaction and condensing based collection method is reported here. Human subjects were recruited to exhale toward the device for 1, 2, 3, and 4 min. The exhaled breath quickly formed into tiny droplets on the hydrophobic surface, which were subsequently scavenged into a 10 µL rolling deionized water droplet. The collected EBC was further analyzed using culturing, DNA stain, Scanning Electron Microscope (SEM), polymerase chain reaction (PCR) and colorimetry (VITEK 2) for bacteria and viruses. Experimental data revealed that bacteria and viruses in EBC can be rapidly collected using the method developed here, with an observed efficiency of 100 µL EBC within 1 min. Culturing, DNA stain, SEM, and qPCR methods all detected high bacterial concentrations up to 7000 CFU/m(3) in exhaled breath, including both viable and dead cells of various types. Sphingomonas paucimobilis and Kocuria variants were found dominant in EBC samples using VITEK 2 system. SEM images revealed that most bacteria in exhaled breath are detected in the size range of 0.5–1.0 µm, which is able to enable them to remain airborne for a longer time, thus presenting a risk for airborne transmission of potential diseases. Using qPCR, influenza A H3N2 viruses were also detected in one EBC sample. Different from other devices restricted solely to condensation, the developed method can be easily achieved both by impaction and condensation in a laboratory and could impact current practice of EBC collection. Nonetheless, the reported work is a proof-of-concept demonstration, and its performance in non-invasive disease diagnosis such as bacterimia and virus infections needs to be further validated including effects of its influencing matrix. |
858 | Seasonal influenza vaccination knowledge, risk perception, health beliefs and vaccination behaviours of nurses | The relationship between knowledge, risk perceptions, health belief towards seasonal influenza and vaccination and the vaccination behaviours of nurses was explored. Qualified nurses attending continuing professional education courses at a large London university between 18 April and 18 October 2010 were surveyed (522/672; response rate 77·7%). Of these, 82·6% worked in hospitals; 37·0% reported receiving seasonal influenza vaccination in the previous season and 44·9% reported never being vaccinated during the last 5 years. All respondents were categorized using two-step cluster analyses into never, occasionally, and continuously vaccinated groups. Nurses vaccinated the season before had higher scores of knowledge and risk perception compared to the unvaccinated (P<0·001). Nurses never vaccinated had the lowest scores of knowledge and risk perception compared to other groups (P<0·001). Nurses' seasonal influenza vaccination behaviours are complex. Knowledge and risk perception predict uptake of vaccination in nurses. |
859 | IFN-γ Signaling to Astrocytes Protects from Autoimmune Mediated Neurological Disability | Demyelination and axonal degeneration are determinants of progressive neurological disability in patients with multiple sclerosis (MS). Cells resident within the central nervous system (CNS) are active participants in development, progression and subsequent control of autoimmune disease; however, their individual contributions are not well understood. Astrocytes, the most abundant CNS cell type, are highly sensitive to environmental cues and are implicated in both detrimental and protective outcomes during autoimmune demyelination. Experimental autoimmune encephalomyelitis (EAE) was induced in transgenic mice expressing signaling defective dominant-negative interferon gamma (IFN-γ) receptors on astrocytes to determine the influence of inflammation on astrocyte activity. Inhibition of IFN-γ signaling to astrocytes did not influence disease incidence, onset, initial progression of symptoms, blood brain barrier (BBB) integrity or the composition of the acute CNS inflammatory response. Nevertheless, increased demyelination at peak acute disease in the absence of IFN-γ signaling to astrocytes correlated with sustained clinical symptoms. Following peak disease, diminished clinical remission, increased mortality and sustained astrocyte activation within the gray matter demonstrate a critical role of IFN-γ signaling to astrocytes in neuroprotection. Diminished disease remission was associated with escalating demyelination, axonal degeneration and sustained inflammation. The CNS infiltrating leukocyte composition was not altered; however, decreased IL-10 and IL-27 correlated with sustained disease. These data indicate that astrocytes play a critical role in limiting CNS autoimmune disease dependent upon a neuroprotective signaling pathway mediated by engagement of IFN-γ receptors. |
860 | Influence of Mabs on PrP(Sc) Formation Using In Vitro and Cell-Free Systems | PrP(Sc) is believed to serve as a template for the conversion of PrP(C) to the abnormal isoform. This process requires contact between the two proteins and implies that there may be critical contact sites that are important for conversion. We hypothesized that antibodies binding to either PrP(c)or PrP(Sc) would hinder or prevent the formation of the PrP(C)–PrP(Sc) complex and thus slow down or prevent the conversion process. Two systems were used to analyze the effect of different antibodies on PrP(Sc) formation: (i) neuroblastoma cells persistently infected with the 22L mouse-adapted scrapie stain, and (ii) protein misfolding cyclic amplification (PMCA), which uses PrP(Sc) as a template or seed, and a series of incubations and sonications, to convert PrP(C) to PrP(Sc). The two systems yielded similar results, in most cases, and demonstrate that PrP-specific monoclonal antibodies (Mabs) vary in their ability to inhibit the PrP(C)–PrP(Sc) conversion process. Based on the numerous and varied Mabs analyzed, the inhibitory effect does not appear to be epitope specific, related to PrP(C) conformation, or to cell membrane localization, but is influenced by the targeted PrP region (amino vs carboxy). |
861 | Predictors and outcome of patients with acute respiratory distress syndrome caused by miliary tuberculosis: a retrospective study in Chongqing, China | BACKGROUND: Miliary tuberculosis (TB) is an uncommon cause of acute respiratory distress syndrome (ARDS) with a high mortality. The aim of the present study was to evaluate the clinical characteristics, predictors and outcome of patients with ARDS caused by miliary TB. METHODS: A retrospective study was conducted among patients with a diagnosis of ARDS with miliary TB in four hospitals from 2006 to 2010. Medical records and laboratory examinations of these patients were taken during the first 24 h of admission. RESULTS: Eighty-five patients with miliary TB developed ARDS, 45 of whom survived (52.9%). The median age was 36.6 ± 12.5 years with 38 males (44.7%). Diabetes mellitus (DM) was the most common underlying disease (18.8%).ICU mortality was 47.1%. The time from admission to anti-tuberculosis therapy was 4.5 ± 2.0 days. Mean duration of mechanical ventilation was 8.5 ± 3.0 days in all patients. Duration of time to diagnosis, time from diagnosis to mechanical ventilation, and time to anti-tuberculosis therapy were significantly shorter in survivors than those in non-survivors. Diabetes mellitus (OR 5.431, 95%CI 1.471-20.049; P = 0.005), ALT (70-100U/L, OR 10.029, 95%CI 2.764-36.389; P = 0.001), AST (>94U/L,OR 8.034, 95%CI 2.200-29.341; P = 0.002), D-dimer (>1.6mg/L, OR 3.167, 95%CI 0.896-11.187; P = 0.042), hemoglobin (<90g/L, OR 14.824, 95%CI 3.713-59.179; P = 0.001), albumin (<25g/L, OR 15.896, 95%CI 3.975-63.566; P = 0.001) were independent predictors of ARDS development in the setting of miliary TB. CONCLUSIONS: Accurate diagnosis, early initiation of anti-tuberculosis therapy and mechanical ventilation are important for the outcome of patients with ARDS caused by miliary TB. DM, ALT, AST, D-dimer, hemoglobin, and albumin are independent predictors of ARDS development in patients with miliary TB. |
862 | Symptomatic Venous Thromboembolism Is a Disease Related to Infection and Immune Dysfunction | The characteristics of human genomics and cellular immune function between clinically symptomatic venous thromboembolism (VTE) and controls were systematically compared to explore the immunologic pathogenesis of VTE. Microarray assay showed the mRNA expressions of genes related to non-specific cellarer immune and cytokines were significantly down-regulated. Abnormal expressions of CD3+, CD4+, CD8+, NK marker CD16+56+, CD19 and aberrant CD4+/CD8+ ratio were detected in 54 among 56 patients. In PE patients, microarray assay revealed the imbalance in the expressions of genes related to the immune system. The expressions of genes related to non-specific immune cells and cytokines were markedly up-regulated and those associated with cellular immune were dramatically down-regulated. In VTE patients, cytological examination indicated the functions of NK cells were significantly compromised, and the antigen recognition and killing function of T cells markedly decreased. The consistence between genomic and cytological examination suggests the symptomatic VTE is closely associated with the infection and immune dysfunction. |
863 | Cedar Virus: A Novel Henipavirus Isolated from Australian Bats | The genus Henipavirus in the family Paramyxoviridae contains two viruses, Hendra virus (HeV) and Nipah virus (NiV) for which pteropid bats act as the main natural reservoir. Each virus also causes serious and commonly lethal infection of people as well as various species of domestic animals, however little is known about the associated mechanisms of pathogenesis. Here, we report the isolation and characterization of a new paramyxovirus from pteropid bats, Cedar virus (CedPV), which shares significant features with the known henipaviruses. The genome size (18,162 nt) and organization of CedPV is very similar to that of HeV and NiV; its nucleocapsid protein displays antigenic cross-reactivity with henipaviruses; and it uses the same receptor molecule (ephrin- B2) for entry during infection. Preliminary challenge studies with CedPV in ferrets and guinea pigs, both susceptible to infection and disease with known henipaviruses, confirmed virus replication and production of neutralizing antibodies although clinical disease was not observed. In this context, it is interesting to note that the major genetic difference between CedPV and HeV or NiV lies within the coding strategy of the P gene, which is known to play an important role in evading the host innate immune system. Unlike HeV, NiV, and almost all known paramyxoviruses, the CedPV P gene lacks both RNA editing and also the coding capacity for the highly conserved V protein. Preliminary study indicated that CedPV infection of human cells induces a more robust IFN-β response than HeV. |
864 | Host Modulators of H1N1 Cytopathogenicity | Influenza A virus infects 5–20% of the population annually, resulting in ∼35,000 deaths and significant morbidity. Current treatments include vaccines and drugs that target viral proteins. However, both of these approaches have limitations, as vaccines require yearly development and the rapid evolution of viral proteins gives rise to drug resistance. In consequence additional intervention strategies, that target host factors required for the viral life cycle, are under investigation. Here we employed arrayed whole-genome siRNA screening strategies to identify cell-autonomous molecular components that are subverted to support H1N1 influenza A virus infection of human bronchial epithelial cells. Integration across relevant public data sets exposed druggable gene products required for epithelial cell infection or required for viral proteins to deflect host cell suicide checkpoint activation. Pharmacological inhibition of representative targets, RGGT and CHEK1, resulted in significant protection against infection of human epithelial cells by the A/WS/33 virus. In addition, chemical inhibition of RGGT partially protected against H5N1 and the 2009 H1N1 pandemic strain. The observations reported here thus contribute to an expanding body of studies directed at decoding vulnerabilities in the command and control networks specified by influenza virulence factors. |
865 | Association of Fcγ Receptor IIB Polymorphism with Cryptococcal Meningitis in HIV-Uninfected Chinese Patients | BACKGROUND: As important regulators of the immune system, the human Fcγ receptors (FcγRs) have been demonstrated to play important roles in the pathogenesis of various infectious diseases. The aim of the present study was to identify the association between FCGR polymorphisms and cryptococcal meningitis. METHODOLOGY/PRINCIPAL FINDINGS: In this case control genetic association study, we genotyped four functional polymorphisms in low-affinity FcγRs, including FCGR2A 131H/R, FCGR3A 158F/V, FCGR3B NA1/NA2, and FCGR2B 232I/T, in 117 patients with cryptococcal meningitis and 190 healthy controls by multiplex SNaPshot technology. Among the 117 patients with cryptococcal meningitis, 59 had predisposing factors. In patients with cryptococcal meningitis, the FCGR2B 232I/I genotype was over-presented (OR = 1.652, 95% CI [1.02–2.67]; P = 0.039) and the FCGR2B 232I/T genotype was under-presented (OR = 0.542, 95% CI [0.33–0.90]; P = 0.016) in comparison with control group. In cryptococcal meningitis patients without predisposing factors, FCGR2B 232I/I genotype was also more frequently detected (OR = 1.958, 95% CI [1.05–3.66]; P = 0.033), and the FCGR2B 232I/T genotype was also less frequently detected (OR = 0.467, 95% CI [0.24–0.91]; P = 0.023) than in controls. No significant difference was found among FCGR2A 131H/R, FCGR3A 158F/V, and FCGR3B NA1/NA2 genotype frequencies between patients and controls. CONCLUSION/SIGNIFICANCE: We found for the first time associations between cryptococcal meningitis and FCGR2B 232I/T genotypes, which suggested that FcγRIIB might play an important role in the central nervous system infection by Cryptococcus in HIV-uninfected individuals. |
866 | Infectious Disease Transmission during Organ and Tissue Transplantation | Infectious disease transmission through organ and tissue transplantation has been associated with severe complications in recipients. Determination of donor-derived infectious risk associated with organ and tissue transplantation is challenging and limited by availability and performance characteristics of current donor epidemiologic screening (e.g., questionnaire) and laboratory testing tools. Common methods and standards for evaluating potential donors of organs and tissues are needed to facilitate effective data collection for assessing the risk for infectious disease transmission. Research programs can use advanced microbiological technologies to define infectious risks posed by pathogens that are known to be transplant transmissible and provide insights into transmission potential of emerging infectious diseases for which transmission characteristics are unknown. Key research needs are explored. Stakeholder collaboration for surveillance and research infrastructure is required to enhance transplant safety. |
867 | Non-HIV Pneumocystis pneumonia: do conventional community-acquired pneumonia guidelines under estimate its severity? | BACKGROUND: Non-HIV Pneumocystis pneumonia (PCP) can occur in immunosuppressed patients having malignancy or on immunosuppressive agents. To classify severity, the A-DROP scale proposed by the Japanese Respiratory Society (JRS), the CURB-65 score of the British Respiratory Society (BTS) and the Pneumonia Severity Index (PSI) of the Infectious Diseases Society of America (IDSA) are widely used in patients with community-acquired pneumonia (CAP) in Japan. To evaluate how correctly these conventional prognostic guidelines for CAP reflect the severity of non-HIV PCP, we retrospectively analyzed 21 patients with non-HIV PCP. METHODS: A total of 21 patients were diagnosed by conventional staining and polymerase chain reaction (PCR) for respiratory samples with chest x-ray and computed tomography (CT) findings. We compared the severity of 21 patients with PCP classified by A-DROP, CURB-65, and PSI. Also, patients’ characteristics, clinical pictures, laboratory results at first visit or admission and intervals from diagnosis to start of specific-PCP therapy were evaluated in both survivor and non-survivor groups. RESULTS: Based on A-DROP, 18 patients were classified as mild or moderate; respiratory failure developed in 15 of these 18 (83.3%), and 7/15 (46.7%) died. Based on CURB-65, 19 patients were classified as mild or moderate; respiratory failure developed in 16/19 (84.2%), and 8 of the 16 (50%) died. In contrast, PSI classified 14 as severe or extremely severe; all of the 14 (100%) developed respiratory failure and 8/14 (57.1%) died. There were no significant differences in laboratory results in these groups. The time between the initial visit and diagnosis, and the time between the initial visit and starting of specific-PCP therapy were statistically shorter in the survivor group than in the non-survivor group. CONCLUSIONS: Conventional prognostic guidelines for CAP could underestimate the severity of non-HIV PCP, resulting in a therapeutic delay resulting in high mortality. The most important factor to improve the mortality of non-HIV PCP is early diagnosis and starting of specific-PCP therapy as soon as possible. |
868 | Proteomic Investigation of Falciparum and Vivax Malaria for Identification of Surrogate Protein Markers | This study was conducted to analyze alterations in the human serum proteome as a consequence of infection by malaria parasites Plasmodium falciparum and P. vivax to obtain mechanistic insights about disease pathogenesis, host immune response, and identification of potential protein markers. Serum samples from patients diagnosed with falciparum malaria (FM) (n = 20), vivax malaria (VM) (n = 17) and healthy controls (HC) (n = 20) were investigated using multiple proteomic techniques and results were validated by employing immunoassay-based approaches. Specificity of the identified malaria related serum markers was evaluated by means of analysis of leptospirosis as a febrile control (FC). Compared to HC, 30 and 31 differentially expressed and statistically significant (p<0.05) serum proteins were identified in FM and VM respectively, and almost half (46.2%) of these proteins were commonly modulated due to both of the plasmodial infections. 13 proteins were found to be differentially expressed in FM compared to VM. Functional pathway analysis involving the identified proteins revealed the modulation of different vital physiological pathways, including acute phase response signaling, chemokine and cytokine signaling, complement cascades and blood coagulation in malaria. A panel of identified proteins consists of six candidates; serum amyloid A, hemopexin, apolipoprotein E, haptoglobin, retinol-binding protein and apolipoprotein A-I was used to build statistical sample class prediction models. By employing PLS-DA and other classification methods the clinical phenotypic classes (FM, VM, FC and HC) were predicted with over 95% prediction accuracy. Individual performance of three classifier proteins; haptoglobin, apolipoprotein A-I and retinol-binding protein in diagnosis of malaria was analyzed using receiver operating characteristic (ROC) curves. The discrimination of FM, VM, FC and HC groups on the basis of differentially expressed serum proteins demonstrates the potential of this analytical approach for the detection of malaria as well as other human diseases. |
869 | Novel Method for Isolation of Murine Clara Cell Secretory Protein-Expressing Cells with Traces of Stemness | Clara cells are non-ciliated, secretory bronchiolar epithelial cells that serve to detoxify harmful inhaled substances. Clara cells also function as stem/progenitor cells for repair in the bronchioles. Clara cell secretory protein (CCSP) is specifically expressed in pulmonary Clara cells and is widely used as a Clara cell marker. In addition CCSP promoter is commonly used to direct gene expression into the lung in transgenic models. The discovery of CCSP immunoreactivity in plasma membranes of airway lining cells prompted us to explore the possibility of enriching Clara cells by flow cytometry. We established a novel and simple method for the isolation of CCSP-expressing cell Clara cells using a combination of mechanical and enzymatic dissociation followed by flow cytometry sorting technology. We showed that ∼25% of dissociated cells from whole lung expressed CCSP. In the resulting preparation, up to 98% of cells expressed CCSP. Notably, we found that several common stem cell markers including CD44, CD133, Sca-1 and Sox2 were expressed in CCSP(+) cells. Moreover, CCSP(+) cells were able to form spheroid colonies in vitro with 0.97‰ efficiency. Parallel studies in vivo confirmed that a small population of CCSP(−)expressing cells in mouse airways also demonstrates stem cell-like properties such as label retention and harboring rare bronchioalveolar stem cells (BASCs) in terminal bronchioles (TBs). We conclude that CCSP(+) cells exhibit a number of stem cell-like features including stem cell marker expression, bronchosphere colony formation and self-renewal ability. Clara cell isolation by flow cytometry sorting is a useful method for investigating the function of primary Clara cells in stem cell research and mouse models. |
870 | Development of Real-Time PCR Array for Simultaneous Detection of Eight Human Blood-Borne Viral Pathogens | BACKGROUND: Real-time PCR array for rapid detection of multiple viral pathogens should be highly useful in cases where the sample volume and the time of testing are limited, i.e. in the eligibility testing of tissue and organ donors. FINDINGS: We developed a real-time PCR array capable of simultaneously detecting eight human viral pathogens: human immunodeficiency virus types 1 and 2 (HIV-1 and -2), hepatitis B virus (HBV), hepatitis C virus (HCV), human T-cell leukemia virus-1 and -2 (HTLV-1 and -2), vaccinia virus (VACV) and West Nile virus (WNV). One hundred twenty (120) primers were designed using a combination of bioinformatics approaches, and, after experimental testing, 24 primer sets targeting eight viral pathogens were selected to set up the array with SYBR Green chemistry. The specificity and sensitivity of the virus-specific primer sets selected for the array were evaluated using analytical panels with known amounts of viruses spiked into human plasma. The array detected: 10 genome equivalents (geq)/ml of HIV-2 and HCV, 50 geq of HIV-1 (subtype B), HBV (genotype A) and WNV. It detected 100–1,000 geq/ml of plasma of HIV-1 subtypes (A – G), group N and CRF (AE and AG) isolates. Further evaluation with a panel consisting of 28 HIV-1 and HIV-2 clinical isolates revealed no cross-reactivity of HIV-1 or HIV-2 specific primers with another type of HIV. All 28 viral isolates were identified with specific primer sets targeting the most conserved genome areas. The PCR array correctly identified viral infections in a panel of 17 previously quantified clinical plasma samples positive for HIV-1, HCV or HBV at as low as several geq per PCR reaction. CONCLUSIONS: The viral array described here demonstrated adequate performance in the testing of donors’ clinical samples. Further improvement in its sensitivity for the broad spectrum of HIV-1 subtypes is under development. |
871 | TcdC Does Not Significantly Repress Toxin Expression in Clostridium difficile 630ΔErm | In the past decade, Clostridium difficile has emerged as an important gut pathogen. Symptoms of C. difficile infection range from mild diarrhea to pseudomembranous colitis, sometimes resulting in colectomy or death. The main virulence factors of C. difficile are toxin A and toxin B. Besides the genes encoding these toxins (tcdA and tcdB), the pathogenicity locus (PaLoc) also contains genes encoding a sigma factor (tcdR) and a putative anti-sigma factor (tcdC). The important role of TcdR as a sigma factor for toxin expression is undisputed, whereas the role of TcdC as an anti-sigma factor, inhibiting toxin expression, is currently the subject of debate. To clarify the role of TcdC in toxin expression, we generated an isogenic ClosTron-based mutant of tcdC in Clostridium difficile strain 630Δ Erm (CT::tcdC) and determined the transcription levels of the PaLoc genes and the expression levels of the toxins in the wild type strain and the tcdC mutant strain. We found only minor differences in transcription levels of the PaLoc genes between the wild type and CT::tcdC strains and total toxin levels did not significantly differ either. These results suggest that in C. difficile 630Δerm TcdC is not a major regulator of toxin expression under the conditions tested. |
872 | Childhood Tuberculosis Presenting with Haemophagocytic Syndrome | Haemophagocytic syndrome is a life threatening complication of systemic infection resulting from an exaggerated immune response to a triggering agent. Prompt recognition and treatment of this disorder can abrogate otherwise high fatality associated with this disorder. A 2 year old girl presented with acute enteritis, developed prolonged fever and organomegaly complicated by multi-organ failure. She fulfilled the diagnostic criteria for haemophagocytic lymphohistiocytosis including bone marrow evidence of haemophagocytosis. In addition she had serological evidence of tubercular infection as well as a positive family history of tuberculosis. She responded rapidly to immunosuppressive therapy and anti-tubercular therapy. Our case illustrates the association of haemophagocytic syndrome with tuberculosis as well as the favourable response obtained with prompt diagnosis and treatment. |
873 | Activation of the Canonical Bone Morphogenetic Protein (BMP) Pathway during Lung Morphogenesis and Adult Lung Tissue Repair | Signaling by Bone Morphogenetic Proteins (BMP) has been implicated in early lung development, adult lung homeostasis and tissue-injury repair. However, the precise mechanism of action and the spatio-temporal pattern of BMP-signaling during these processes remains inadequately described. To address this, we have utilized a transgenic line harboring a BMP-responsive eGFP-reporter allele (BRE-eGFP) to construct the first detailed spatiotemporal map of canonical BMP-pathway activation during lung development, homeostasis and adult-lung injury repair. We demonstrate that during the pseudoglandular stage, when branching morphogenesis progresses in the developing lung, canonical BMP-pathway is active mainly in the vascular network and the sub-epithelial smooth muscle layer of the proximal airways. Activation of the BMP-pathway becomes evident in epithelial compartments only after embryonic day (E) 14.5 primarily in cells negative for epithelial-lineage markers, located in the proximal portion of the airway-tree, clusters adjacent to neuro-epithelial-bodies (NEBs) and in a substantial portion of alveolar epithelial cells. The pathway becomes activated in isolated E12.5 mesenchyme-free distal epithelial buds cultured in Matrigel suggesting that absence of reporter activity in these regions stems from a dynamic cross-talk between endoderm and mesenchyme. Epithelial cells with activated BMP-pathway are enriched in progenitors capable of forming colonies in three-dimensional Matrigel cultures. As lung morphogenesis approaches completion, eGFP-expression declines and in adult lung its expression is barely detectable. However, upon tissue-injury, either with naphthalene or bleomycin, the canonical BMP-pathways is re-activated, in bronchial or alveolar epithelial cells respectively, in a manner reminiscent to early lung development and in tissue areas where reparatory progenitor cells reside. Our studies illustrate the dynamic activation of canonical BMP-pathway during lung development and adult lung tissue-repair and highlight its involvement in two important processes, namely, the early development of the pulmonary vasculature and the management of epithelial progenitor pools both during lung development and repair of adult lung tissue-injury. |
874 | Pulmonary sequelae in a patient recovered from swine flu | The pandemic of swine flu (H1N1) influenza spread to involve the whole world rapidly. Many patients manifested a mild clinical illness but some developed pneumonia and respiratory failure. High mortality was observed in patients with severe disease. Among survivors, studies are limited. Ground-glass opacities on a high-resolution computerized tomography scan and reduced diffusion capacity were noted after 3 months in a study. But long-term complications in patients with swine flu pneumonia have not been studied well. We are presenting an unusual case of swine flu pneumonia who developed interstitial lung disease after recovery. |
875 | Dengue Virus Serotype 2 Blocks Extracellular Signal-Regulated Kinase and Nuclear Factor-κB Activation to Downregulate Cytokine Production | BACKGROUND: Dengue virus (DENV) infection is the most common mosquito-borne viral disease threatening human health around the world. Type I interferon (IFN) and cytokine production are crucial in the innate immune system. We previously reported that DENV serotype 2 (DENV-2) induced low levels of interferon regulatory factor 3 and NF-κB activation, thus leading to reduced production of IFN-β in the early phase of infection. Here, we determined whether DENV infection not only hampers type I IFN activation but also cytokine production triggered by Toll-like receptor (TLR) signaling. METHODOLOGY/PRINCIPAL FINDINGS: We used quantitative RT-PCR and found that only low levels of IFN-β and inflammatory cytokines such as interleukin 10 (IL-10), IL-12 and tumor necrosis factor α (TNFα) mRNA were detected in DENV-2–infected bone-marrow–derived dendritic cells. Furthermore, DENV-2 infection repressed cytokine production triggered by TLR signaling. To elucidate the molecular mechanisms underlying this suppression event, we measured NF-κB activation by p65 nuclear translocation and luciferase reporter assay and found that NF-κB activation triggered by TLR ligands was blocked by DENV-2 infection. As well, extracellular signal-regulated kinase (ERK) activity was suppressed by DENV-2 infection. CONCLUSIONS/SIGNIFICANCE: To downregulate the host innate immunity, DENV-2 by itself is a weak inducer of type I IFN and cytokines, furthermore DENV-2 can also block the TLR-triggered ERK–NF-κB activation and cytokine production. |
876 | High Influenza A Virus Infection Rates in Mallards Bred for Hunting in the Camargue, South of France | During the last decade, the role of wildlife in emerging pathogen transmission to domestic animals has often been pointed out. Conversely, far less attention has been paid to pathogen transmission from domestic animals to wildlife. Here, we focus on the case of game restocking, which implies the release of millions of animals worldwide each year. We conducted a 2-year study in the Camargue (Southern France) to investigate the influence of hand-reared Mallard releases on avian influenza virus dynamics in surrounding wildlife. We sampled Mallards (cloacal swabs) from several game duck facilities in 2009 and 2010 before their release. A very high (99%) infection rate caused by an H10N7 strain was detected in the game bird facility we sampled in 2009. We did not detect this strain in shot ducks we sampled, neither during the 2008/2009 nor the 2009/2010 hunting seasons. In 2010 infection rates ranged from 0 to 24% in hand-reared ducks. The 2009 H10N7 strain was fully sequenced. It results from multiple reassortment events between Eurasian low pathogenic strains. Interestingly, H10N7 strains had previously caused human infections in Egypt and Australia. The H10 and N7 segments we sequenced were clearly distinct from the Australian ones but they belonged to the same large cluster as the Egyptian ones. We did not observe any mutation linked to increased virulence, transmission to mammals, or antiviral resistance in the H10N7 strain we identified. Our results indicate that the potential role of hand-reared Mallards in influenza virus epizootics must be taken into account given the likely risk of viral exchange between game bird facilities and wild habitats, owing to duck rearing conditions. Measures implemented to limit transmission from wildlife to domestic animals as well as measures to control transmission from domestic animals to wild ones need to be equally reinforced. |
877 | Establishment of a Reverse Genetics System for Studying Human Bocavirus in Human Airway Epithelia | Human bocavirus 1 (HBoV1) has been identified as one of the etiological agents of wheezing in young children with acute respiratory-tract infections. In this study, we have obtained the sequence of a full-length HBoV1 genome (including both termini) using viral DNA extracted from a nasopharyngeal aspirate of an infected patient, cloned the full-length HBoV1 genome, and demonstrated DNA replication, encapsidation of the ssDNA genome, and release of the HBoV1 virions from human embryonic kidney 293 cells. The HBoV1 virions generated from this cell line-based production system exhibits a typical icosahedral structure of approximately 26 nm in diameter, and is capable of productively infecting polarized primary human airway epithelia (HAE) from the apical surface. Infected HAE showed hallmarks of lung airway-tract injury, including disruption of the tight junction barrier, loss of cilia and epithelial cell hypertrophy. Notably, polarized HAE cultured from an immortalized airway epithelial cell line, CuFi-8 (originally derived from a cystic fibrosis patient), also supported productive infection of HBoV1. Thus, we have established a reverse genetics system and generated the first cell line-based culture system for the study of HBoV1 infection, which will significantly advance the study of HBoV1 replication and pathogenesis. |
878 | Personal, Occupational, and Public Health Perspectives on Dealing with the First Case of Influenza A (H1N1) in the United Arab Emirates | New epidemics of infectious diseases often involve health care workers. In this short communication we present a case report of a health care professional who became the first case of influenza H1N1 virus to be notified in the United Arab Emirates. There are several issues related to workplace considerations and general public health, including preventive measures, the need for isolation of the patient, dealing with contacts, return to work, and communication with the workforce. |
879 | Human Anti-CCR4 Minibody Gene Transfer for the Treatment of Cutaneous T-Cell Lymphoma | BACKGROUND: Although several therapeutic options have become available for patients with Cutaneous T-cell Lymphoma (CTCL), no therapy has been curative. Recent studies have demonstrated that CTCL cells overexpress the CC chemokine receptor 4 (CCR4). METHODOLOGY/PRINCIPAL FINDINGS: In this study, a xenograft model of CTCL was established and a recombinant adeno-associated viral serotype 8 (AAV8) vector expressing a humanized single-chain variable fragment (scFv)-Fc fusion (scFvFc or “minibody”) of anti-CCR4 monoclonal antibody (mAb) h1567 was evaluated for curative treatment. Human CCR4(+) tumor-bearing mice treated once with intravenous infusion of AAV8 virions encoding the h1567 (AAV8-h1567) minibody showed anti-tumor activity in vivo and increased survival. The AAV8-h1567 minibody notably increased the number of tumor-infiltrating Ly-6G(+) FcγRIIIa(CD16A)(+) murine neutrophils in the tumor xenografts over that of AAV8-control minibody treated mice. Furthermore, in CCR4(+) tumor-bearing mice co-treated with AAV8-h1567 minibody and infused with human peripheral blood mononuclear cells (PBMCs), marked tumor infiltration of human CD16A(+) CD56(+) NK cells was observed. The h1567 minibody also induced in vitro ADCC activity through both mouse neutrophils and human NK cells. CONCLUSIONS/SIGNIFICANCE: Overall, our data demonstrate that the in vivo anti-tumor activity of h1567 minibody is mediated, at least in part, through CD16A(+) immune effector cell ADCC mechanisms. These data further demonstrate the utility of the AAV-minibody gene transfer system in the rapid evaluation of candidate anti-tumor mAbs and the potency of h1567 as a potential novel therapy for CTCL. |
880 | The VNTR Polymorphism of the DC-SIGNR Gene and Susceptibility to HIV-1 Infection: A Meta-Analysis | BACKGROUND: Dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin related (DC-SIGNR) can bind to the human immunodeficiency virus-1 (HIV-1) gp120 envelope glycoprotein and is thus important for the host-pathogen interaction in HIV-1 infection. Studies of the association between the variable number tandem repeat (VNTR) polymorphism of the DC-SIGNR gene and HIV-1 susceptibility have produced controversial results. METHODS AND FINDINGS: We conducted a meta-analysis of the data contained in the literature to clarify these findings. In total, 10 studies consisting of 2683 HIV-1 patients and 3263 controls (2130 healthy controls and 1133 HIV-1 exposed but seronegative (HESN) controls) were included. Odds ratios (ORs) with 95% confidence intervals (95% CIs) were assessed in the main analyses. Further stratified analyses by ethnicity and sample size were performed. By dividing the controls into two groups, healthy controls and HIV-1 exposed but seronegative (HESN) controls, we explored different genetic models to detect any association between the VNTR polymorphism and predisposition to HIV-1 infection. The results showed that the 5-repeat allele carriers (OR = 0.84, 95% CI = 0.73–0.96) and the 5/5 homozygous (OR = 0.68, 95% CI = 0.50–0.93) had significantly reduced risk when using the HIV-1 exposed but seronegative (HESN) as controls. The stratified analyses by ethnicity and sample size confirmed these findings. However, a low to moderate degree of heterogeneity was also found across studies. CONCLUSIONS: Our findings demonstrate that the VNTR polymorphism of the DC-SIGNR gene is associated with a moderate effect on host susceptibility to HIV-1 infection. Similar to the 32-bp deletion in the chemokine receptor-5 gene (CCR5Δ32), the DC-SIGNR VNTR 5-repeat allele might have a role in resistance to HIV infection, particularly in Asian populations. |
881 | Toll-like receptors are critical for clearance of Brucella and play different roles in development of adaptive immunity following aerosol challenge in mice | Brucella spp. cause undulant fever in humans and brucellosis in variety of other animals. Both innate and adaptive immunity have been shown to be important in controlling Brucella infection. Toll-like receptors (TLRs) represent a group of pattern recognition receptors (PRRs) that play critical roles in the host innate immune response, as well as development of adaptive immunity. In the current report, we investigated the role of TLR signaling in the clearance of Brucella and development of adaptive immunity in TLR2(−/−), TLR4(−/−), or MyD88(−/−) mice following aerosol exposure to B. melitensis 16 M. Consistent with previous reports, MyD88 is required for efficient clearance of Brucella from all three organs (lung, spleen, and liver). The results reveal Th2-skewed immune responses in TLR2(−/−) mice late in infection and support a TLR2 requirement for efficient clearance of Brucella from the lungs, but not from the spleen or liver. Similarly, TLR4 is required for efficient clearance of Brucella from the lung, but exhibits a minor contribution to clearance from the spleen and no demonstrable contribution to clearance from the liver. Lymphocyte proliferation assays suggest that the TLRs are not involved in the development of cell-mediated memory response to Brucella antigen. Antibody detection reveals that TLR2 and 4 are required to generate early antigen-specific IgG, but not during the late stages of infection. TLR2 and 4 are only transiently required for IgM production and not at all for IgA production. In contrast, MyD88 is essential for antigen specific IgG production late in infection, but is not required for IgM generation over the course of infection. Surprisingly, despite the prominent role for MyD88 in clearance from all tissues, MyD88-knockout mice express significantly higher levels of serum IgA. These results confirm the important role of MyD88 in controlling infection in the spleen while providing evidence of a prominent contribution to protection in other tissues. In addition, although TLR4 and TLR2 contribute little to control of spleen infection, a significant contribution to clearance of lung infection is described. |
882 | Diagnostic Devices for Isothermal Nucleic Acid Amplification | Since the development of the polymerase chain reaction (PCR) technique, genomic information has been retrievable from lesser amounts of DNA than previously possible. PCR-based amplifications require high-precision instruments to perform temperature cycling reactions; further, they are cumbersome for routine clinical use. However, the use of isothermal approaches can eliminate many complications associated with thermocycling. The application of diagnostic devices for isothermal DNA amplification has recently been studied extensively. In this paper, we describe the basic concepts of several isothermal amplification approaches and review recent progress in diagnostic device development. |
883 | Clinical characteristics of pediatric hospitalizations associated with 2009 pandemic influenza A (H1N1) in Northern Bavaria, Germany | BACKGROUND: The 2009 pandemic influenza A (H1N1) (PIA) virus infected large parts of the pediatric population with a wide clinical spectrum and an initially unknown complication rate. The aims of our study were to define clinical characteristics and outcome of pandemic influenza A (H1N1) 2009-associated hospitalizations (PIAH) in children <18 years of age. All hospitalized cases of children <18 years of age with laboratory-confirmed pandemic influenza A (H1N1) 2009 in the region of Wuerzburg (Northern Bavaria, Germany) between July 2009 and March 2010 were identified. For these children a medical chart review was performed to determine their clinical characteristics and complications. RESULTS: Between July 2009 and March 2010, 94 PIAH (62% males) occurred in children <18 years of age, with a median age of 7 years (IQR: 3–12 years). Underlying diseases and predisposing factors were documented in 40 (43%) children; obesity (n = 12, 30%), asthma (n = 10, 25%) and neurologic disorders (n = 8, 20%) were most frequently reported. Sixteen (17%) children received oxygen supplementation; three (3%) children required mechanical ventilation. Six (6%) children were admitted to an intensive care unit, four of them with underlying chronic diseases. CONCLUSIONS: Most PIAH demonstrated a benign course of disease. However, six children (6%) needed treatment at an intensive care unit for severe complications. |
884 | Pip6-PMO, A New Generation of Peptide-oligonucleotide Conjugates With Improved Cardiac Exon Skipping Activity for DMD Treatment | Antisense oligonucleotides (AOs) are currently the most promising therapeutic intervention for Duchenne muscular dystrophy (DMD). AOs modulate dystrophin pre-mRNA splicing, thereby specifically restoring the dystrophin reading frame and generating a truncated but semifunctional dystrophin protein. Challenges in the development of this approach are the relatively poor systemic AO delivery and inefficient dystrophin correction in affected non-skeletal muscle tissues, including the heart. We have previously reported impressive heart activity including high-splicing efficiency and dystrophin restoration following a single administration of an arginine-rich cell-penetrating peptide (CPPs) conjugated to a phosphorodiamidate morpholino oligonucleotide (PMO): Pip5e-PMO. However, the mechanisms underlying this activity are poorly understood. Here, we report studies involving single dose administration (12.5 mg/kg) of derivatives of Pip5e-PMO, consecutively assigned as Pip6-PMOs. These peptide-PMOs comprise alterations to the central hydrophobic core of the Pip5e peptide and illustrate that certain changes to the peptide sequence improves its activity; however, partial deletions within the hydrophobic core abolish its efficiency. Our data indicate that the hydrophobic core of the Pip sequences is critical for PMO delivery to the heart and that specific modifications to this region can enhance activity further. The results have implications for therapeutic PMO development for DMD. |
885 | Clinical characteristics and outcome of Penicillium marneffei infection among HIV-infected patients in northern Vietnam | OBJECTIVE: This study reports the clinical characteristics and outcome of HIV-associated Penicilliummarneffei infection in northern Vietnam. METHODS: We conducted a retrospective chart review of all patients with laboratory confirmed Penicilliummarneffei infection admitted to the National Hospital for Tropical Diseases in Hanoi, Vietnam, between July 2006 and September 2009. RESULTS: 127 patients with P. marneffei infection were identified. All were HIV-infected; median CD4+ T-cell count was 24 cells/μl (IQR:12-48); 76% were men. Common clinical features were fever (92.9%), skin lesions (82.6%), hepatomegaly (61.4%), lymphadenopathy (40.2%), weight loss (59.1%) and cough (49.6%). Concurrent opportunistic infections were present in 22.0%; half of those had tuberculosis. Initial treatment regimens were: itraconazole or ketoconazole capsule (77.2%), amphotericin B (20.5%), and fluconazole (1.6%). In-hospital mortality was 12.6% and showed no significant difference in patients treated with itraconazole (or ketoconazole) and amphotericin B (p = 0.43). Dyspnea, ascites, and increased LDH level were independent predictors of mortality. No seasonality was observed. CONCLUSION: The clinical features, treatments and outcomes of HIV-associated P. marneffei infection in northern Vietnam are similar to those reported in other endemic regions. Dyspnea was an important predictor of mortality. More patients were treated with itraconazole than amphotericin B and no significant difference in treatment outcome was observed. It would be of clinical value to compare the efficacy of oral itraconazole and amphotericin B in a clinical trial. |
886 | Prospective application of clinician-performed lung ultrasonography during the 2009 H1N1 influenza A pandemic: distinguishing viral from bacterial pneumonia | BACKGROUND: Emergency department visits quadrupled with the initial onset and surge during the 2009 H1N1 influenza pandemic in New York City from April to June 2009. This time period was unique in that >90% of the circulating virus was surveyed to be the novel 2009 H1N1 influenza A according to the New York City Department of Health. We describe our experience using lung ultrasound in a case series of patients with respiratory symptoms requiring chest X-ray during the initial onset and surge of the 2009 H1N1 influenza pandemic. METHODS: We describe a case series of patients from a prospective observational cohort study of lung ultrasound, enrolling patients requiring chest X-ray for suspected pneumonia that coincided with the onset and surge of the 2009 H1N1 influenza pandemic. RESULTS: Twenty pandemic 2009 H1N1 influenza patients requiring chest X-ray were enrolled during this time period. Median age was 6.7 years. Lung ultrasound via modified Bedside Lung Ultrasound in Emergency protocol assisted in the identification of viral pneumonia (n = 15; 75%), viral pneumonia with superimposed bacterial pneumonia (n = 7; 35%), isolated bacterial pneumonia only (n = 1; 5%), and no findings of viral or bacterial pneumonia (n = 4; 20%) in this cohort of patients. Based on 54 observations, interobserver agreement for distinguishing viral from bacterial pneumonia using lung ultrasound was ĸ = 0.82 (0.63 to 0.99). CONCLUSIONS: Lung ultrasound may be used to distinguish viral from bacterial pneumonia. Lung ultrasound may be useful during epidemics or pandemics of acute respiratory illnesses for rapid point-of-care triage and management of patients. |
887 | Serum Levels of Gelatinase Associated Lipocalin as Indicator of the Inflammatory Status in Coronary Artery Disease | Background. Atherosclerosis is a chronic inflammatory disease and the acute clinical manifestations represent acute on chronic inflammation. Neutrophil gelatinase-associated lipocalin (NGAL) is found in the granules of human neutrophils, with many diverse functions. The aim of this study was to evaluate the hypothesis that levels NGAL in blood may reflect the inflammatory process in various stages of coronary artery disease. Methods. We studied 140 patients, with SA 40, UA 35, NSTEMI 40, and STEMI 25, and 20 healthy controls. Serum NGAL was measured upon admission and before coronary angiography. Results. Significant differences were observed in median serum-NGAL(ng/mL) between patients with SA (79.23 (IQR, 37.50–100.32)), when compared with UA (108.00 (68.34–177.59)), NSTEMI (166.49 (109.24–247.20)), and STEMI (178.63 (111.18–305.92)) patients and controls (50.31 (44.30–69.78)) with significant incremental value from SA to STEMI. We observed a positive and significant correlation between serum-NGAL and hs-CRP (spearman coefficient rho = 0.685, P < 0.0001) as well as with neutrophil counts (r = 0.511, P < 0.0001). Conclusions. In patients with coronary artery disease serum levels of NGAL increase and reflect the degree of inflammatory process. In patients with acute coronary syndromes, serum levels of NGAL have high negative predictive value and reflecting the inflammatory status could show the severity of coronary clinical syndrome. |
888 | Activity based protein profiling to detect serine hydrolase alterations in virus infected cells | Activity-based protein profiling (ABPP) is a newly emerging technique that uses active site-directed probes to monitor the functional status of enzymes. Serine hydrolases are one of the largest families of enzymes in mammals. More than 200 serine hydrolases have been identified, but little is known about their specific roles. Serine hydrolases are involved in a variety of physiological functions, including digestion, immune response, blood coagulation, and reproduction. ABPP has been used recently to investigate host–virus interactions and to understand the molecular pathogenesis of virus infections. Monitoring the altered serine hydrolases during viral infection gives insight into the catalytic activity of these enzymes that will help to identify novel targets for diagnostic and therapeutic application. This review presents the usefulness of ABPP in detecting and analyzing functional annotation of host cell serine hydrolases as a result of host–virus interaction. |
889 | Murphy's law—if anything can go wrong, it will: Problems in phage electron microscopy | The quality of bacteriophage electron microscopy appears to be on a downward course since the 1980s. This coincides with the introduction of digital electron microscopes and a general lowering of standards, possibly due to the disappearance of several world-class electron microscopists The most important problem seems to be poor contrast. Positive staining is frequently not recognized as an undesirable artifact. Phage parts, bacterial debris, and aberrant or damaged phage particles may be misdiagnosed as bacterial viruses. Digital electron microscopes often seem to be operated without magnification control because this is difficult and inconvenient. In summary, most phage electron microscopy problems may be attributed to human failure. Journals are a last-ditch defense and have a heavy responsibility in selecting competent reviewers and rejecting, or not, unsatisfactory articles. |
890 | Herbal Products: Benefits, Limits, and Applications in Chronic Liver Disease | Complementary and alternative medicine soughts and encompasses a wide range of approaches; its use begun in ancient China at the time of Xia dynasty and in India during the Vedic period, but thanks to its long-lasting curative effect, easy availability, natural way of healing, and poor side-effects it is gaining importance throughout the world in clinical practice. We conducted a review describing the effects and the limits of using herbal products in chronic liver disease, focusing our attention on those most known, such as quercetin or curcumin. We tried to describe their pharmacokinetics, biological properties, and their beneficial effects (as antioxidant role) in metabolic, alcoholic, and viral hepatitis (considering that oxidative stress is the common pathway of chronic liver diseases of different etiology). The main limit of applicability of CAM comes from the lacking of randomized, placebo-controlled clinical trials giving a real proof of efficacy of those products, so that anecdotal success and personal experience are frequently the driving force for acceptance of CAM in the population. |
891 | Identification of pneumonia and influenza deaths using the death certificate pipeline | BACKGROUND: Death records are a rich source of data, which can be used to assist with public surveillance and/or decision support. However, to use this type of data for such purposes it has to be transformed into a coded format to make it computable. Because the cause of death in the certificates is reported as free text, encoding the data is currently the single largest barrier of using death certificates for surveillance. Therefore, the purpose of this study was to demonstrate the feasibility of using a pipeline, composed of a detection rule and a natural language processor, for the real time encoding of death certificates using the identification of pneumonia and influenza cases as an example and demonstrating that its accuracy is comparable to existing methods. RESULTS: A Death Certificates Pipeline (DCP) was developed to automatically code death certificates and identify pneumonia and influenza cases. The pipeline used MetaMap to code death certificates from the Utah Department of Health for the year 2008. The output of MetaMap was then accessed by detection rules which flagged pneumonia and influenza cases based on the Centers of Disease and Control and Prevention (CDC) case definition. The output from the DCP was compared with the current method used by the CDC and with a keyword search. Recall, precision, positive predictive value and F-measure with respect to the CDC method were calculated for the two other methods considered here. The two different techniques compared here with the CDC method showed the following recall/ precision results: DCP: 0.998/0.98 and keyword searching: 0.96/0.96. The F-measure were 0.99 and 0.96 respectively (DCP and keyword searching). Both the keyword and the DCP can run in interactive form with modest computer resources, but DCP showed superior performance. CONCLUSION: The pipeline proposed here for coding death certificates and the detection of cases is feasible and can be extended to other conditions. This method provides an alternative that allows for coding free-text death certificates in real time that may increase its utilization not only in the public health domain but also for biomedical researchers and developers. TRIAL REGISTRATION: This study did not involved any clinical trials. |
892 | Functional Analysis of Rift Valley Fever Virus NSs Encoding a Partial Truncation | Rift Valley fever virus (RVFV), belongs to genus Phlebovirus of the family Bunyaviridae, causes high rates of abortion and fetal malformation in infected ruminants as well as causing neurological disorders, blindness, or lethal hemorrhagic fever in humans. RVFV is classified as a category A priority pathogen and a select agent in the U.S., and currently there are no therapeutics available for RVF patients. NSs protein, a major virulence factor of RVFV, inhibits host transcription including interferon (IFN)-β mRNA synthesis and promotes degradation of dsRNA-dependent protein kinase (PKR). NSs self-associates at the C-terminus 17 aa., while NSs at aa.210–230 binds to Sin3A-associated protein (SAP30) to inhibit the activation of IFN-β promoter. Thus, we hypothesize that NSs function(s) can be abolished by truncation of specific domains, and co-expression of nonfunctional NSs with intact NSs will result in the attenuation of NSs function by dominant-negative effect. Unexpectedly, we found that RVFV NSs truncated at aa. 6–30, 31–55, 56–80, 81–105, 106–130, 131–155, 156–180, 181–205, 206–230, 231–248 or 249–265 lack functions of IFN–β mRNA synthesis inhibition and degradation of PKR. Truncated NSs were less stable in infected cells, while nuclear localization was inhibited in NSs lacking either of aa.81–105, 106–130, 131–155, 156–180, 181–205, 206–230 or 231–248. Furthermore, none of truncated NSs had exhibited significant dominant-negative functions for NSs-mediated IFN-β suppression or PKR degradation upon co-expression in cells infected with RVFV. We also found that any of truncated NSs except for intact NSs does not interact with RVFV NSs even in the presence of intact C-terminus self-association domain. Our results suggest that conformational integrity of NSs is important for the stability, cellular localization and biological functions of RVFV NSs, and the co-expression of truncated NSs does not exhibit dominant-negative phenotype. |
893 | Predictive and Reactive Distribution of Vaccines and Antivirals during Cross-Regional Pandemic Outbreaks | As recently pointed out by the Institute of Medicine, the existing pandemic mitigation models lack the dynamic decision support capability. We develop a large-scale simulation-driven optimization model for generating dynamic predictive distribution of vaccines and antivirals over a network of regional pandemic outbreaks. The model incorporates measures of morbidity, mortality, and social distancing, translated into the cost of lost productivity and medical expenses. The performance of the strategy is compared to that of the reactive myopic policy, using a sample outbreak in Fla, USA, with an affected population of over four millions. The comparison is implemented at different levels of vaccine and antiviral availability and administration capacity. Sensitivity analysis is performed to assess the impact of variability of some critical factors on policy performance. The model is intended to support public health policy making for effective distribution of limited mitigation resources. |
894 | A Novel Vaccine Using Nanoparticle Platform to Present Immunogenic M2e against Avian Influenza Infection | Using peptide nanoparticle technology, we have designed two novel vaccine constructs representing M2e in monomeric (Mono-M2e) and tetrameric (Tetra-M2e) forms. Groups of specific pathogen free (SPF) chickens were immunized intramuscularly with Mono-M2e or Tetra-M2e with and without an adjuvant. Two weeks after the second boost, chickens were challenged with 107.2 EID50 of H5N2 low pathogenicity avian influenza (LPAI) virus. M2e-specific antibody responses to each of the vaccine constructs were tested by ELISA. Vaccinated chickens exhibited increased M2e-specific IgG responses for each of the constructs as compared to a non-vaccinated group. However, the vaccine construct Tetra-M2e elicited a significantly higher antibody response when it was used with an adjuvant. On the other hand, virus neutralization assays indicated that immune protection is not by way of neutralizing antibodies. The level of protection was evaluated using quantitative real time PCR at 4, 6, and 8 days post-challenge with H5N2 LPAI by measuring virus shedding from trachea and cloaca. The Tetra-M2e with adjuvant offered statistically significant (P < 0.05) protection against subtype H5N2 LPAI by reduction of the AI virus shedding. The results suggest that the self-assembling polypeptide nanoparticle shows promise as a potential platform for a development of a vaccine against AI. |
895 | Factors Associated with Increased Risk Perception of Pandemic Influenza in Australia | The aim of this study was to assess factors associated with increased risk perception of pandemic influenza in Australia. The sample consisted of 2081 Australian adults aged 16 years and older who completed a short three item pandemic influenza question module which was incorporated into the NSW Health Adult Population Health Survey during the first quarter of 2007. After adjusting for covariates, multivariate analysis indicated that those living in rural regions were significantly more likely to perceive a high risk that a pandemic influenza would occur, while those with poor self-rated health perceived both a high likelihood of pandemic and high concern that self/family would be directly affected were such an event to occur. Those who spoke a language other than English at home and those on low incomes and younger people (16–24 years) were significantly more likely to have changed the way they lived their lives due to the possibility of pandemic influenza, compared to those who spoke only English at home, middle-high income earners, and older age groups, respectively. This data provides an Australian population baseline against which the risk perceptions of demographic subgroups regarding the current, and potential future pandemics, can be compared and monitored. |
896 | Blow Flies Were One of the Possible Candidates for Transmission of Highly Pathogenic H5N1 Avian Influenza Virus during the 2004 Outbreaks in Japan | The 2003-2004 H5N1 highly pathogenic avian influenza (HPAI) outbreaks in Japan were the first such outbreaks in 79 years in Japan. Epidemic outbreaks have been occurring in Southeast Asia, with the most recent in 2010. Knowledge of the transmission route responsible for the HPAI outbreaks in these countries remains elusive. Our studies strongly suggested that field and laboratory studies focusing on mechanical transmission by blow flies should be considered to control H5N1 avian influenza outbreaks, in particular in epidemic areas, where there are high densities of different fly species throughout the year. In this paper, we review these field and laboratory entomological studies and discuss the possibility of blow flies transmitting H5N1 viruses. |
897 | Examining the knowledge, attitudes and practices of domestic and international university students towards seasonal and pandemic influenza | BACKGROUND: Prior to the availability of the specific pandemic vaccine, strategies to mitigate the impact of the disease typically involved antiviral treatment and “non-pharmaceutical” community interventions. However, compliance with these strategies is linked to risk perceptions, perceived severity and perceived effectiveness of the strategies. In 2010, we undertook a study to examine the knowledge, attitudes, risk perceptions, practices and barriers towards influenza and infection control strategies amongst domestic and international university students. METHODS: A study using qualitative methods that incorporated 20 semi-structured interviews was undertaken with domestic and international undergraduate and postgraduate university students based at one university in Sydney, Australia. Participants were invited to discuss their perceptions of influenza (seasonal vs. pandemic) in terms of perceived severity and impact, and attitudes towards infection control measures including hand-washing and the use of social distancing, isolation or cough etiquette. RESULTS: While participants were generally knowledgeable about influenza transmission, they were unable to accurately define what ‘pandemic influenza’ meant. While avian flu or SARS were mistaken as examples of past pandemics, almost all participants were able to associate the recent “swine flu” situation as an example of a pandemic event. Not surprisingly, it was uncommon for participants to identify university students as being at risk of catching pandemic influenza. Amongst those interviewed, it was felt that ‘students’ were capable of fighting off any illness. The participant’s nominated hand washing as the most feasible and acceptable compared with social distancing and mask use. CONCLUSIONS: Given the high levels of interaction that occurs in a university setting, it is really important that students are informed about disease transmission and about risk of infection. It may be necessary to emphasize that pandemic influenza could pose a real threat to them, that it is important to protect oneself from infection and that infection control measures can be effective. |
898 | Use of functional gene arrays for elucidating in situ biodegradation | Microarrays have revolutionized the study of microbiology by providing a high-throughput method for examining thousands of genes with a single test and overcome the limitations of many culture-independent approaches. Functional gene arrays (FGA) probe a wide range of genes involved in a variety of functions of interest to microbial ecology (e.g., carbon degradation, N fixation, metal resistance) from many different microorganisms, cultured and uncultured. The most comprehensive FGA to date is the GeoChip array, which targets tens of thousands of genes involved in the geochemical cycling of carbon, nitrogen, phosphorus, and sulfur, metal resistance and reduction, energy processing, antibiotic resistance and contaminant degradation as well as phylogenetic information (gyrB). Since the development of GeoChips, many studies have been performed using this FGA and have shown it to be a powerful tool for rapid, sensitive, and specific examination of microbial communities in a high-throughput manner. As such, the GeoChip is well-suited for linking geochemical processes with microbial community function and structure. This technology has been used successfully to examine microbial communities before, during, and after in situ bioremediation at a variety of contaminated sites. These studies have expanded our understanding of biodegradation and bioremediation processes and the associated microorganisms and environmental conditions responsible. This review provides an overview of FGA development with a focus on the GeoChip and highlights specific GeoChip studies involving in situ bioremediation. |
899 | Functional Studies of ssDNA Binding Ability of MarR Family Protein TcaR from Staphylococcus epidermidis | The negative transcription regulator of the ica locus, TcaR, regulates proteins involved in the biosynthesis of poly-N-acetylglucosamine (PNAG). Absence of TcaR increases PNAG production and promotes biofilm formation in Staphylococci. Previously, the 3D structure of TcaR in its apo form and its complex structure with several antibiotics have been analyzed. However, the detailed mechanism of multiple antibiotic resistance regulator (MarR) family proteins such as TcaR is unclear and only restricted on the binding ability of double-strand DNA (dsDNA). Here we show by electrophoretic mobility shift assay (EMSA), electron microscopy (EM), circular dichroism (CD), and Biacore analysis that TcaR can interact strongly with single-stranded DNA (ssDNA), thereby identifying a new role in MarR family proteins. Moreover, we show that TcaR preferentially binds 33-mer ssDNA over double-stranded DNA and inhibits viral ssDNA replication. In contrast, such ssDNA binding properties were not observed for other MarR family protein and TetR family protein, suggesting that the results from our studies are not an artifact due to simple charge interactions between TcaR and ssDNA. Overall, these results suggest a novel role for TcaR in regulation of DNA replication. We anticipate that the results of this work will extend our understanding of MarR family protein and broaden the development of new therapeutic strategies for Staphylococci. |
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